Heavy Metal Effects on Glia

Heavy Metal Effects on Glia Evelyn Tiffany-Castiglioni,* Marie E. Legare, Lora A. Schneider, Edward D. Harris, Rola Barhoumi, Jan Zmudzki, Yongchang Qian, and Robert C. Burghardt

Introduction Heavy metals can contribute to neurologic and mental dysfunction, either as a result of metabolic trace metal imbalances, as in the case of brain copper (Cu) deficiency in Menkes' disease (1), or as a result of central nervous system (CNS) exposure to environmental toxicants, such as lead (Pb). Most of the research efforts from our laboratory concerning interactions of heavy metals with glia have focused on the effects of Pb on astroglia in culture. We have also investigated some interactions of Cu and other metals with these cells. This chapter will describe the methods used by the authors to measure heavy metal accumulation by rat astroglial cells in culture, as well as the effects of metal exposure on cell function. Methods described will include atomic absorption spectroscopy, determination of influx and efflux kinetics constants, and use of cellular fluorescence imaging techniques for analysis of cell functions. Data from this laboratory indicate that neural cells in culture respond to a variety of toxic or viral insults by a limited number of functional alterations, including disruptions of cytosolic glutathione content, mitochondrial membrane potential, C a 2+ homeostasis, and gap junctional intercellular communication (GJIC). Therefore we shall describe the imaging techniques we use to analyze those events. Inorganic lead is recognized to be a cause of neurobehavioral deficits in children at blood Pb levels exceeding 10-15 /xg/dl (2). Two goals of lead neurotoxicity research are to define mechanisms of Pb uptake and tolerance in CNS cells that accumulate Pb and to identify molecular and cellular alterations that underlie behavioral deficits. Cell and tissue cultures are practical tools with which to pursue these goals, offering such advantages over in vivo models as defined cell types, an extracellular environment that can be precisely manipulated, and direct observation. Cell culture studies of Pb neurotoxicity have been reviewed recently (3). In early cell and tissue culture studies of Pb neurotoxicity, investigators attempted to identify organ and

* To whom correspondence should be addressed. Methods in Neurosciences, Volume 30

Copyright 9 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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tissue targets of Pb toxicity and employed rudimentary measurements of cytotoxicity, such as viability and cell counts. Pb doses studied were typically high and of short duration. The collective data from these studies suggest differential sensitivity to Pb toxicity among various types of cultured neural cells, ranked as follows from most to least sensitive: myelinating cells (oligodendroglia and Schwann cells), neurons, and astroglia. In addition, astroglia were shown to take up and store large amounts of Pb intracellularly, a phenomenon resembling the Pb-sequestering ability hypothesized for mature astroglia in vivo. Subsequent studies have been more specific in their objectives, seeking to identify organelle and enzyme targets of Pb. Prelethal subcellular effects, such as blockage of ion channels and inhibition of enzymes by Pb, were measured as toxic end points, and the effects of low, more toxicologically relevant, Pb levels were addressed. Current research is now attempting to characterize alterations in discrete molecular targets, particularly those whose effects in the cell may be metabolically amplified. Putative cellular defense mechanisms are an important focus of this research effort, particularly in astroglia, which can survive and function in the presence of high intracellular lead concentrations. Given the slow turnover of Pb in the brain, mechanisms for tolerance are of considerable importance. Astroglial cultures prepared from immature rodent brain are very well characterized, possessing many of the features of astroglia in vivo (4). Several procedures have been described for culturing astroglia. We use a modification of the method of McCarthy (5) to prepare primary astroglial cultures from the cerebral hemispheres of 0 to 3-day-old Spraque-Dawley rat pups. Cultures produced in this manner have previously been shown by immunocytochemical staining for glial fibrillary acidic protein (GFAP) to be nearly pure astroglia (6). Primary cultures, first-passage cultures, and glial cell lines such as the C6 rat glioma (American Type Culture Collection, Rockville, MD) are all amenable to the analytical methods described below. Analysis of Metal Content" Atomic Absorption Spectroscopy

Rationale for Analysis by Atomic Absorption Spectroscopy Several studies have shown that astroglia accumulate Pb from the culture medium and store it intracellularly (reviewed in Ref. 3). This finding is in agreement with morphological observations in vivo that astroglia take up Pb (7, 8). Pb accumulation by astroglia in culture is accompanied by transient alterations in intracellular Fe and Cu levels (6). Furthermore, astroglia in vitro also accumulate Fe from the culture medium when exposed to high extracellular concentrations (6). These findings suggest a role for astroglia

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in heavy metal sequestration in cases of abnormally high exposure to certain metals. The most commonly used method for trace element determination is atomic absorption spectroscopy (AAS). AAS operates on the principal that atoms of an element absorb radiation from an atomic emission light source at characteristic ground-state energy levels. As a result, the decrease in intensity is directly proportional to the concentration of the element in the sample. Atomization of the sample is necessary for elemental detection. Two major methods of atomization are available: the flame method and the furnace method, the latter of which is also called electrothermal atomization (ETA). The flame method requires a sample volume of at least 1 ml and can detect lead down to the level of parts per million (ppm). The ETA method, on the other hand, can operate with a smaller sample volume (1-100/A) and can detect at the level of parts per billion (ppb). We use ETA-AAS because of its more sensitive detection limit; furthermore, sample preparation for this method is amenable to cell culture. Other analytical methods are available for measuring metals in biological samples, such as inductively coupled plasma atomic emission spectroscopy (ICP-AES) and neutron activation analysis (NAA). The main advantage of ICP-AES and NAA is that multielement analyses can simultaneously be performed with the same sample. However, there are several constraints with these methods that may reduce their application, particularly for routine procedures, including cost of analysis, complicated preparation procedures, and limited detection ability for some elements (methods reviewed in Refs. 9 and 10).

A t o m i c Absorption M e t h o d s Primary or first-passage cultures of astroglia are seeded in T-75 tissue culture flasks (Corning, Oneonta, NY). The seeding density we use for first-passage astroglia is 2 x 10 6 cells/flask. C6 rat glioma cells have also been studied successfully. We typically culture astroglia in Waymouth' s 705/1 MD medium (GIBCO/BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS; various vendors, depending on growth characteristics of the serum lot), and C6 cells in a mixture (1:1, v/v) of Dulbecco's modified Eagle's medium/Ham's F12 medium (DMEM/F12; GIBCO/BRL) supplemented with 10% FBS. Cultures are exposed to Pb in cell culture medium at desired concentrations for lengths of time that vary from less than 1 day to several weeks, after which the cells are harvested from the flasks by the following steps. First, the medium is removed and the cell monolayer is washed one time in situ with 4-5 ml HEPES-buffered saline solution containing EDTA (HBS) by gently tilting the flask from side to side. HBS consists of the

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following components (g/liter): NaCI, 8.0; KC1, 0.4; N a z H P O 4 , 0.1; HEPES buffer, 2.38; dextrose, 1.0; and EDTA, 0.29225 (Sigma, St. Louis, MO). The resulting solution is adjusted to pH 7.2-7.4 and filter sterilized (0.2 /xm VacuCap-90; Gelman Sciences, Ann Arbor, MI). Next, the HBS solution is removed and the cells are dislodged with trypsin. For trypsinization, we add 1.5-2.0 ml trypsin, 0.25%, to the flask, tilting it to bathe all the cells. Trypsin is prepared from 2.5% stock solution (Sigma) freshly diluted 1:9 with HBS. The flask is then incubated at 37~ for 3-5 minutes. Next we add 10 ml HBS solution to the flask, dislodging the cells into the solution by means of a pipette or by rapping the flask on the counter. The suspended cells are collected by pipette into one 15-ml disposable polystyrene centrifuge tube per flask. The cells are centrifuged at 800-1000 g for 5 minutes at room temperature to form a pellet. The cells are then washed twice in the same tubes by resuspending the pellet with a pipette in 10 ml fresh HBS, centrifuging, and removing the HBS with a Pasteur pipette connected to a vacuum hose and bottle. After the second wash, and prior to the last centrifugation, three small aliquots of well-suspended cells (total about 300/zl) are counted with a hemocytometer, and an average cell number per flask is calculated. Results are expressed as nanograms Pb/2 • 106 cells, although other methods of expression may also be useful (e.g., nanograms Pb per unit of protein or DNA). After the final centrifugation step, the HBS is aspirated with a Pasteur pipette and the cell pellet is allowed to dry overnight in the uncapped tubes placed on their sides in the tissue culture hood under fluorescent light. Once dried, the samples may be immediately processed for ETA-AAS or capped and stored at -20~ It is best to process and analyze as soon as possible to prevent loss of metal from leeching into the tube. The cell pellet is now ready for digestion and subsequent atomic absorption spectroscopy. Concentrated nitric acid (200/zl; Ultrex, J. T. Baker, Phillipsburg, NJ) is added to each tube and the suspension is vortexed for 20 seconds, then allowed to sit overnight at room temperature. The digested cells are diluted with 1.8 ml of matrix modifiers. The matrix modifiers contain 0.5% nitric acid and 1% ammonium phosphate in a 2:1 ratio (v/v). The diluted cell solution is vortexed for 20 seconds and centrifuged for 5-6 minutes at 2000 rpm (800 g). Total lead, copper, iron, and zinc are measured by atomic absorption spectroscopy with a Thermo Jarrell Ash Smith-Heiftje 12 spectrometer with furnace atomizer, model 188. Determination is by injection of 10-20/zl of digestion solution with drying, ashing, and atomization in accordance with optimum parameters for each element as suggested by the "Methods Manual for Furnace Operation," Vol. II (11). The parameters, such as ashing times and temperatures and slit width, are not given in this chapter because they vary with type of instrumentation used. All materials that come in contact with the samples, such as flasks and tubes, must be Pb

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free. We therefore recommend the use of disposable plasticware and glass Pasteur pipettes for all procedures.

Interpretation and Limitations of Lead Measurement by Atomic Absorption Spectroscopy The limit of detection for Pb in our laboratory by the ETA-AAS method described is 5/zg/1 (ppb). The technique may be insufficient for some applications, such as determinations of influx and efflux kinetics or detection of Pb levels in cells exposed to very low levels of Pb (e.g., <0.1 /xM total Pb in the medium). Furthermore, a large number of cells (about 4 x 106) is needed for each determination, even in the case of astroglia and C6 glioma cells, which readily accumulate Pb in culture. Therefore, the technique is not amenable to measurements of Pb in rat oligodendroglia or neurons, which are much more difficult to culture in large numbers. The technique measures total Pb (or other metal) concentration, but does not provide a measurement of the form in which the metal is found (e.g., free ion, protein bound, salt precipitate), nor does it indicate the specific site of lead in the cell (e.g., nuclei, mitochondria, lysosomes).

Analysis of Metal Transport" Influx and Effiux Kinetics C o n s t a n t s

Rationale for Measurement of Kinetics Constants Several possible mechanisms can be invoked for metal entry into astroglia, such as receptor-mediated transport (vis-~t-vis Fe 2§ transport by transferrin), endocytosis and/or pinocytosis, ion channels, or an anion-exchange transport system. These areas might be fruitfully explored by characterizing the kinetics of metal transport into astroglia in the presence of various chemical blockers or metabolic inhibitors. Our investigations of kinetics constants for metal transport in astroglia have thus far focused on copper. As previously mentioned, copper accumulates in cultured astroglial cells exposed to Pb. Intracellular accumulation of copper has also been described for fibroblasts from patients with Menkes' disease. One of the main clinical manifestations in Menkes' disease is neurological degeneration (1). In this lethal genetic disorder, copper metabolism is greatly disrupted, resulting in Cu accumulation by the intestinal epithelium and Cu deficiency in the brain. The intracellular Cu content of Menkes' fibroblasts is more than five times that of normal cells (12). Astroglial cells cultured from

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the macular mouse, an animal model of Menkes' disease, also accumulate an excessive amount of copper (13). The overall Cu deficiency in brain, accompanied by Cu accumulation in astroglia, suggests a defect in the transport or release of Cu from astroglia to neurons. Thus, the astroglial cell is of interest for its propensity to accumulate Cu in both Menkes' disease and lead toxicity. To date, the mechanism of copper transport into astroglia is not understood. Consequently we have no knowledge of transport parameters, particularly the kinetic constants that relate to the rates of uptake and efflux. Here we describe a method that uses 67CH to determine the K m and Vmax for Cu in cultured astroglial cells.

Methods f o r Measurement o f Kinetics Constants Time Dependence of 67Cu(II) Uptake Glial cells (first-passage astroglia or C6 glioma cells) are seeded at high density in 35-mm tissue culture dishes and grown for 2-3 days to a final cell density of about 3 x 10 6 cells/dish. Prior to the analysis, the medium is removed from the cultures and the cultures are washed with 2 ml of Dulbecco's phosphate-buffered saline (DPBS, Irvine Scientific, Santa Ana, CA) at room temperature for 30 seconds. The cultures are refed with 2 ml of a mixture (1 : 1, v/v) of Dulbecco's modified Eagle's medium/Ham's F12 medium. This medium is serum free to minimize interference by plasma proteins that bind copper. A final concentration of 50 nM 67CHC12 (Brookhaven National Laboratory, Raton, NY; specific activity 256 Ci/mmol), carrier-free, is then added to the cultures. The radioactive medium is removed after various incubation times (e.g., 10, 20, and 50 minutes at 37~ and the cells are washed with 2 ml of 150 mM NaC1 (adjusted to pH 4.0 with 0.1M HC1) at room temperature for 30 seconds. The cells are harvested by scraping the dish bottom with a cell scraper after adding 1 ml of 0.5 N NaOH. The cell lysate is completely transferred to a 4-ml vial for counting the quantity of 67Cu(II) retained in the cells with a gamma counter. The uptake of 67C11 is expressed as picomoles Cu/mg protein. Protein is assayed with bicinchoninic acid (BCA; Pierce, Rockford, IL) according to Pierce's assay protocol. A typical time-course analysis shows a progressive uptake for the 50-minute incubation period. cells can be used to No uptake is observed at 4~ The 67CH in 4~ correct for radioactivity that adheres to the cell membrane.

Determination of Kinetic Parameters (Km and Vmax): Uptake of 67Cu(II) The cells are plated as described above and washed with 2 ml DPBS for 30 seconds at room temperature. Fresh, serum-free DMEM/F12 medium containing the equivalent of 10, 20, 50, 100, and 200 nM of 67CuCI2 is carefully

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pipetted over the cell layer, 2 ml/dish. After a 10-minute incubation at 37~ the radioactive medium is removed and cells are washed with 2 ml of 150 mM NaC1 (adjusted to pH 4.0) at room temperature for 30 seconds. Copper67 uptake for each concentration of 67Cu is expressed as picomoles Cu/mg protein/minute. A double reciprocal plot of velocity v e r s u s 67Cu concentration is determined to asses the apparent K m and gmax parameters. These data are shown in Fig. 1. A straight-line relationship is obtained by plotting 1/pmol Cu/mg protein/minute v e r s u s 1/[67Cu]. Values for apparent K m and gma x ar e calculated on the basis of the straight-line relationship of the Michaelis-Menten equation: llv-

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which graphically is equivalent to y = b + m x , where b is the intersecting point on the y axis (1/Vmax), and m is the slope, equal to K m / V m a x . The calculation of x when y equals zero sets x equal to - 1 / K m, which is an alternative and perhaps more convenient method for determining the K m parameter. Using the equations, one is able to derive values of K m -- 512 nM and Vmax = 3.57 pmol Cu/mg protein/minute for Cu transport into astroglial cells. Moreover, the linearity of the plot suggests the cells contain either

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a single binding site for copper in the membrane or multiple sites with identical binding affinities.

Time Dependence of 67Cu(II) Efflux Cultures are prepared as described, and washed with 2 ml DPBS, after which 2 ml of fresh DMEM/F12 serum-free medium containing 50 nM 67CUC12 is carefully pipetted over the cells. After a 50-minute incubation at 37~ the radioactive medium is removed and the cells are rinsed gently with 2 ml DPBS at room temperature for 30 seconds. Fresh DMEM/F12 medium (2 ml) is then pipetted over the cells and incubation is allowed to continue. After 5, 15, or 30 minutes of incubation at 37~ the cells are collected as above with 0.5 M NaOH and a cell scraper, and 67Cu retained in the cells is determined and expressed as picomoles Cu/mg protein. Copper-67 appearing in the medium with time is also monitored. Effluxing ceases in about 30 minutes. Any 67Cu remaining within the cells at 50 minutes is, therefore, regarded as nonexportable. The difference between cell-retained 67Cu at 50 minutes and time zero is used to calculate the exported fraction.

Determination of Kinetic Parameters (K m and Vmax):Efflux of 6ZCu(II) Plated cells are preloaded with 10, 20, 50, 100, or 200 nM of 67CUC12 in fresh DMEM/F12 serum-free medium at 37~ for 50 minutes. According to the Darwish assumption (14), the concentration of copper in the cell at the efflux site is equivalent to the concentration of copper in the medium once equilibrium has been established. After the incubation, the radioactive medium is removed and the cells are washed with 2 ml DPBS at room temperature for 30 seconds. Fresh DMEM/F12 medium (2 ml) is layered over the cells as before. Cell-retained 67Cu is determined at time zero and after a 5minute incubation at 37~ The efflux velocity is obtained from the difference between the two readings for each 67Cu load and expressed as picomoles Cu/mg protein/minute. Graphic analysis of the velocity versus 67Cu concentrations in the medium gives the kinetic curve of 67Cu eff~ux. The values for Km and Vmaxof 67Cu efflux are determined with a double-reciprocal plot (see preceding). The results of the kinetic analysis are shown in Fig. 2. Based on the values shown, astroglial cells have an efflux Km = 67.7 nM and an efflux gmax = 0.677 pmol Cu/mg protein/minute.

Interpretation and Limitations o f Kinetics Constants Determinations The above procedure estimates Km and Vmax transport constants by standard kinetic analysis and employs standard assumptions. Rates of uptake must be performed rapidly, preferably when only unidirectional flow of 67Cu into

[10] HEAVY METAL EFFECTS ON GLIA

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FIG. 2 Double-reciprocal plot of copper efflux in cultured C6 rat glioma cells. For this plot, y = 1.47 + 100.15x; r 2 = 1.00. Kinetics constants for copper efflux are K m = 67.7 nM and Vmax = 0.677 pmol Cu/mg protein/minute. Each point represents the mean of duplicate determinations. the cell is occurring. Binding of 67Cu to the membrane must also occur rapidly, but depends on the concentration of 67CHin the medium, i.e., below the level that would saturate the membrane binding sites. Transport is viewed as a two-step process with binding preceding the actual activation of the mobilizing factor. The method gives no insight into how the transport mechanism or carrier is functioning. It is essential that the 67Cu in the medium have free and open access to the membrane carrier and that other competing ligands be eliminated from the medium. Because serum contains albumin, and albumin has at least one high-affinity site for copper on the protein, the albumin must be removed. Cells, therefore, are suspended in serum-free medium for the duration of exposure to 67CH. Our interpretation of the data presented in Figs. 1 and 2 is that the import and export of copper by astroglia require two systems. The efflux system appears eight times more sensitive. This conclusion can be interpreted to mean that cells export copper with a greater affinity than they take it up. The imbalance is met by having Vmax for efflux only one-fifth as rapid (at saturation) as input. Because of the strong binding affinity of the efflux system, internal systems that require copper for metabolic function must compete with a highly efficient efflux system designed to keep the cell in homeostasis and to protect the cell from transient high environmental exposure. The remainder of the chapter will address methods for analyzing disruptions in cell homeostasis that result from exposure to metals in culture.

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A n a l y s i s o f Cell F u n c t i o n : C e l l u l a r F l u o r e s c e n c e I m a g i n g

Rationale for Use of Imaging Techniques Over the past several years, the convergence of several technologies has led to the development of new experimental approaches for investigating mechanisms of chemical toxicity. Innovations in cell culture strategies and a renaissance in light microscopy centered around fluorescence techniques have led to the development of a new generation of in vitro toxicity assays based on the quantification of fluorescent probes in living cells. The authors have been exploiting the powerful new technology of microscopic image analysis with vital fluorescent probes and interactive laser cytometry for the development of highly sensitive, mechanistically relevant assays for neurotoxicants. This technology has many potential advantages over traditional in vitro toxicity assays. First, the assays, which are based on the quantitation of fluorescent probes in individual cells and cultures, are noninvasive and are typically carried out with living cells in the culture dish. Second, the assays are highly sensitive and quantitative. Third, data can be collected from both individual cells and cell populations. This feature is valuable for screening out variant cells (such as those in mitosis) that may respond differently from the majority of cells to a toxin. Fourth, in many cases repeated measurements can be taken from individual cells as well as from cultures. Appropriate controls may also be run in the same culture dish because of the amenability of the system to repeated measurements. Fifth, the types of measurements obtained are highly mechanistic, and thus provide not only a screening device for the presence of a toxicant, but also information on the mechanisms of toxic action. Sixth, once assays have been developed and validated with an instrument such as the Meridian ACAS interactive laser cytometer (as described further in the next section), they should be readily adaptable to automated cytofluorometric assay systems.

Fluorescent Probes and Analytical Instrumentation Fluorescent probes have been created (and continue to evolve) that are designed to react with biomolecules under conditions of relatively low temperature (i.e., temperatures compatible with living cells) and near-neutral pH (15). Many fluorescent indicators of cellular function are uniquely suitable for probing living cells because of a combination of five properties (16): specificity, the ability to detect the probe in a complex mixture of biomolecules; sensitivity, the potential for detection of few molecules in a given volume; spectroscopy, differences in absorption or emission caused by sensi-

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tivity to their immediate physicochemical environment; temporal resolution, the potential for fluorescence measurements over time points longer than the excitation/emission of the fluorescent probe, i.e., longer than 10-s seconds; and spatial resolution, defined by the resolving power of the microscope lens used to image the fluorescent probe. Many of the probes, such as the acetoxymethyl (AM) ester derivatives of a number of fluorescent indicators, may be noninvasively delivered (without microinjection or damage to the plasma membrane) into cells because of their membrane permeability properties. After entry into the cell the ester derivatives are subsequently cleaved by nonspecific cytosolic esterase activity to yield charged, membrane-impermeant probes. Other noninvasively delivered probes are membrane permeable but partition into cells based on charge, or are nonfluorescent until covalently bound to molecules within the cytoplasm. Coinciding with advances in fluorescent probe technology are developments in analytical microscopy instrumentation that permit the analysis of diverse molecular targets on or within cells. Technical improvements in microscopy include the development of lasers with emission lines suitable for use with many fluorescent probes, computer automation and control over the intensity and duration of sample illumination, and digital imaging photometric systems coupled with computer-controUed stage positioning, all of which combine to permit spatially resolved fluorescence signals. Modern fluorescence detection systems provide a means to balance the need for sufficient illumination with sensitivity, permitting excitation irradiance levels low enough to avoid damage to the sample while providing detectability equivalent to micromolar concentrations of fluorophores. There are a number of commercially available instruments capable of exciting and detecting fluorescent signals from living cells. The following description is restricted to our experience with an instrument manufactured by Meridian Instruments, Inc. (Okemos, MI), referred to as an ACAS 570 Interactive Laser Cytometer (ACAS is an acronym referring to the Adherent Cell Analysis and Sorting capabilities of the instrument). Relevant features of the instrument have been previously described (17) and include a Coherent Innova 90-5 5 W argon ion laser that produces ultraviolet (UV) illumination in the range of 351.1-363.8 nm and several visible lines throughout the range of 457.9-528.7 nm. The selected laser illumination line is directed through the rear illumination port of an Olympus IMT-2 inverted microscope equipped for epifluorescence. Fluorescence excitation with minimal destruction of fluorophores is optimized by regulation of the intensity and duration of the laser spot. Attenuation of the laser beam is controlled with two separate components. Neutral-density filters are inserted into the optical path to reduce beam intensity. The second component of beam attenuation employs an acoustooptic modulator (AOM). The AOM is used to separate the zero

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and first diffracted order of light. Only the first diffracted order light is admitted to the optical system and the amount of first-order light is regulated by activation of the AOM. By application of a high-frequency, high-voltage signal to the AOM, changes in the Bragg angle of the crystal diverts the firstorder beam from the optical axis to attenuate the beam with potentially rapid (_<2 ~sec) on/off transitions. Sample image scanning is obtained by stage stepper motors, which allows the laser beam to be kept along the center of the optical system. Emitted fluorescence is passed through a dichroic element and barrier filter, and is detected and amplified by a photomultiplier tube. The output of the fluorescence-measuring circuits is digitized with a 12-bit analog-to-digital converter and transfers this to the computer under software control. Individual image scans are held in a digital frame store and are readily available for computer analysis. A diagram of the Meridian ACAS 570 is shown in Fig. 3. The ACAS instrumentation has recently been upgraded (Ultima) by the incorporation of a Coherent Enterprise laser capable of providing simultaneous or sequential UV (351.1-363.8 nm) and visible (488 or 514 nm) illumination. In addition to motorized X and Y stage scanning, combination of Xaxis galvanometric mirror and Y-axis stage scanning provides more rapid imaging capabilities. Both the ACAS 570 and Ultima instruments are equipped with confocal capabilities, which add a third spatial dimension (i.e., volume analysis) to fluorescence imaging. Even without confocal capabilities, the additional dimension of time (i.e., kinetics) combined with image analysis of living cells expands the analytical potential of the instruments dramatically. Although the quantitative analyses described in the following sections have been adapted for use with the ACAS 570, the methods are also valid for other digital imaging fluorescence instruments. Additional general considerations of this technology for in vitro toxicology testing have been reviewed

(18).

Analysis of Cytosolic Glutathione Content and Mitochondrial Membrane Potential

Rationale for Analyzing Glutathione Content and Mitochondrial Membrane Potential In view of the ability of astroglia to take up Pb from the culture medium and store it intracellularly, coupled with their resistance to overt Pb toxicity, it would be reasonable to postulate mechanisms of Pb tolerance in these cells.

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FIG. 3 Generalized configuration of Meridian ACAS Interactive Laser Cytometer. The diagram illustrates the major components of the instrumentation, including the host computer, laser illumination, microscope, motorized stage, and detector systems. The instrument control system is a microcomputer-based unit that also supports fluorescence intensity data acquisition and analysis. The argon ion laser can be tuned to provide one of the several useful wavelengths of light (UV or visible) that are used to excite fluorescent probes on or within cells. The laser beam is attenuated by an acoustooptic modulator (AOM) that can regulate the intensity and duration of laser illumination (duty cycle). Neutral-density filters (NDF) are also used to reduce the intensity (amplitude) of irradiation as needed. Laser light enters the inverted microscope through the epiillumination port of the microscope and, after passing through an excitation filter (EF), is reflected into the objective lens and onto cells on the stage of the microscope by a dichroic mirror (DM). Because the objective lens is fixed in position, fluorescent images are generated by moving the stage of the microscope in X and Y directions with precision stepper motors. Another stepper motor can control Z axis position for confocal imaging applications. Emitted fluorescence is collected by the microscope objective (OB) lens and passes through the dichroic mirror due to its longer wavelength and proceeds to the detector, which contains photomultiplier tubes (PMT). Isolation filters (IF) and the dichroic mirror divide light into separate wavelength bands, each of which can then be amplified for fluorescence intensity images. Dashed lines indicate the light path. Solid lines indicate electrical circuits. FSM, Front surface mirror; BF, barrier filter; VC, video camera.

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The astroglial cell is an excellent candidate for examining the concept of tolerance, i.e., adaptation by a cell to the presence of Pb by adjustment of cellular homeostatic mechanisms. In this section, the measurement of intracellular glutathione (GSH) is considered as a potential indicator of cell tolerance to the accumulated Pb. Another cellular function, the maintenance of a high electrochemical membrane potential in mitochondria, is considered as a site for cell damage that may ensue from the failure of the GSH system to protect the cell, and also as a direct target for Pb-induced damage. Intracellular GSH is an important component of cellular homeostasis. This abundant tripeptide, which is synthesized from constituent amino acids (glutamate, cysteine, and glycine), comprises the principal component of a cellular detoxification system, capable of scavenging reactive oxygen species and maintaining the redox state of cellular thiols (19). Astroglia are of particular interest with regard to GSH function because they appear to be involved in GSH compartmentation in brain (20). Chemically induced GSH depletion has been shown to cause mitochondrial degeneration in brain cells, apparently because GSH-dependent reactions are critical for reducing the significant levels of hydrogen peroxide produced in mitochondria (21). It may therefore be useful to measure mitochondrial membrane potential at the same time points for which one measures GSH levels. Mitochondria, the organelles that generate cellular ATP, are postulated targets for Pb-induced injury in astroglia (22) and other cells (reviewed in Ref. 23). The inner mitochondrial membrane contains the enzymes for electron transport and oxidative phosphorylation. An electrochemical gradient, which can be detected as a membrane potential difference and a pH gradient, exists across this membrane to couple energy released during electron transport to the phosphorylation of ADP to ATP. Chemically induced dissipation of the electrochemical gradient would decrease the rate of oxidative phosphorylation and deplete cellular energy levels. Temporal correlations can be studied by fluorescence cytometry for Pb-induced alterations in GSH content and mitochondrial membrane potential.

Methods for Glutathione Measurement The ACAS 570 described above is used for the quantification of cellular fluorescence. Primary or first-passage cultures of astroglia have been used for these assays, and are examined in culture on days that correspond temporally with postnatal development ages of interest. Monochlorobimane (mBC1; Molecular Probes, Eugene, OR) is currently the fluorescent probe of choice for intracellular GSH measurement due to its low reactivity to GSH and

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[10] HEAVY METAL EFFECTS ON GLIA

other thiols, and its ability to form a fluorescent adduct with GSH in a reaction catalyzed by glutathione S-transferase (GST) (reviewed in Ref. 24). In this assay, cells are initially plated in T-75 tissue culture flasks, in which they are treated with Pb at desired concentrations and exposure times. Because the fluorescent adduct of mBCI requires ultraviolet excitation, cells must be subcultured on poly(L-lysine)-coated coverglass chamber dishes (Nunc, Naperville, IL) for at least 12-24 hours prior to analysis. Cultures are washed once with CaZ+/MgZ+-free phosphate-buffered saline (pH 7.4) and then loaded with 40 IxM mBC1 (stock solutions are 10 mM in ethanol) in serum-free, phenol red-free medium. Cultures are then washed four times with serum-free medium without phenol red and analyzed. In all fluorescence imaging assays carried out in our laboratory, we use HEPES-buffered DMEM/F12 medium without serum or phenol red (Sigma) because serum may interfere with dye loading and phenol red may interfere with fluorescence light detection. HEPES adequately buffers the medium at pH 7.2-7.4 in the absence of CO2 for the time period (usually 20-30 minutes) during which the culture is on the microscope stage. Because GST activity can vary from one cell type to another, loading parameters must be optimized by the performance of a kinetic analysis of probe loading into cells. Typically two culture dishes per treatment are loaded on the stage and the fluorescence intensity in clusters of about 30 cells is recorded at 1-minute intervals. Once loading kinetics are determined, fluorescence data may be analyzed by means of a curve-fitting regression analysis program and extrapolated to identify equilibrium loading and the rate constant from the equation F (t) = Feq(1 -

e-kt),

where F (t) is the fluorescence at any time, t, Feq is the fluorescence intensity at equilibrium (i.e., GSH level), and k is the estimated rate constant for mBC1 conjugation to GSH (i.e., k = GST activity) (25). Emitted fluorescence (461 nm) is detected with a barrier filter (BP 485/45) with the ACAS 570 at an excitation wavelength of 351-363 nm. Figure 4 shows a digital image of astroglia labeled with mBC1. Typical analysis of cells is performed by defining the area occupied by individual cells. Borders of the cell are identified by drawing a polygon around the area to be analyzed. The integrated and average fluorescence intensity of each cell can then be determined. Background fluorescence detection values of dishes containing serum-free medium with and without cells are used to set threshold sensitivity for the photomultipliers. Control GSH levels are determined by scanning at least 10 cells in four areas from each control dish. Two or more dishes per treatment group are tested in each experiment. GSH values are expressed

150

PARADIGMSOF NEURAL INJURY Color -

Values -

3880t

-

2813

-

2626

-

2440

-

2253

~ -

2867

-

1 8 8 8

- 1694

-~

i

1587 1320

1134 947 761 574 388 281 151

FIG. 4 Digital image of cellular monochlorobimane fluorescence; this image was reproduced from the pseudocolor intensity image on black and white film. The pseudocolor scale provides a visual index of fluorescence intensity within each area of the cell. In this image, lighter areas within cells reflect higher fluorescence intensity (i.e., more GSH-mBC1 conjugate). The image shows a control culture of astroglia, which exhibits a characteristic mBC1 fluorescence pattern identifying normal cytoplasmic levels of GSH. as the mean + S E M for each treatment group and can cally for the analysis of treatment effects. Figure 5 two concentrations of Pb on G S H content in cultured expressed as a percentage of control fluorescence in alterations as a function of time (26).

be compared statistishows the effects of astroglia. Values are order to detect G S H

Methods for Measurement of Mitochondrial Membrane Potential The potentiometric fluorescent dye rhodamine 123 (Molecular Probes, Eugene, OR) is used in this assay to provide a relative assessment of electrochemical potential across the mitochondrial membrane. The incorporation of rhodamine 123 is dependent on the maintenance of an electrochemical potential across the mitochondrial membrane (27), and dissipation of the

151

[10] HEAVY METAL EFFECTS ON GLIA 200 I-I control !~1 0.1 pMPb

m 1.0 pM Pb

,=_,

100

I

0 0 tO e~

0.3

!

i

1

1

2

3

6

9

days of treatment

FIG. 5 Astroglial glutathione levels after low-level lead acetate treatment plotted as a percentage of control glutathione levels. Astroglial cultures corresponding in age to postnatal day 21 were treated daily with 0, 0.1, or 1.0/zM lead acetate in Waymouth's 705/1 medium with 10% FBS. After an initial decrease in the astroglial content of glutathione in response to Pb treatment, glutathione content increased compared to control values in a dose-dependent manner. ,, Differs significantly from control, same day (p < 0.01). **, Differs significantly from control and other Pbtreated groups, same day (p < 0.01). Reproduced from Ref. 26, with permission.

electrochemical potential is indicated by a decrease in fluorescence (28). Cells previously treated with Pb in T-75 flasks are subcultured at low density (75,000 to 100,000 cells per dish) into 35-mm plastic tissue culture dishes at least 12 hours prior to assay. Mitochondrial loading with rhodamine 123 is performed by washing cultures in situ once with PBS, and then incubating the cultures with 5 /zg/ml rhodamine 123 in serum-free medium (prepared from 2 mg/ml stock in ethanol). Incubation time is based on kinetic analysis of rhodamine 123 loading, which is typically 30 minutes in astroglia. Cells are then washed four times in serum-free medium without phenol red before scanning on the ACAS 570 (488 nm excitation wavelength). Up to eight different areas of the dish can be used to record mitochondrial fluorescence intensity, as well as several dishes per treatment group. The intensity of

152

PARADIGMS OF NEURAL INJURY 200 [ ] control [ ] 0.1 pM Pb

9 1.0 pM Pb

l-

I1) O

o -o

_~

100

T

-r

-1-

tO 0 tO L_

(I)

I

0.5

1

6

12

14

days of treatment

FIG. 6 Mitochondrial membrane potential in astroglia after low-level lead acetate treatment as a percentage of control values. Astroglial cultures corresponding in age to postnatal day 21 were treated with Pb as described for Fig. 5. A significant, nearly maximal decrease in mitochondrial membrane potential was seen after a 1day exposure to 1 /xM Pb. By day 12 of treatment, both doses of Pb resulted in a decrease in mitochondrial membrane potential to the same degree. ,, Differs significantly from control, same day (p < 0.01). Reproduced from Ref. 26, with permission. (Additional timepoints have been added to the previously published figure.)

labeling is quantified as a function of metal dose, time of treatment, and length of posttreatment recovery. The average fluorescence intensity is recorded from at least 100 cells per treatment group. Data are treated as described for GSH. Figure 6 shows the effect of Pb on mitochondrial membrane potential in astroglia as a function of treatment time.

Interpretation and Limitations of Glutathione and Mitochondrial Membrane Potential Measurements Analysis of the fluorescence intensity of the G S H - m B C 1 conjugate by digital fluorescence imaging is a useful assay of cellular GSH content. Other thiolspecific probes have been described that are suitable for visible-wavelength laser excitation, including 5-chloromethylfluorescein diacetate (CMFDA) and 5-chloromethyleosin diacetate (CMEDA) (29). C M F D A and C M E D A

[10] HEAVY METAL EFFECTS ON GLIA

153

are not as specific for GSH as mBC1, and therefore these visible probes may be more suitable for monitoring the total level of free intracellular thiol than GSH specifically. Both mBC1 and CMFDA are nontoxic agents that may also be used intentionally to deplete GSH within cells (25). With the interactive laser cytometer, there is no signal overlap of the two probes and therefore it is possible to deplete available GSH with one fluorescent probe and monitor new synthesis with the other at the appropriate wavelength. Fluorescence intensity values of the conjugated GSH are then monitored at appropriate intervals (ranging from 1 to 5 minutes over 1 hour). The assay for mitochondrial membrane potential does not distinguish between toxic insults that act directly on mitochondria and extramitochondrial events that damage mitochondrial membrane integrity, such as decreased GSH concentrations or increased intracellular C a 2+ concentrations. Therefore, mitochondrial membrane potential should be interpreted in the context of other indicators of cellular injury. Thus the data shown in Fig. 5 indicate a transient decrease in cytosolic GSH concentration in astroglia exposed to 0.1 to 1.0/zM lead acetate, followed by recovery to normal levels, and then a subsequent elevation to levels in excess of normal. Within the same time period (Fig. 6), mitochondrial membrane potential decreases and remains decreased even after GSH levels have recovered. This result suggests that additional mechanisms are involved in the toxicity of Pb at the later time points. The relationship between GSH depletion and decreased mitochondrial membrane potential has not yet been clarified in this system but could be approached by testing the ability of exogenous GSH or a membranepermeant GSH ester to protect mitochondria from Pb-induced damage. In any case, the period of GSH depletion in astroglia may represent a period of vulnerability to cell injury, both to the astroglia and to neurons that depend on them for amino acids and other supporting functions. A n a l y s i s o f I n t r a c e l l u l a r C a 2+ C o n t e n t

Rationale for Measuring [Ca2+] C a 2+ has well-established roles as a second messenger in signal transduction. The extracellular C a 2+ concentration is high, typically 1.2-1.3 mM. In contrast, the Ca 2+ concentration in the cytoplasm is narrowly buffered to around 10 -7 M by C a 2+ homeostatic mechanisms, including plasma membrane transport systems and CaZ+-sequestering systems. These mechanisms are important potential targets for the toxic effects of Pb and other metals. Although Pb is considered a nonphysiologic metal, the ionized form (Pb 2+) apparently can enter a number of metabolic pathways by replacing other ions, particu-

154

PARADIGMS OF NEURAL INJURY larly Ca 2+, which is a divalent cation with a similar hydrated diameter. The interaction of Pb 2+ and Ca 2+ at cellular sites has recently been reviewed (3,

30). This interaction takes place at three sites: the plasma membrane, where Pb 2+ and Ca 2+ compete for transport systems that regulate their entry or exit, such as Ca 2+ channels and the CaZ+-ATPase pump; intracellular Ca 2+ storage depots, such as the endoplasmic reticulum, mitochondria, and Ca 2+binding proteins; and effector proteins whose activity is regulated by Ca 2+,

such as protein kinase C and calmodulin. One of the earliest and most discrete toxic effects of Pb common to different cell types and numerous cellular processes is perturbation of the Ca 2+ intracellular signaling system. Pb is hypothesized to affect Ca 2+ signaling by two mechanisms: replacement of Ca 2+ or elevation of intracellular free Ca 2+ levels. A variety of fluorescent probes has been developed to detect very small, rapid changes in intracellular free calcium concentrations, [Ca2+]i, including indo-1, fluo-3, fura-2, and quin-2 (31). These probes can be loaded noninvasively into living cells as membrane-permeant acetoxymethoxy derivatives and can indicate changes in [Ca2+]i on a millisecond time scale because of a characteristic change in their individual spectra on binding to Ca 2+. When cells are treated with agents that alter Ca 2+ homeostasis in a predictable fashion, such as Ca 2+ ionophores, sophisticated analysis of Ca 2+ homeostatic mechanisms is possible. The choice of probes is based on the wavelength selection capabilities of the available instrumentation and the type of analysis being performed. Indo- 1 is well-suited for measuring small, rapid changes in [Ca2+]i with instruments that generate a single excitation wavelength (such as the ACAS 570). Indo-1 requires UV excitation (351-363 nm) but provides the advantage of permitting measurement of [Ca2+]i because there is a shift in the emission spectrum from 485 to 405 nm on binding Ca 2+. The two emission wavelengths are separated with a dichroic mirror and are monitored simultaneously with separate detectors. Intracellular [Ca 2+] is calculated by comparing the ratio of emissions to that of a standard curve. Ratiometric analysis of emission wavelength pairs is independent of absolute changes in fluorescence intensity at a given wavelength. Thus, quantification of [Ca2+]i is independent of the extent of dye loading, leakage, cell thickness, or photobleaching. In addition, indo-1 has great sensitivity below 1/zM. Fluo-3 has two major advantages, its ease of use due to its visible excitation at 488 nm and emission at 520 nm and its great sensitivity in the micromolar range. Fluo-3 is limited, however, in its ability to m e a s u r e [Ca2+]i, because of its single emission wavelength. Quantitative measurements are possible, however, if cells can be uniformly loaded. Fura-2 requires the use of dual excitation wavelengths in the UV range (380 and 340 nm), which is not available with the ACAS 570. Ca 2+ binding shifts the excitation spectrum about 30 nm to the shorter wavelengths,

[10] HEAVY METAL E F F E C T S ON GLIA

155

so that the ratio of intensities obtained from 340/380-nm excitation pairs provides a good measure of [Ca2+]i . Quin-2, which has less specificity for C a 2+ than newer generation probes, is most efficiently excited at relatively shorter wavelengths (340 nm), lacks the brightness of newer probes, and does not show the useful CaZ+-induced wavelength shift in either excitation or emission spectrum needed to generate a ratio signal (31). The similarity of physical and chemical properties between Pb 2+ and C a 2+ that allows them to interact with the same cellular substrates unfortunately may also allow both cations to interact with the same fluorescent probes. Though the specificity of newer generation Ca 2+ probes over other metals is generally good, for those probes with which Pb 2+ interactions have been studied, the results have shown a strong interaction with Pb 2+. Solutions to the technical problems arising from this circumstance are still under development, and will be examined below. The description of methodology in this section is limited to the use of indo-1 for the analysis of intracellular [Ca 2+] in cultured astroglia.

Method for Measuring [Ca2+] The Ca2+-sensitive fluorophore indo-1 (Molecular Probes) is used to quantify intracellular C a 2+ in astroglia with the ACAS 570. Because the excitation wavelength of indo-1 is in the UV range, cells are subcultured onto a glass substrate 12-48 hours prior to analysis, just as described for the measurement of glutathione content. Cells are noninvasively labeled with the acetoxymethyl ester of indo-1. A stock solution of 1 mM indo-1/AM is prepared in dimethyl sulfoxide (DMSO) and diluted with serum-free, phenol red-free medium to 1 /zM (0.05% final DMSO concentration) for loading cells in culture dishes. Typically, loading of astroglial cells with indo-1 requires 1 hour. Following incubations, cells are washed once with PBS followed by three washes with serum-free, phenol red-free medium. Cells are then scanned with an excitation wavelength of 351.1-363.8 nm and emitted fluorescence is monitored simultaneously at 485 and 405 nm. Figure 7 shows a paired emission wavelength scan of astroglial cells in culture labeled with indo-1. Fluorescence intensity values are then compared to a calibration curve generated by monitoring fluorescence of indo-1 free acid and Ca 2+ added to known concentrations in a physiological buffer solution (10 mM MOPS, 115 mM KC1, 20 mM NaCI, 1 mM M g S O 4 , and 1 mM EGTA). A standard 0.1 M calcium solution (Orion) is used to generate the calibration curve. Fluorescence measurements are collected from 10 cells per dish and four culture dishes for each experimental treatment group.

156

PARADIGMS OF NEURAL INJURY Col

Detector

1

Data

or

Values

-

2500

-

2362

t

-:

.....

-

2224

-

-

2087

-

-

1 9 4 9

-

-

1 8 1 2

-

-

1674

-

-

1537

-

-

1399

-

-

1261

-

-

1 1 2 4

-

-

986

-

-

849

-

-

711

-

-

574

-

-

436

-

-

2995

-

....

Detector

2

Data

........... ....

FIG. 7 One of a temporal series of digital fluorescence images of the Ca 2+-sensitive ratiometric probe, indo- 1, excited with UV light (351-363 nm) and recorded simultaneously at 485 nm (detector 1, image at left) 405 nm (detector 2, image at right). Unbound indo-1 emits at 485 nm and CaZ+-bound indo-1 emits at 405 nm. By computing the ratioed fluorescence (detector 2/detector 1 = 405/485), quantification of free Ca 2+ within the cell can be determined. Direct quantification of Ca 2+ content in cells is then performed by comparing the ratio of emissions to that generated in a standard curve. More rapid detection of changes in intracellular [Ca2+] can also be performed by monitoring ratioed fluorescence from line scans through cells or by monitoring ratioed fluorescence changes within a single point in a cell.

Interpretation and Limitations of[Ca e+] Measurements The image analysis procedure discussed provides a method for measuring basal [Ca2+]~ with indo-1 that is rapid, noninvasive, and can be applied to many cells simultaneously. In addition, the same procedures may be used to measure the ability of cells to recover from deliberate perturbations in Ca 2+ homeostasis as a function of treatment with a toxic metal. For example, the Ca 2+ ionophore ionomycin (final concentration 1.0 ~ M in <0.05% DMSO) causes a rapid influx of Ca 2+ into the cytosol, which peaks within 30 seconds after addition to the medium (Fig. 8). [Ca2+]i returns to basal levels within several minutes in normal astroglia as a result of Ca2+-ATPases pumping Ca 2+ out of the cell or into organellar depots. The basal level of indo-1 ratioed fluorescence ( F 0) and the peak level of ratioed fluorescence (Fp) in control

157

[10] HEAVY METAL E F F E C T S ON GLIA 1.2 1.1

1.0

0.9

0.8

0.6

t i i i I i i i i ! i i i i i i i i i i i i i i i i I

0

1O0

200

300

400

500

Calcium (Units) vs. Time (sec)

600

FIG. 8 An illustration of changes in indo-1 ratioed fluorescence over time within a single astroglial cell. Basal indo-1 fluorescence was recorded for the first 60 seconds. Next, 1 /xM of the Ca 2+ ionophore, ionomycin, was added at the time indicated by the vertical line, resulting in a rapid increase in intracellular [Ca2+], which returned to baseline over the next 6-7 minutes. Although the data shown are for a single cell, recordings from multiple cells are typically collected simultaneously.

cells can be expressed as a ratio (Fp/F0) and used as a reference point for altered C a 2+ homeostasis. Previous work has shown that this ratio is a more sensitive indicator of cell injury than [Ca2+]i, because a significant depression of Fp/Fo can be detected in toxin-treated cells before significant changes in [Ca2+]i are observed (32). A second modified application of the indo-1 procedure is to add the ionophore to cultures in calcium-free Tris-buffered saline, rather than medium, in order to assess fluxes of internal calcium stores. A third application is to a s s e s s C a 2+ permeability across cell membranes by measuring [Ca2+]i immediately after the addition to the culture of agents that open or block C a 2+ channels, as well as agents that release or prevent the release of C a 2+ from intracellular depots. In each case cellular responses can be compared between control cultures and cultures treated with metals as a potential indication of cell injury. As suggested earlier, the presence of Pb in cells presents special problems when measurements of [Ca2+]i are attempted with fluorescent probes. In the case of indo-1, we have recently found that Pb 2+ reacts strongly with indo-1 under the experimental conditions described (Fig. 9). However, the emission

158

PARADIGMS OF NEURAL INJURY .6.5

I

0 O9

.4 0

CD

mM

.3

TPEN uM

uM

EK

.2 .1 0.0

i

0.4

0.8 1.2 1 5 i0 0 CONCENTRATION

i

i

i

I00

FIG. 9 A ratiometric calibration curve that examines indo-1 fluorescence during sequential addition of increasing concentrations of Ca 2+, Pb 2+, and the heavy metal chelating agent N, N, N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). The ratio of bound-to-unbound indo-1 fluorescence was monitored during addition of increasing [Ca 2+] (0.2-1.2 mM in 0.2 mM increments) until saturation. Once saturation of indo-1 fluorescence was obtained, Pb 2+ was added (1.0, 5, and 10/zM). Addition of Pb 2+ resulted in an increase in the 405/485 fluorescence ratio. Subsequent addition of TPEN (100/xM) returned the fluorescence ratio to the pre-Pb 2+ treatment level, i.e., the Ca 2+ saturation level.

spectra of pb2+-indo-1 and CaZ+-indo-1 differ sufficiently from each other that it may be possible to detect Pb 2+ entry into cells with indo-1 (33). Several experimental approaches have been used to analyze PbZ+-Ca 2+ interactions in cells, particularly the perturbation of Ca 2+ stores and the ability of Pb 2+ to act as a Ca 2+ surrogate. Before the advent of methods to investigate changes in intracellular Ca 2+ concentrations by the use of fluorescent probes, radioisotopes were used to label internal divalent cation pools (both Ca 2+ and Pb 2+) and their efflux was measured. This method was not sensitive to rapid, quite small changes in cytosolic [Pb 2+] or [Ca2+]. More recently, two CaZ+-binding indicators, 5F-1,2-bis(o-aminophenoxy)ethaneN,N,N',N'-tetraacetic acid (5F-BAPTA) and fura-2, have been used to measure changes in both intracellular Ca 2+ and Pb 2+. 5F-BAPTA binds many metals, but each produces a different chemical shift that is detectable by 19F nuclear magnetic resonance (NMR) spectroscopy. As previously mentioned, fura-2 is a commonly used Ca 2+ indicator that can be analyzed by dualexcitation spectrofluorometry because the excitation spectra of the free fura2 molecule and its Ca 2+ complex are different. 5F-BAPTA, like fura-2, may

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H E A V Y M E T A L E F F E C T S ON GLIA

159

be introduced into living cells as membrane-permeable acetoxymethoxy derivatives. Schanne et al. (34) exploited the capacity of 19F NMR spectroscopy to quantitate distinct signals simultaneously emitted by complexes formed between the 5F-BAPTA and C a 2+ or Pb 2+ in 5F-BAPTA-Ioaded cells. These investigators demonstrated the twofold elevation of [Ca2+]i in a mouse neural cell line after exposure for 2 hours to 5/xM lead acetate, with a concomitant increase of [pb2+]i to 30 pM. This study has been the only one to date to m e a s u r e [pb2+]i and to demonstrate that [Ca2+]i is sensitive to Pb treatment in a cell line of neural origin. 19F NMR is an attractive technique for measuring intracellular [pb2+], except for the considerable expense of the technology, which limits the availability of the required instrumentation. Tomsig and Suszkiw (35) used fluorescence cytometry with fura-2 to explore whether subsequent Pb-induced events are mediated by [Ca2+]i or by [PbZ+]i, given that Pb treatment elevates intracellular C a 2+. These investigators showed that Pb 2+ can trigger the release of norepinephrine from isolated bovine chromaffin cells, apparently acting as a potent Ca 2+ surrogate. Cells were loaded with fura-2, which binds C a 2+ with high affinity (Kd = 2 x 10 -7 M ) , but was also shown to bind 1 : 1 with Pb 2+ at a much higher affinity (Kd = 4 x 10 -12 M ; Kd values are from Ref. 30). The excitation spectra of fura-2 in the presence of saturating concentrations of either Pb 2+ or C a 2+ are largely overlapping but sufficiently distinct to be analyzed by recording the 340/380-nm ratio of fluorescence. Spectra recorded from fura-2-1oaded cells that were incubated in Pb 2+ buffer solutions indicated the entry of Pb 2+ into the cells at a level (1-10 pM) proportional to extracellular [pb2+]. Extracellular Pb 2+ also triggered [3H]norepinephrine release from intact fura-2 loaded cells. The possibility that Pb 2+ induces secretion through recruitment of Ca 2+ from internal stores in intact cells could not be excluded by this experimental technique. Furthermore, Simons (30) has subsequently shown that fura-2 also forms a complex with Z n 2+ (K d = 1 x 10 -9 M ) with a fluorescence excitation spectrum similar to that of Pb 2+ and a stronger fluorescence yield. Nevertheless, fura-2 should be useful for measuring changes in intracellular [pb2+], though not for determining absolute Pb 2+ values.

Analysis of Gap Junctional Intercellular Communication

Rationale for Measuring Gap Junctional Intercellular Communication Gap junctions are communication channels that allow the diffusion of small molecules (up to 1000 Da, or 1.5 nm in diameter) or electrical signals from the cytoplasm of one cell to another. A gap junction is composed of aggregates

160

PARADIGMS OF N E U R A L INJURY

of connexons located on the plasma membranes of two closely apposed adjacent cells. Each connexon consists of six polypeptides (connexins) that span the plasma membrane and line a central pore. The connexons of two cells align coaxially to form a cytoplasmic continuity between the interiors of apposed cells, thus facilitating direct intercellular communication. The effects of neurotoxicants on gap junctional intercellular communication (GJIC) are of interest because of the importance of gap junctional contact in cellular homeostasis. Astroglial gap junctions are believed to be important in the regulation of ionic environment (36) and cellular signal transduction (37), and as participants in a pulsatile calcium communication network throughout the central nervous system (CNS) (38). Therefore, gap junctional communication may be used in chemical signaling as well as in a metabolic buffering capacity by passage of low molecular weight substances from one cell to another. Most gap junction channels are rapidly closed by cytoplasmic acidification (36) and by nonphysiological levels of [ C a 2 + ] i . Closure of gap junctions is thought to provide a general mechanism to seal off unhealthy or injured cells from healthy members of a physiologically coupled cellular community (39).

Method for Measurements of Gap Junctional Intercellular Communication Gap junctional intercellular communication can be measured by methods based on the transfer of low molecular weight fluorescent dyes between adjacent, communicating cells. The method described is a modification of the fluorescence recovery after photobleaching (FRAP) technique (40). FRAP applied to gap junctions (gap FRAP) makes use of 5-carboxyfluorescein diacetate (CFDA; Molecular Probes) for loading cells. CFDA is a nonfluorescent, membrane-permeable dye that is hydrolyzed in the cytoplasm by nonspecific esterases to yield a fluorescent, membrane-impermeant dye, 5carboxyfluorescein (CF). Cells are subcultured at a medium density (to yield a growth pattern consisting of small groups or pairs of cells) into 35-mm plastic tissue culture dishes. After 12-24 hours GJIC is measured by dye coupling in the culture dish with the ACAS 570. Loading of the cells with dye is performed by incubating the cultures in situ with 10/~g/ml CFDA (prepared from a stock solution that is 2 mg/ml in DMSO) in HEPES-buffered medium without serum or phenol red for 10 minutes at 37~ Cultures are then washed four times with serum-free medium without phenol red. A microscopic field containing aggregates of cells is selected for analysis. Several abutting cells in

[10] HEAVY METAL E F F E C T S ON GLIA

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groups or in pairs are selected from each field for monitoring of fluorescence transfer at an excitation wavelength of 488 nm. Cellular fluorescence is imaged by scanning the cells on the motorized stage of the microscope in a two-dimensional raster pattern. Laser strength for scanning is adjusted to a level sufficient to excite CF fluorescence without causing significant photobleaching of the cells. One cell is photobleached in each pair or group by a series of higher intensity point bleaches produced by focusing the 488-nm argon ion laser beam through the microscope objective. The point bleaches photochemically reduce the amount of CF fluorescence in the cell. Laser strength of the point bleaches and number of bleaches per cell are carefully controlled so as to reduce fluorescence enough to measure recovery without causing visible cell damage at the light microscopic level. Isolated cells, which should not recover fluorescence, may be bleached as negative controls and other groups of cells left unbleached and demarcated as positive controls. A series of five postbleach image scans is generated at 1-minute intervals to measure subsequent redistribution of intracellular fluorescence through gap junctions. Figure 10 shows a typical analysis for astroglial-astroglial GJIC in culture. At least three analyses from each of three dishes per treatment group are conducted. Data (percentage of the prebleach fluorescence level recovered) are expressed as the mean _+ SEM. Fluorescence recovery can be compared between treatment groups at user-determined time points after photobleaching to assess the approximate level of communication. Dose responses can be determined in this manner. In experiments with astroglial cultures, we do not exceed 4 minutes postbleaching because of artifacts generated in subsequent measurements. Figure 11 shows a comparison of the effects of two Pb concentrations on GJIC in cultured astrog|ia, where Pb was found to produce no change from control values. Treatment groups can be compared more precisely by use of a curve-fitting regression analysis to extrapolate fluorescence recovery over time and thus determine the rate constant for fluorescence recovery. The rate constant k may be obtained from the following equation: F (t) = Feq(1 - e -kt) + F (0), where t is time after photobleaching, Feq is the percentage of fluorescence recovery of the bleached cell at equilibrium, e is the constant (the base of the natural system of logarithms), and F (0) is the percentage of fluorescence intensity immediately after photobleaching. The value of Feq depends on the number of cells in contact with each other and the initial level of photobleaching, F (0) (18).

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PARADIGMS OF N E U R A L INJURY

FIG. 10 Analysis of gap junction-mediated intercellular communication by fluorescence recovery after photobleaching (gap FRAP). The pseudocolor images produced by the ACAS 570 were reproduced on black and white film. Upper left: Image of cellular carboxyfluorescein fluorescence in an astroglial culture prior to photobleaching. Lighter areas within the cells reflect higher fluorescence intensity. Upper right: Cellular fluorescence intensity immediately after photobleaching cells 1 and 2. Cell 3 was selected as a nonphotobleached control to monitor potential background photobleaching from fluorescence image scanning. Cellular fluorescence was recorded after 4 minutes (lower left) and revealed an increase in fluorescence in cells 1 and2 that was contributed by contacting cells via gap junctional contacts. Lower right: Kinetics of fluorescence recovery for the three cells. Note the increase in fluorescence in cells 1 and 2 over time, whereas the unbleached control cell 3 did not lose fluorescence.

Interpretation of Gap Junctional Intercellular Communication Measurements The experimental approach for direct monitoring of cell-cell communication by means of gap FRAP offers many advantages over other direct physiological methods of junction permeability measurement. Unlike the " s c r a p e load-

[10]

163

H E A V Y M E T A L E F F E C T S ON G L I A 40

] / /

I"1

control

9 II.IMPb 30

E"

8 (D tQ) O (/)

20

c O ',1--

10'

0

1

2

3

4

time (minutes after photobleaching

FIG. l 1 Gap FRAP analysis of astroglial gap junctional communication after exposure to lead acetate (1.0/~M). No effect on junctional cell-cell communication was seen after 5 days of repeated daily exposure to Pb, either in terms of total recovery of fluorescence or percentage of recovery 1-4 minutes after photobleaching. Reproduced from Ref. 26, with permission.

ing" method with Lucifer Yellow or fluorescent dye microinjection, gap FRAP is noninvasive. Furthermore, there is no loss of temporal resolution as with microinjection methods. Dual voltage clamping is a very sensitive method for evaluating junctional communication, but its use is restricted to the measurement of the electrical properties of gap junctions in paired cells. Perhaps the greatest advantages of the gap FRAP method are that appropriate controls can be run in the same microscopic field of scanning (and hence the same culture dish) and that multiple measurements can be conducted on the same cell without traumatic manipulations. As many as five consecutive gap FRAP assays on the same cells is possible (41). As shown in Fig. 11, GJIC in astroglial cultures is not affected by exposure over several days to micromolar Pb concentrations. This finding is consistent with an earlier report that several heavy metals at sublethal concentrations (Ni, Cd, Pb, and Cr) are not potent inhibitors of communication in Syrian hamster embryo primary cultures (42). However, we have also found that

164

PARADIGMS OF N E U R A L INJURY 50,

40

> o O tD

rr

30

20

10 -

=

Fe4 Fe5 control

0

Time (rain) FIG. 12 Gap FRAP analysis of astroglial gap junctional communication after exposure to 0, l0 (FeS), or 100/~M (Fe4) FeC12 . In this experiment, cells were exposed daily for 7 days to the Fe dose indicated. For each datum 15-19 cells were measured. Error bars represent standard errors. At each time point measured (1-4 minutes) the mean percentage of recovery was significantly higher in the Fe4 group than in the other two groups. The rate constant (k) for diffusion of CFDA into the photobleached cell was calculated, which is proportional to the permeability of the channel. The k value was calculated to be 0.3425, 0.3784, and 0.5275 for the 0, FeS, and Fe4 groups, respectively. Thus the permeability of the gap junctional channels was increased by over 40% in cells treated with 100/zM Fe (Fe4) compared to the other treatment groups (p < 0.01).

Fe up-regulates gap junctional communication, as shown in Fig. 12. The dose of Fe (100/~M) selected was that which produces the same amount of metabolic injury in astroglia as 1 /~M Pb [as measured by reduction of glutamine synthetase (glutamate-ammonia ligase) specific activity]. The finding that Pb and Fe affect GJIC differently supports the concept that metals damage cells by distinct pathways, leaving a unique set of molecular end points, which we term their "signatures." It should be possible to develop a battery of sensitive assays to detect and characterize these unique signatures, particularly by the use of cellular fluorescence imaging techniques.

[10] HEAVY METAL EFFECTS ON GLIA

165

Acknowledgments Work in the laboratories of E.T.-C., R.C.B., and E.D.H. has been supported by grants from the NIH (R01-ES05871, R01-HD26182, and R01-HD29959) and an NIH Superfund Grant to Dr. Stephen Safe (P42-ES04917). M.E.L. and L.A.S. are recipients of Physician Scientist Awards (K11-ES00251 and K11-ES00279) from the NIH. We thank Jeff Bowen and Michelle Nyberg for excellent assistance in the preparation of the manuscript.

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