Comparison of the effects of inorganic lead on the growth and differentiation of cultured rat cortical and hippocampal neurons

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Comparison of the effects of inorganic lead on the growth and differentiation of cultured rat cortical and hippocampal neurons
Kern, Marcey Kay
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xii, 102 leaves : illustrations, photographs ; 29 cm


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Lead -- Toxicology ( lcsh )
Lead -- Physiological effect ( lcsh )
Rats -- Effects of heavy metals on ( lcsh )
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )


Includes bibliographical references.
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Submitted in partial fulfillment of the requirements for the degree, Master of Arts, Biology.
Statement of Responsibility:
by Marcey Kay Kern.

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University of Colorado Denver
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Auraria Library
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28286331 ( OCLC )
LD1190.L45 1992m .K47 ( lcc )


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COMPARISON OF THE EFFECTS OF INORGANIC LEAD ON THE GROWTH AND DIFFERENTIATION OF CULTURED RAT CORTICAL AND HIPPOCAMPAL NEURONS by Marcey Kay Kern B.S., Colorado State University, 1985 A thesis submitted to the Faculty of the Graduate School of the University of Colorado at Denver in partial fulfillment of the requirements for the degree of Master of Arts Biology 1992


This thesis for the Master of Arts degree by Marcey Kay Kern has been approved for the Department of Biology by Teresa Audesirk Gerald Audesirk


Kern, Marcey Kay (M.A., Biology) Comparison of the Effects of Inorganic Lead on the Growth and Differentiation of Cultured Rat Cortical and Hippocampal Neurons Thesis directed by Associate Professor Teresa Audesirk ABSTRACT Nervous systems exposed to lead display a wide range of morphological, cognitive, and behavioral abnormalities. The present study was designed to: 1) study the effects of inorganic lead (0.1 nM to 1 mM concentration range) on several growth parameters of cultured rat cortical neurons, and compare these to previous findings from our laboratory on rat hippocampal cells exposed to similar lead concentrations in a 2% fetal calf serum medium; 2) examine the morphological effects of lead on both cortical and hippocampal cells in a 10% fetal calf serum medium; and 3) explore the feasibility of using a membrane-permeant heavy metal chelator, N,N,N' ,N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), to measure intracellular calcium employing the calcium-sensitive indicator fura-2 and the Quantex QX -7 imaging system, in order to elucidate mechanisms of lead neurotoxicity. The effects of lead tended to be multimodal and concentration dependent. Neurite initiation was the most sensitive parameter, showing inhibition at both high and low lead concentrations. Media with 10% fetal calf serum


enhanced several growth parameters, but not all, and tended to reduce lead's toxic effects for both hippocampal and cortical cells. Lead affected both cell types. However, hippocampal cells displayed an increased sensitivity to lead in axon length, dendrite length, and survival rates. Initial results indicated that TPEN may be a promising tool for measuring intracellular calcium using the fura-2 indicator dye in lead exposed cells. This abstract accurately represents the content of the candidate's thesis. I recommend its publication. Signed Teresa Audesirk IV


To My Supportive Family


ACKNOWLEDGEMENTS I am sincerely grateful to Dr. Teresa Audesirk for her guidance, help, and support throughout this entire project. I would like to thank Dr. Gerald Audesirk and Dr. Alan Brockway for their valuable time and suggestions regarding this work. I would also like to acknowledge Charlie Ferguson, David Shugarts and Leigh Cabell-Kluch for their assistance and help, without which much of this work would not have been possible. This work was supported by a grant from the National Institutes of Environmental Health Sciences. Vl


CONTENTS Figures ..... .Xl Tables .... . . . . . . . Xll CHAPTER 1. INTRODUCTION . . . . . . . 1 General Lead Overview/Thesis Arrangement .... .. 1 High Dose Lead Neurotoxicity .. 2 Low Dose Lead Neurotoxicity . . . . . ... 3 In Vivo Lead Neurotoxicity Models . . . . 5 In Vitro Assays for Lead Neurotoxicity . . . . 10 Mechanisms of Lead Neurotoxicity . . . . .13 Interference with [Ca2+]; Regulation by Lead .. ......... 14 Effects of Lead on Plasma Membrane Regulation of Calcium ......................... 15 Endoplasmic Reticulum Regulation of Calcium .......................... 21 Effects of Lead on the Endoplasmic Reticulum .................................. 22 Mitochondrial Calcium Regulation .................. 23 Effects of Lead on Mitochondrial Calcium Stores .............................. 24 Intracellular Effects of Lead ........................ 25 Lead and Neurotransmitter Release . . . . 26


Effects of Lead on Protein Kinase C. . . . 28 Effects of Lead on Calmodulin . . . . 30 2. CELL CULTURE EXPERIMENTS ................... 31 Introduction/Purpose of Experiments . . . . 31 Materials and Methods . . . . . . .. 32 Cell Isolation . . . . . . . 32 Cell Cryopreservation . . . . . . 33 Cell Culture Methods . . . . . . .. 33 Assessment of Growth Parameters . . . . 35 Cell Culture Statistical Analysis . . . . 37 Results . . . . . . . . . 38 Percent Cell Survival . . . . . . 38 Percent Cells Initiating Neurites . . . . 38 Mean Axon Length . . . . . . 41 Number of Branches Per Axon . ............. 45 Number of Dendrites Per Cell . .............. 45 Mean Dendrite Length . . . . . . 48 Other Parameters . . . . . . 48 Discussion . . . . . . . . 50 2% versus 10 % FCS Medium Comparison ............ 50 Vlll


Comparison of Survival and Initiation. . . . Cellular Mechanisms That Regulate Initiation, Dendrite Production, and Axonal Branching . . . Axon and Dendrite Elongation . . . 55 58 60 Overall Cell Type Comparison . . . . . 62 Future Experiments . . . . . . . 64 3. FURA/TPEN EXPERIMENTS . . . . . 66 Fura Introduction . . . . . . . 66 The Use of TPEN as a Tool to Measure [Ca2+]; ........... 68 Purpose of the Experiment . . ........ 70 Fura Materials and Methods . . . . . 71 Fura-2 Loading of Cells ....................... 71 TPEN Experimental Procedure ................... 72 Imaging System Operation ...................... 74 Data Analysis of Images ....................... 75 Fura-2 Imaging Statistical Analysis ................................. 76 Fura Results . . . . . . . . 76 Assessment of TPEN/Lead Binding ................. 76 TPEN/Lead Perfusion Experiments . . . . 78 IX


Fura Discussion . . . . . . . 81 TPEN/Lead Binding Assay . . . . . 81 TPEN and Lead Perfusion Experiments . . . . . . . 82 Future Experiments . . . . . . ... 87 4. FINAL SUMMARY/CONCLUSIONS ................. 89 LITERATURE CITED . . . . . . . 91 X


FIGURES Figure 1. The effects of lead on cell survival . . . . 39 2. The effects of lead on the percentage of living cells that produced neurites . . . . 40 3. Photomicrographs of cortical neurons ................. 42 4. Photomicrographs of hippocampal neurons ............... 43 5. The effects of lead on mean axon length . . . . 44 6. The effects of lead on the number of branches per axon . . . . . . . 46 7. The effects of lead on the number of dendrites per cell . . . . . . . 4 7 8. The effects of lead on mean dendrite length ............. .49 9. Perfusion of hippocampal cells in HKCl medium . . . . . . 79 10. Perfusion of hippocampal cells with TPEN medium . . . . . . 79 11. Perfusion of hippocampal cells with lead medium . . . . . . 80 12. Perfusion of hippocampal cells with TPEN/lead medium ....................... 80 XI


TABLES Tables 1. Lead Effects on Hippocampal and Cortical Neurons . .. 51 2. TPEN/Lead Binding Assessment .................... 77 Xll


CHAPTER 1 INTRODUCTION General Lead Overview/Thesis Arrangement The heavy metal lead has long been utilized in the human history. The Egyptians found it useful for ornaments, cosmetics, and figurines. The Greeks were aware of lead's toxicity, but still used it to line wine storage vessels. The Romans used lead in their vast water systems (see review by Needleman and Landrigan, 1981). Today lead is still used, although not as frequently, since there has been increasing awareness of lead's toxicity. Lead in large doses is very toxic, and it is suspected that there may be no concentration threshold to lead's deleterious effects on the brain (for a review see Audesirk, 1985). The Centers for Disease Control (CDC) has set the acceptable blood lead level at 10-15 ug/ dL, reduced from 30-40 ug/dL from the 1970's (Needleman and Landrigan, 1981; Tiffany Castiglioni et al., 1989). If the blood level is above 10-15 ug/ dL, treatment (chelation therapy) may be recommended, since several studies suggest that higher lead doses result in deficits in cognitive and sensorimotor development in children (see review by Needleman and Bellinger, 1991).


In this introduction I will discuss lead's behavioral and morphological (brain) effects in children, in vivo and in vitro models for studying lead neurotoxicity, and some of the proposed mechanisms of lead neurotoxicity. For simplicity and ease of reading, this thesis is divided into two main parts, which reflect the research areas. The first part of the thesis includes the effects of lead on cultured rat cortical neurons. This part contains a separate introduction, methods, results, and discussion section. The second part includes preliminary research on the feasibility of using a heavy metal chelator, N,N,N' ,N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), in order to measure intracellular calcium in the presence of lead. This part contains a separate introduction, methods, results, and discussion section. Finally, in an attempt to unify and relate the two parts of the thesis, I will conclude with a summary of the research. High Dose Lead Neurotoxicity Lead exposure can have devastating effects on the peripheral and central nervous systems. I will focus on the central nervous system for this introduction because it is relevant to my thesis research (for a review of studies on the effects of lead on the peripheral nervous system, see Audesirk, 1985). Also, most of the studies mentioned in this introduction will be concerned with the effects of inorganic lead. 2


Acute childhood lead encephalopathy, characterized by seizures and stupor, may result from blood lead levels in excess of 80-100 ug lead/ dl blood (Tiffany-Castiglioni et al., 1989). Such massive doses of lead result in clearly manifested symptoms such as permanent mental retardation and can eventually result in death if left untreated (Pentschew, 1965; Scott and Lew, 1986). The cellular basis of lead induced encephalopathy is not clearly understood, but is believed to be due to neuronal as well as neuroglial malfunctioning. Childhood encephalopathy is characterized, in humans and rats, by edema and hemorrhage of the cerebellum and cerebral cortex, which may be the result of increased capillary permeability of the immature blood-brain barrier (damaged by lead), which may in turn allow more lead to be absorbed into the brain (Clasen et al., 1974; Goldstein et al., 1974; Holtzman et al., 1987; Press, 1977; see review by Tiffany Castiglioni et al., 1989). Low Dose Lead Neurotoxicity Current research has focused on the detrimental effects of low dose, chronic lead exposure. It is believed that subclinical lead exposure may cause mental deficits such as low I .. Q., hyperactivity, and poor fine motor control. Particularly susceptible to low doses of lead are young children whose nervous systems have not fully developed (see reviews by 3


Needleman and Bellinger, 1991; Needleman, 1983; Needleman and Landrigan, 1981). Numerous studies show that lead exerts toxic effects on childhood behavior and brain development (Needleman and Bellinger, 1991). Interpretation and comparison of these studies is sometimes difficult due to confounding variables such as how lead exposure was measured (hair, tooth or blood analysis), the type of psychological testing (there are many different tests), and background variability of test subjects (e.g., differences in social class, education, medical history and parental education)(Lee and Moore, 1990). Needleman et al. (1979) found a correlation between children with high lead ( > 200ppm) levels in dentine and decreased classroom performance. Needleman et al. ( 1990) in a follow-up study ( 11 years later) of the dentine level study, found that behavior such as poor reading skills and poor hand-eye coordination persisted as the children aged. Chronic low level lead exposure has also been shown to be related to hyperactivity in children (David, 1974). Also, Bellinger et al. (1984) have shown that there is an impairment in sensory motor development of children exposed to low doses of lead prenatally. Alternately, Dietrich et al. (1990) found that infants with high blood levels did not display any behaviorial abnormalities during the first two years of life. The authors 4


caution that although there were no effects of lead observed in the two year olds, the possibility that lead may affect later brain development does exist. Several studies using rats as models provide further evidence that lead at low doses causes behavioral defects (Crofton et al., 1980; Overmann, 1977; Petit and Alfano, 1979). Crofton et al. (1980) showed that exploratory and locomotor behavior was delayed in neonatal rats exposed to lead via the milk of dams. Overmann ( 1977) found that neonatal rats exposed to lead via intubation displayed an increase in motor activity (i.e., jiggle cages) and poor motor coordination (i.e., wheel rotation test). Petit and Alfano (1979) observed increases in activity levels of neonatal rats exposed to lead through the milk of dams. They also noted that the blood lead levels measured in the rats were higher than what is reported in children with behavioral abnormalities. This suggests that the rat may be more tolerant to higher doses of lead than children. Numerous other studies of both animal models and humans have concluded that lead at low doses should be of concern (see reviews by Davis and Svendsgaard, 1987; Marlowe, 1985; Lee and Moore, 1990). In Vivo Lead Neurotoxicity Models Much research has been done using rat or animal models that substantiate the toxic effects of lead on behavior (Petit et al., 1983). This 5


research has revealed that low doses of lead affect the morphology and development of the brain, particularly the hippocampus and cerebral cortex. For example, Alfano et al. (1982) found a significant reduction in the length and width of the hippocampal mossy fiber pathway in neonatal rats administered lead carbonate (PbC03 ) through maternal milk of dams fed 4% PbC03 However, they found that between the ages of 25 and 60 days, the lead exposed rats displayed an increase in width and length in the mossy fiber pathway similar to what was seen with control rats. Mossy fibers are axons of the dentate granule cells of the dentate gyrus (region in the hippocampus) that extend into a different region of the hippocampus (the CA3 region, which primarily contains pyramidal cells). It was suggested by the study that proper functioning of the hippocampus (which is responsible for memory and learning), was disrupted due to the improper communication between the dentate gyrus and the CA3 region. It was also suggested that the reason the older animals (age 25-60 days) did not display any effects of lead was due to the maturity of the mossy fiber pathway at this stage of development. Therefore, as the animal matured and the mossy fiber pathway matured, the pathway was more capable of withstanding the effects of lead, or could recover from the effects more so than an immature animal's mossy fiber pathway. 6


Changes in dendritic branching have also been observed. For example, hippocampal granule cells of neonatal rats exposed to lead via the milk of dams fed 4% and 0.4% PbC03 displayed an increase in dendritic branching at 20 urn from the cell body, but a decrease in dendritic branching at 160 urn from the cell body and a decrease in length of the entire dendritic field (Alfano and Petit, 1982). The normal dendritic development of the dentate gyrus occurs with afferent axon growth (to the CA3 region), an increase in dendrite length and an increase in dendrite branching distal to the cell body. Therefore, in lead exposed rats, the normal development of dendrites (elongation and branching) was reduced. Alfano and Petit (1982) suggested that this disruption in dendritic development may hinder the normal communication of cells in the hippocampus. Newborn rats exposed to lead via maternal milk of dams (fed 0.1% lead carbonate), then subsequently exposed to lead in drinking water (0.1% lead carbonate), displayed a significant decrease in the density of dendritic spines of hippocampal pyramidal neurons (Kiraly and Jones, 1982). There have also been reports of lead affecting the morphology of the cerebral cortex. Petit and LeBoutillier (1979) found that neonatal rats exposed to lead via maternal milk of dams (fed 4% lead carbonate in diet), displayed a reduced neocortical brain weight and decreased dendritic branching of pyramidal cells at 80-100 urn from the cell body. 7


Additionally, they found a decrease in synapse number in the occipital cortex and a decrease in the length of the apical dendrite of pyramidal cells. A decrease in synapse formation may disrupt the normal communication between cells. Averill and Needleman ( 1980) noted a decreased synaptogenesis in the frontal cortex of rat pups exposed to lead through maternal milk. Another study involving neonatal rats exposed to lead revealed that a reduction in synaptic density in the cerebral cortex occurred, and it was suggested that this was due to lead's effects on cerebral energy metabolism (McCauley et a!., 1979). Finally, Krigman et a!. (1974) found a decreased number of synapses and reduction of the thickness and weight of gray matter in the cerebral cortex of rats exposed to lead via maternal milk of dams (fed 4% lead carbonate in their diet). Normal development of the cerebral cortex involves a progression of glial cell influx and neural cell body enlargement with dendritic and axonal branching and finally synapse formation. Krigman eta!. (1974) proposed that the observed decrease in cerebral cortical weight was due to smaller cell bodies and decreased axonal and dendritic branching in animals exposed to "lead. As can be seen by the above studies, the main areas of concern have been the hippocampus and cerebral cortex, because behavioral abnormalities are associated with malfunctioning of these areas (Petit et a!., 8


1983). Other brain regions have been examined. For example, McConnell and Berry (1979) examined cerebellar Purkinje cells in rats exposed to lead and found a decrease in dendritic length and branching. However, the hippocampus and cerebral cortex have been the main regions of morphological studies. Another factor in lead affecting the hippocampus is that lead tends to preferentially accumulate in the hippocampus. It is also possible that since lead accumulates in the hippocampus, this may make it particularly susceptible to lead toxicity. For example, Fjerdingstad et al. (1974) tested the lead content of hippocampi dissected from both halves of rat brains. Using atomic absorption analysis, they found that the hippocampus contained 10 times more lead than the rest of the brain (measurements made on dried brains). Scheuhammer and Cherian (1982) found that although other brain regions accumulated lead (i.e., amygdala, cerebral cortex, cerebellum, and striatum), the hippocampus had the highest content of lead in rats maintained on a lead supplemented diet for several weeks. Collins et al. (1982) also found that lead tended to collect in the hippocampus, but factors such as lead dosage and exposure duration and time may affect the accumulation of lead in the various brain regions. For example, small doses of lead (0.025 mg/kg) administered for 8 weeks caused an increase in hippocampal lead accumulation of 127%. High lead 9


doses (0.1 mg/kg) administered for 4 weeks caused an increase in lead accumulation in the hippocampus and cerebral cortex. However, administration of high doses of lead for 8 weeks resulted in elevated distributions of lead within various brain regions (hippocampus, midbrain, cerebral cortex, and striatum). Another similar study showed that the hippocampus, cerebellum, striatum and hypothalamus of suckling rats had higher lead concentrations than the cerebral cortex (Kishi et a!., 1982). Also, the cerebral cortex, cerebellum and medulla oblongata of suckling rats had a higher accumulation of lead than those of adult rats, perhaps owing to an underdeveloped blood-brain barrier incapable of excluding lead (Kishi et al., 1982). Finally, a postmortem study of adult humans exposed to low doses of lead revealed that the hippocampus and amygdala had higher concentrations of lead than the cerebral cortex (Grandjean, 1978). Therefore, one particular region of interest in regards to lead toxicity has been the hippocampus, although it is likely that other brain regions are affected by lead as well. In Vitro Assays for Lead Neurotoxicity When attempting to elucidate mechanisms underlying lead neurotoxicity, one can study its effects on isolated cells rather than the whole organism, which involves many complicating interactions. The 10


disadvantage of studying human subjects is that lead exposure cannot be controlled. Therefore, animal models are routinely employed. The problem with animal models is that is is difficult to examine the effects and mechanisms of lead toxicity on isolated cells. To circumvent some of the problems with in vivo models, in vitro studies are used. The precise sites of lead's actions can be more readily discerned in a controlled environment. One disadvantage to using cell culture methods is that lead toxicity does involve interactions. For example, glial cells normally protect neuronal cells by secreting and taking up substances. Lead may indirectly affect neurons by disrupting glial cell functions (Holtzman et al., 1987; Tiffany-Castiglioni et al., 1989). Therefore, glial cells may buffer lead neurotoxicity in vivo, but may or may not be present in vitro. Also, exposure times in culture are often limited to days and weeks, which may not correlate with human exposure durations of years (see review by Audesirk, 1985). There have been in vitro studies on the effects of lead on brain morphology and development using several different cell types. Audesirk et al. ( 1989) studied the effects of inorganic and organic (triethyl) lead on cultured chick embryonic neurons and Lymnaea stagnalis (pond snail) neurons. They found that in chick neurons, inorganic lead ( 100-1000 uM) decreased the number of cells that initiated neurites, and that lead did not 11


affect neurite length or the number of neurites per cell. The snail neurons displayed a decrease in the number of cells that initiated neurites upon exposure to 10 uM lead. In another study Audesirk et al. (1991) found that rat hippocampal neurons displayed a multimodal, dose dependent response to inorganic lead. Hippocampal cells exposed to high (100-1000 uM) and low (25-500 nM) doses of lead showed a decrease in the percentage of cells that initiated neurites. Also, the axon lengths and axon branches of hippocampal cells were increased at higher doses of lead ( 1001000 uM). Finally, they saw an increase in the number of dendrites in rat hippocampal cells. N1E-115 neuroblastoma cells displayed an increase in neurite length at high and low doses of lead, while B-50 neuroblastoma cells showed no effects. The initiation rate for the N 1E-115 neuroblastoma cells was increased at high and low doses of lead, while the B-50 neuroblastoma cells were not affected. Audesirk et al. ( 1991) postulated that the differences in lead's effects were due to the cell type and its ability to sequester, expel or compensate for lead exposure. Or, alternatively, that different cell types had different mechanisms of differentiation. The study by Audesirk et al. (1991) was designed to examine the effects of lead on neuronal development, and showed that lead had dramatic effects at what are considered to be low doses (nanomolar range). Also, the dose dependent multimodal effects observed are not unknown, since others have 12


shown similar responses to lead in vivo (Slomianka et al., 1989). For example, suckling rats exposed to lead (via maternal milk of dams fed 109872 ppm lead) displayed varying effects on the hippocampus. The CA3 region showed a reduction in thickness when exposed to low doses of lead (218 ppm). At higher doses ( 436-872 ppm) the CA3 region no longer displayed any significant effects. Mechanisms of Lead Neurotoxicity So far I have mentioned numerous behavioral, in vivo, and in vitro morphological studies of the effects of lead. Another aim of current research is to elucidate the mechanisms of lead neurotoxicity in order to understand its effects which in tum will allow for better treatment and more realistic exposure allowances. It should be noted that the cellular mechanisms of lead's action are not well understood, and that many different mechanisms and complicated interactions may be involved. Basically, it is believed that lead exerts its effects by either interfering with or mimicking calcium and calcium regulated processes (Audesirk, 1985; Bressler and Goldstein, 1991; Pounds,1984). Intracellular calcium ([Ca2+]J regulation is very for many cellular processes including development, learning and memory, synaptic transmission and cytoskeletal integrity. Calcium has many potential intracellular targets. Calcium 13


directly regulates phospholipase C, phospholipase A2 ion channels, protein kinase C, and calpain. Additionally, calcium indirectly affects proteins (i.e., CAM kinase II, adenylate cyclase and calcineurin) via calmodulin (Kennedy, 1989). Any disruption in calcium regulation could be very detrimental (Pounds and Rosen, 1988; Bondy 1989). Interference with fCa2+l; Regulation by Lead At this point it is pertinent to discuss some basic principles of intracellular calcium [Ca2+]; regulation, and how lead may impact the processes of calcium regulation. The intracellular concentration of calcium [Ca2+]; of a normal resting cell is approximately 107 M, with the extracellular concentration being much higher {approximately 103 M) (Bondy, 1989). Therefore, the cell must have mechanisms to regulate [Ca2+]; since there is a large calcium concentration gradient. The plasma membrane, inner mitochondrial membrane, and endoplasmic reticulum are all involved in internal calcium regulation (Pounds and Rosen, 1988; Carafoli, 1987). Each component has both different and similar mechanisms to regulate [Ca2+];. For example, the plasma membrane has calcium permeable channels, sodium/calcium exchange proteins and Ca2+ -A TPase pumps. The endoplasmic reticulum has Ca2+ -A TPases, and mitochondria have calcium 14


uniporters and sodium/calcium exchange proteins. I will describe the mechanisms of [Ca2+]i regulation, and how lead possibly disrupts each mechanism. For a comprehensive review on the mechanisms of intracellular calcium regulation of cultured hippocampal cells see Frank (1991). Effects of Lead on Plasma Membrane Regulation of Calcium The plasma membrane is probably the most important site of [Ca2+l regulation (Carafoli, 1987). The main mechanisms of calcium regulation at the plasma membrane are through calcium channels, sodium/calcium exchangers and calcium ATPase pumps (Kostyuk and Tepikin, 1991). Calcium Channels. Voltage sensitive calcium channels (VSCC) can simplistically be grouped into 3 types: L, T, and N. These are transmembrane ion channels that allow for calcium to flow inward upon voltage activation (membrane depolarization) (Audesirk, 1990; Tsien et al., 1988). The T-type (transient) channel has a low threshold for activation (i.e., activated by membrane potentials near resting potential) and then inactivates rapidly during sustained depolarization. The L-type (long lasting) dihydropyridine-sensitive channel activates at a more positive potential and undergoes little inactivation during sustained depolarization. Finally, the N-type (neither L nor T) channel opens with large 15


depolarizations and inactivates fairly rapidly during long lasting depolarizations (Audesirk, 1990; Kostyuk and Tepikin, 1991). Another type of calcium channel is the receptor operated calcium channel (ROC). These channels operate by binding with a ligand, which facilitates calcium channel opening, thereby allowing calcium influx (Miller, 1987). TheN-methyl D-aspartate (NMDA) receptor is an example of a ROC. When the amino acid glutamate binds to the NMDA receptor, the channel opens and calcium ions can enter, and perhaps voltage dependent channels also open. Normally, the NMDA receptor is blocked by magnesium. Upon depolarization, which occurs upon receptor binding and channel activation, the magnesium blockage is relieved. The NMDA receptor is thought to be important for long term potentiation (memory) (McCormick, 1990). Effects of lead on calcium channels. Various electrophysiological studies using voltage clamp techniques on several cell types have revealed that lead can inhibit or block calcium influx through VSCC. Free lead competes with calcium for the VSCC and blocks calcium's entry (Audesirk and Audesirk, in press). For example, in snail neurons (Lymnaea stagnalis), lead acetate concentrations of 0.25-14 uM inhibited calcium influx through VSCC (Audesirk, 1987). Another study by Audesirk and Audesirk ( 1989) showed that pond snail neurons displayed a reduction of 16


calcium current through channels due to nanomolar free lead concentrations, but the effect varied depending on the cell type. Lead also has been shown to be a competitive calcium channel blocker in Aplysia neurons (Busselberg et a/., 1991). Perhaps more relevant to humans are similar studies of calcium channels in vertebrate neurons. For example, free lead at concentrations of 20 nM-14 uM inhibited the Tand Ltype channels in N1E-115 mouse neuroblastoma cells, with the L-type channel being somewhat more sensitive (Audesirk and Audesirk, 1991). Oortgiesen et al. (1990) also found that N1E-115 neuroblastoma cells showed L-and T-type channel blockage when exposed to lead. Audesirk and Audesirk (in press) showed that lead was somewhat more selective against L-type than N-type calcium channels in cultured rat hippocampal neurons. In yet another cell type, SH-SY5Y human neuroblastoma cells, lead blocked both the Nand Ltype calcium channels but the free lead concentration was not measured (Reuveny and Narahashi, 1991). Evans et al. (1991) found that lead blocked Land T-type calcium currents in rat dorsal root ganglion cells. It can be concluded that lead has varying inhibitory effects on the different cell types as well as on different channel types (Audesirk, in press). It has also been shown that while lead can bind to and block calcium channels, it can also enter the cell through calcium channels. Simons and 17


Pocock (1987) observed that lead entered through the calcium channels of bovine adrenal medullary cells. Furthermore, they observed that lead's entrance was blocked by the L-type channel blocker, D-600. Another study performed on bovine chromaffin cells measured the intracellular lead concentration by the use of the fura-2 indicator dye (Tomsig and Suszkiw, 1990). When lead binds with this indicator, it emits a slightly different wavelength than the calcium/fura complex. In principle this allows one to estimate the lead concentration. Tomsig and Suszkiw (1990) found that when the chromaffin cells were exposed to micromolar free lead concentrations, the intracellular lead concentration was estimated to be in the picomolar range. They found that when the cells were depolarized in a high potassium chloride medium, lead entry was enhanced although still in the picomolar range. In another study similar to the one mentioned above, Tomsig and Suszkiw (1991) confirmed that lead entered through calcium channels by demonstrating that lead's entrance was enhanced by the calcium channel agonist Bay K8644 and blocked by the L-channel blocker, nifedipine. It should be noted that in these experiments, the calcium concentration was assumed to remain constant upon lead entry. But it has been shown that lead may increase intracellular calcium (Schanne et al., 1989a,b). Therefore, the true lead concentration may not be accurately measured by the technique of Tomsig and Suszkiw (1990). 18


The above studies show that lead not only blocks calcium channels, but also enters into the cell cytosol through calcium channels. Sodium/calcium exchanger. The sodium/calcium (Na + /Ca2+) exchange protein of the plasma membrane is bi-directional, meaning that calcium can be transported into or out of the cell, depending on the relative calcium and sodium concentrations (Blaustein, 1988). The exchanger normally operates by using the sodium gradient (electrochemical gradient) across the membrane to power the expulsion of calcium out of the cell (Blaustein, 1988). The efflux of calcium occurs when there is a high external sodium concentration, and conversely influx of calcium occurs when extracellular sodium is reduced or intracellular sodium is increased. This allows for calcium to be exchanged for sodium. The stoichiometry for ion movement is 3 sodiums: 1 calcium (Carafoli, 1987). The exchanger has a lower affinity (K.J =0.5-1 uM) for calcium than the Ca2+ -A TPase pump, but a higher exchange rate than the Ca2+ATPase pump discussed below. Kd is a dissociation constant and can be defined as the affinity of the exchanger to bind with calcium, or the concentration of calcium at which the binding of the exchanger is at half of its maximum capacity. If the Kd is a low number (low concentration), then the affinity for binding is high. 19


So the sodium/calcium exchanger does play a role in cellular calcium regulation, the importance depending on the cell type examined (i.e., excitable cells display greater importance) (Blaustein, 1988). Effects of Lead on the Na+ /Ca2+ exchanger. To date, there is not much available information on the effects of lead on the sodium/calcium exchanger (Simons, in press). However, since lead is known to interfere with calcium related processes, for example by blocking VSCC, it is entirely possible that lead may exert some effect on the sodium/calcium exchanger. It is possible that lead may mimic calcium and enter or exit through the exchanger. In this manner, lead could also disrupt the calcium concentration by competing with calcium and indirectly decreasing or increasing [Ca2+]i (Pounds, 1984). Calcium Pump (Ca2+ -A TPase). Ca2+-A TPase is another important calcium regulating mechanism located in the plasma membrane. It may be the major pathway for calcium transport across the membrane (Nicholls, 1986). The pump is an enzyme with a high affinity for calcium, compared to the sodium/calcium exchanger, but with a lower transport capacity (Blaustein, 1988). The pump uses ATP hydrolysis to remove calcium from the cell in a 1:1 ratio (i.e., 1 ATP: 1 Ca2+). The Ca2+-ATPase pump is also activated directly by the calcium binding protein calmodulin. 20


Calmodulin increases the affinity of the pump for calcium thereby, increasing calcium transport (Carafoli, 1987). Effects of Lead on the Ca2+ ATPase pump. There have been some studies on the effects of lead on the calcium pump of the plasma membrane of various cell types. However, none of the cell types used were nerve cells. Simons (1988) showed that in human red blood cell ghosts, lead can substitute for calcium and is transported out of the cell via the calcium pump. Lead had a higher affinity for the pump (I<.J = 5 x 10 -7M) compared to calcium (Kd= 1 x 1Q6M). In this way, lead may be expelled out of the cell instead of calcium, causing an increase of [Ca2+];. A study involving the use of erythrocytes showed that lead (6 X 10"6 M) inhibited the calcium pump at several different calcium and calmodulin concentrations, while stimulating the pump at lower concentrations of lead (Mas-Oliva, 1989). Therefore, if lead is interfering with the Ca2+ -ATPase, it could conceivably cause an increase or decrease in [Ca2+];. Endoplasmic Reticulum Regulation of Calcium The endoplasmic reticulum (ER) is another component in the regulation of [Ca2+];. It is believed that the ER is a major contributor to calcium sequestration (Carafoli, 1987). The ability of the ER to sequester calcium comes from a Ca2+ -ATPase similar to the one described above for the 21


plasma membrane (Meldolesi et al., 1988). The ER and plasma membrane do differ in that thapsigargin inhibits the ER Ca2+-ATPase from taking in calcium and also causes calcium release, but does not inhibit the plasma membrane Ca2+ -ATPase pump activity (Moore et al., 1991; Thastrup et al., 1990). Also, the ER Ca2+ -ATPases are calmodulin-independent and exchange two Ca2+ for the hydrolysis of 1 ATP (Blaustein, 1988). Just as the ER can take up calcium, it has the ability to release calcium from its stores. The mechanism of calcium's release involves inositol triphosphate (IP3). IP3 and diacylglycerol (DAG) are products of the breakdown of phosphoinositol biphosphate The binding of a ligand to a membrane receptor activates a G protein which in turn activates phospholipase C (PLC) which actually breaks down the PIP2 The IP3 is formed, then binds to receptors on the ER membrane causing calcium to be released into the cytosol (Nahorski, 1988). Effects of Lead on the Endoplasmic Reticulum There are no studies on the effects of lead on the ER of nerve cells. However, some indirect evidence using other cell types indicates that lead may interfere with the ER's storage of calcium (Simons, in press). One study, by Pounds and Mittlestaedt (1983), found that in rat hepatocytes, calcium and lead are treated in the same manner by the ER. For example, 22


upon hormonal stimulation (see receptor binding, above), both calcium and lead were mobilized from intracellular stores. Of course, any applications mentioned above under Ca2+ pump of the plasma membrane may apply to the ER as well. For example, it is possible that intracellular lead is sequestered by the ER at the expense of calcium, thereby causing an increase in [Ca2+]i. Since the ER is an important site of calcium regulation, more studies involving lead's effects on the ER should be performed (Rosen and Pounds, 1989). Mitochondrial Calcium Regulation The inner mitochondrial membrane also has the ability to regulate [Ca2+l (Carafoli, 1987). One mechanism by which mitochondria regulate calcium is through a sodium/calcium exchanger similar to that mentioned previously for the plasma membrane. Like the ER, mitochondria have the ability to take up calcium when the concentration is too high. The mechanism of sequestration is due to a calcium uniport. The uniporter, located in the inner mitochondrial membrane, operates by the use of the chemiosmotic mechanism which links the electron transport chain to ATP synthesis, to move calcium into mitochondrial matrix. There is a charge differential between the mitochondrial matrix and intermembrane space. This is created by 23


hydrogen ions being pumped into the space from the electron transport chain. This membrane potential allows the transport of positively charged calcium ions through the uniport into the negatively charged mitochondrial matrix (Nicholls, 1986). Effects of Lead on Mitochondrial Calcium Stores There have been several studies on the effects of lead on mitochondrial [Ca2+]; regulation. Goldstein (1977), showed that exposure to 5 uM lead resulted in a 50% reduction of calcium uptake by mitochondria isolated from rat brains. They reported that this concentration of lead was five times less than the concentration of lead measured in a previous study of the brains of rats afflicted with lead encephalopathy. Therefore, in this instance, lead would be expected to cause an increase in [Ca2+];, although since isolated mitochondria were used, the [Ca2+]; of intact cells was not measured. In another in vitro assay, lead was shown to cause calcium to be released from mitochondria isolated from kidney cells (Kapoor and van Rossum, 1984). They found that lead inhibited calcium uptake by isolated mitochondria, and displaced the calcium already present in the mitochondria. One conflicting report on rat osteoblasts stated that lead increased mitochondrial calcium stores (Rosen and Pounds, 1989). This could reflect different functions of kidney versus bone cells. Therefore, it 24


is possible that lead may exert diverse effects on the capacity of the mitochondria to regulate [Ca2+]i (Simons, in press). Intracellular Effects of Lead As stated previously, lead is known to enter cells via calcium channels. Once inside the cell, lead may interfere with regulation of intracellular calcium, or mimic calcium in calcium regulated processes, or inhibit calcium regulated processes. For example, Schanne et al. (1989a) using 19F-NMR techniques, observed that in cultured osteoblast cells, 5 uM and 25 uM lead concentrations caused a 50% and 120% increase respectively in [Ca2+]i from resting [Ca2+L over a 5 hour exposure time. The intracellular lead concentration was measured to be approximately 29 pM with the extracellular lead concentration being 25 uM. Schanne et al. (1989b) found similar results using NG108-15 cells, a neuroblastoma x glioma hybrid cell line. In this study the cells were exposed to 5 uM lead for 2 hours and they displayed a 2 fold increase in calcium concentration from the resting [Ca2+li The intracellular lead concentration was measured to be 30 pM. Consistent with the above studies are fura2 studies used to measure lead inside the cell. Tomsig and Suszkiw (1990) (using questionable assumptions described earlier) found that bovine chromaffin cells exposed to micromolar free lead concentrations had 1 o-11 25


to 10-12 M lead concentrations inside the cell. Also, bovine chromaffin cells exposed to 0.5 uM free lead had 2.5 20 pM lead inside the cell depending on the potassium chloride concentration of the medium (Tomsig and Suszkiw, 1991). Once lead is inside the cell, it may exert effects by increasing calcium, perhaps by the mechanisms mentioned thus far (i.e., effects on mitochondria, ER and plasma membrane). It is also possible that lead may act as a calcium surrogate in many calcium regulated processes. For example, lead may bind with calcium binding proteins such as calmodulin or protein kinase C and therefore cause disruption of normal processes (Pounds, 1984). Some of these calcium binding proteins will be examined. However, since calcium interactions with proteins are numerous, I will not discuss all of the interactions, and there may be more interactions of lead and these proteins yet to be discovered. Lead and Neurotransmitter Release Transmitter release from neurons involves many steps, including: transmitter synthesis; transmitter packaging into synaptic vesicles; depolarization of the presynaptic terminal via an action potential; opening of voltage-sensitive calcium channels, allowing for calcium entry; calcium interactions with proteins that promote vesicle fusion with the membrane; 26


exocytosis and release of transmitter into the synaptic terminal; and binding of the transmitter to the postsynaptic membrane receptors (Shepherd and Koch, 1990). Lead has been shown to interfere with various steps of this process (for a review see Audesirk, 1985). This is deleterious because transmitter release is not only important in the normal functioning of nerve cells (cell cell communication), but it is also important in the guidance and pathfinding of developing neurons (Dodd and Jesse!, 1988). In rat synaptosomal preparations, lead exposure induces spontaneous release of transmitter, but inhibits the normal, depolarization-evoked release of transmitter (Minnema eta/., 1988; Oudar et al., 1989; Shao and Suszkiw, 1991). Also, studies involving neuromuscular junctions show similar effects in that depolarization-evoked transmitter release is inhibited by lead (Cooper and Manalis, 1984; Kober and Cooper, 1976). The postulated mechanism is that lead blocks VSCC, which blocks calcium entry that normally takes place during depolarization-evoked transmitter release. Once inside the cell, lead induces spontaneous release of transmitter either by substituting for calcium in its action with substances that promote vesicle fusion and exocytosis, or increasing [Ca2+]; by the mechanisms mentioned previously (Minnema et al., 1988; Oudar et al., 1989; Shao and Suszkiw; 1991). The permeabilized synaptosome studies 27


(in the absence of calcium in the medium), suggest a direct effect of lead on transmitter release (Shao and Suszkiw, 1991). Effects of Lead on Protein Kinase C Protein kinase C (PKC) is a major regulatory, calcium and phospholipid-dependent enzyme that phosphorylates many substrates (Nishizuka, 1988). PKC has been implicated in a variety of processes such as cell growth and division, secretion and exocytosis, gene expression (early immediate genes such as c-fos) and modulation of ion channels (Nishizuka, 1988). PKC is itself regulated by diacylglycerol. Recall from the earlier discussion of the ER, that upon receptor binding and G protein stimulation, PLC is stimulated to hydrolyze PIP2 into IP3 and DAG. DAG then activates PKC which enables PKC to phosphorylate proteins (Altman, 1988). PKC may be an important regulator in long-term potentiation, which is important in memory and learning processes (Bressler and Goldstein, 1991). PKC also has been shown to affect the growth of neurons in various ways. Felipo et a!. ( 1990) found that PKC inhibition stimulated differentiation in Neuro 2A neuroblastoma cells. Tsuda et al. ( 1989) discovered a similar stimulation of differentiation by the inhibition of PKC 28


on cerebellar and N18TG2 neuroblastoma cells. But, in cerebellar granule cells (Cambray-Deakin et al., 1990), and in chick ganglia (Hsu eta/., 1989), PKC stimulation resulted in neurite outgrowth. Lead has been found to stimulate PKC at picomolar concentrations more effectively than calcium in a cell-free biochemical assay (Markovac and Goldstein, 1988). However, other investigators have found that heavy metals including lead inhibit PKC activity to some degree (Saijoh et al., 1988; Speizer eta/., 1989). Speizer eta/. (1989) found that purified PKC isolated from S49 lymphoma cells was inhibited by high doses (3 uM 300 uM) of heavy metals (cadmium, copper, mercury and zinc). Saijoh eta/. (1988) also used purified PKC from mouse brains to show that PKC was inhibited by lead in the micromolar to millimolar range. Some of the discrepancies in these studies could arise from the fact that different lead concentrations were used. Lison et a/. (1990) used intact macrophages exposed to 1-lOOuM lead for 15 minutes and found that PKC was inhibited by the lOOuM lead but not significantly affected by the 1 uM lead. In intact cells it may be possible that lead is indirectly stimulating PKC via an increase in calcium (Lison et a/., 1990), or once PKC is activated by lead directly, down regulation of PKC may occur due to proteases located at the cell membrane (Nishizuka, 1988). Therefore, if lead disrupts PKC 29


activity, neuronal growth and learning and memory processes may be disrupted. Effects of Lead on Calmodulin Calmodulin is a calcium binding protein that mediates many calcium related processes inside the cell (Cheung, 1984). Many enzymes are regulated by calmodulin including adenylate cyclase, Ca2+-ATPase, phosphodiesterase, and some protein kinases. Calmodulin binds calcium at four different binding sites, and requires at least three sites bound for activation. Therefore, if the [Ca2+]; increases, this will favor calmodulin's activation (Cheung, 1984). Recent studies indicate that heavy metals, including micromolar concentrations of lead, can substitute for calcium and bind to calmodulin, causing its activation (Chao et al., 1984; Cheung, 1984). However, as stated earlier, picomolar concentrations of lead are found in the cells, and this is probably not a high enough concentration to activate calmodulin (Simons, in press). It is plausible that lead may indirectly activate calmodulin by increasing [Ca2+]i (Simons, in press). 30


CHAPTER 2 CELL CULTURE EXPERIMENTS Introduction/Purpose of Experiments The first part of my thesis research includes characterization of the morphological effects of lead on cultured cortical neurons. As described in Chapter I, there is reason to believe that lead affects the morphological development of the hippocampus and other brain areas such as the cerebral cortex (Petit et al., 1983). Therefore, the first objective is to examine the effects of lead (concentrations ranging from 0.1 nM -1 mM) on cultured rat cortical neurons (motor cortex area) in a 2% fetal calf serum (FCS) medium, and compare these results to those obtained previously with hippocampal cells (Audesirk et al., 1991). The second objective of the first part of this work is to examine the effects of lead on the growth of both motor cortex (cortical) and hippocampal neurons in a 10% FCS medium (using similar lead concentrations, 0.1 nM-1 mM). The rationale for examining effects in a higher FCS containing medium, is that the increase in FCS is necessary for optimum growth of cortical cells. Since increased FCS may interact with lead, the experiment was repeated on hippocampal cells using 10% FCS.


Components in the FCS have been shown to bind with lead (Audesirk, unpublished data). The increase in FCS may (or may not) contribute to neuronal viability and growth. Also, it is possible that FCS may bind lead, making it less accessible to the cells. Therefore, the second part of the experiment involved comparing the effects of lead on cortical and hippocampal cells grown in 2% FCS and 10% FCS medium. Materials and Methods Cell Isolation Fetal E-18 (embryonic day 18) rats were obtained from timed-pregnant Sprague-Dawley rats (Harlan Sprague-Dawley). Pregnant rats were sacrificed by cervical dislocation following C02 anesthesia. The fetal brains were removed and the hippocampal and cortical (motor cortex) regions were dissected using sterile techniques. The hippocampal and cortical (motor cortex) regions were dissociated according to the technique described by Banker and Cowan (1977). The tissues were placed in cold Mg2+-and Ca2+ -free Hank's balanced salt solution (HBSS, Sigma) with 10 mM HEPES buffer and 1% antibiotic/antimycotic solution (Sigma). The brain regions were incubated at 37 oc in HBSS with trypsin (2 mg/ml) for 15 minutes. The HBSS with trypsin was removed and the brain regions were placed in HBSS with 32


trypsin inhibitor (2 mg/ml) for 5 minutes at 37 oc. Cells were dissociated by trituration in MEM, then cryopreserved as described below. Cell Cr:yopreservation Cryopreservation methods were slightly modified from Mattson and Kater (1988). The HBSS with trypsin inhibitor was removed and replaced with modified Eagle's Minimum Essential Medium (MEM, Sigma) containing Earle's salts, L-glutamine and 25 mM HEPES. The medium was supplemented with 10 mM sodium bicarbonate, 2% glucose, 1 mM sodium pyruvate, 15 mM potassium chloride (final concentration of 20 mM KCl), 1% antibiotic/antimycotic, 10% heat-inactivated fetal calf serum (FCS) and 8% dimethylsulfoxide (DMSO) as a cryopreservant. Cells were triturated by gentle pipetting with a fire polished pipette. Cells were counted using a hemacytometer and dispensed in 0.25 ml quantities (containing approximately 2 million cells) into 1.8 ml freezing vials. Cells were frozen at -70 oc in 1 inch thick styrofoam containers that slowed the freezing process, so as not to disrupt the cells. Usually, cells were stored no longer than 3 months in the freezer. Cell Culture Methods Cortical and hippocampal cells were thawed rapidly by adding 1 ml of warm 37 oc medium. The neurons were plated onto grided, poly-0-Iysine 33


coated 35 mm plastic dishes. The dishes were coated with poly-D-lysine by soaking in a 1 mg/ml solution for 5 minutes, then rinsed with deionized water (DI H20) and dried. Cells were plated in 2 ml of culture medium at a density of 200,000 cells/dish. The cells usually do not contact one another at this density. The culture medium was the same as for cryopreservation, except that 0.2% glucose was used and no DMSO was added. Also, either 2% or 10% FCS was added depending on the experiment. Cortical neurons in media containing 2% FCS were exposed to lead chloride at concentrations ranging from 0 (control with 3.3% DI H20), 0.1 nM, 1 nM, 10 nM, 25 nM, 100 nM, 1 uM, 100 uM, and 1 mM. Hippocampal cells were studied previously in similar concentrations in 2% FCS medium (Audesirk et al., 1991). It was deemed unnecessary to repeat the entire experiment, so only one concentration was retested (1 mM PbC12 ) on hippocampal cells. The results of this experiment were in accordance with previous results (Audesirk et al., 1991). Initiation of cortical cell neurites was found to be significantly lower in 2% than 10% FCS containing medium. Therefore, the experiment was repeated with both cell types in 10% FCS medium, using a subset of lead concentrations. The concentrations used for hippocampal cells were 0 (control with 3.3% DI H20), 1 nM, 25 nM, 100 nM, 1 uM, 1000 uM. 34


The concentrations used for cortical neurons were 0 (control with 3.3% DI H20), 0.1 nM, 1 nM, 25 nM, 100 nM, 1 uM, 100 uM, 1000 uM. All cells were cultured at 37 oc in a humidified, 5% C02 atmosphere for 2-4 hours to allow for cell attachment. The medium was replaced with fresh medium with the same PbC12 concentrations to remove any DMSO from the initial freezing medium. A survival count of the cells was performed at this time (see below). The cells were then incubated for an additional 2 days after which time growth parameters were measured as described below. Assessment of Growth Parameters All dishes were coded (labelled alphabetically) to avoid any experimenter bias during measurement. The code was deciphered only after all dishes of an experiment had been measured. After 2 days of incubation, several parameters of growth and differentiation were measured using a digitizing tablet and morphometric software (Sigmascan; Jandel Scientific): 1) Percent survival is defined as a percentage of cells alive at 4 hours that have remained alive at 48 hours. This was obtained by counting the number of phase bright and dark, fairly round cells with and without 35


neurites in a total of four microscopic camera fields (using the same four fields at 4 and 48 hours). 2) Percent of living cells inititating neurites was measured. This was obtained by counting field by field until at least 30 cells alive and 10 cells growing neurites were counted. Counting was stopped at a maximum of 50 living cells, even if 10 growing cells had not been found. The following measurements were made on pyramidal-like cells only, which are defined as cells that have one process at least two times as long as any other process (Mattson and Kater, 1988). 3) Number of axonal cells as a fraction of the number of growing cells were counted. This is a count of the number of cells that have axons (a process twice as long as any other process) as opposed to those cells whose dendrites or axons cannot be distinguished morphologically. 4) The mean axon length was measured. This is the sum of all axon lengths divided by the number of axonal cells. 5) Mean number of branches per axon were measured. 6) Mean number of dendrites per cell were measured. 7) Mean dendrite length per axonal cell was measured. 8) Mean number of branches per dendrite were measured. Usually, one experiment consisted of two control dishes (no lead) and several concentrations of PbC12 (two dishes each). Each experiment was 36


repeated at least four times. Therefore, 8 replicate culture dishes were averaged to produce a data point. For an experiment to be acceptable, certain criteria had to be met: 1) survival of control cells had to be at least 35% + 10%; 2) initiation of control cells had to be 40% + 10% for the hippocampal cells in 2% and 10% FCS medium and cortical neurons in 10% FCS, and 35% + 10% for the cortical cells in 2% FCS. If the average of the two controls did not meet these criteria, the experiment was rejected. Cell Culture Statistical Analysis Statistical analysis for the cell culture experiments was performed using a three-way analysis of variance (ANOV A) that took into account interactions between media composition, cell type, and lead concentration. The Dunnett's test was then used for comparison of each experimental mean to its control. Two-tailed Student's t-tests were used to compare control data between different cell types and media. Each data point represents the mean of at least 8 replicates. 37


Results Percent Cell Survival In medium containing 2% FCS, survival of control cortical neurons was significantly higher than that of control hippocampal neurons (p < 0.001; Fig. I, top). The cortical neurons did not display any significant difference from the controls at any lead concentration tested (Fig. I, upper left). In contrast, the hippocampal neurons showed a significant increase in survival at 1 nM PbC12 while a decreased survival rate was observed at 1 uM and 10 uM PbC12 (Fig. 1, upper right). In 10% FCS medium, control cortical neurons showed a significant decrease in survival over those grown in the 2% FCS (84% down to 74%, p < 0.01). The increased FCS content did not significantly change control hippocampal neuron survival. Over the range of lead concentrations tested in the 10% FCS medium, there were no significant effects of lead on survival for either cell type (Fig. 1, bottom). Percent Cells Initiating Neurites Cortical and hippocampal neurons showed similar responses to lead, with both high (100-1000 uM) and low (0.1-500 nM) lead concentrations inhibiting initiation (Fig. 2, top). Photomicrographs depicting cortical and 38


0 > ::J Ul c > ::J Ul 100 90 80 70 60 50 40 JO 90 80 70 60 50 40 JO rat cortical cells (2% fcs) rat cortical cells ( 10% fcs) 10-10 10-8 1o-1 10-7 10-1 10-' 1o- 10-J PbCI2 Concentration (M) rat hippocampal cells (2% fcs) 90 80 70 60 50 40 JO 10-10 10-8 10-1 10-7 1o- 10-' 10- 10-J rat hippocampal cells ( 10% fcs) 100 90 80 70 60 50 40 JO PbCI2 Concentration (M) Fig. 1. The effects of lead on cell survival. The vertical axis represents a percentage of cells that were alive at 4 hr vs. 48 hr. Asterisks indicate statistical significance (p < .05). The dashed line represents the control level. Data points are means s.e.m. 39


Ul Q) u rot cortical cells (2% fcs) 70 60 50 40 .JO 20 10 10_10 1o- 10-1 10-7 1o- 10-' 1o- 10-3 rot cortical cells ( 10% fcs) 70 60 50 40 .JO 20 10 0 10-10 10-1 10-1 10-7 1o- 10-' 10- 10-3 PbCI2 Concentration (M) rat hippocampal cells (2% fcs) rot hippocampal cells ( 10% fcs) 70 60 50 40 .JO 20 10 10-10 10-1 10-1 10-7 1o- 10-' 1o- 1o-3 PbCI2 Concentration (M) Fig. 2. The effects of lead on the percentage of living cells that produced neurites. Asterisks indicate statistical significance (p < .05). The dashed line represents the control level. Data points are means s.e.m. 40


hippocampal cells exposed to lead and controls are shown in Figs. 3 and 4. In 2% FCS medium, the control hippocampal neurons showed a significantly higher initiation rate than the cortical neuron controls (p

c----. -..... 0 .., ..... \:. !} (I -. .. .:' .., u 0 6 e Ji' ..... ,_, Fig. 3. Photomicrographs of cortical neurons. Cells exposed to 1000 uM lead (top) and control cortical cells (bottom) at 48 hrs in culture. 42


.......-.._,.. .: -.:. 0 0 .... .. \ ,z 0 0 0 ... ;) 0 .-p ) .... ;;) (j) > -.:_, "t.i Fig. 4. Photomicrographs of hippocampal neurons. Cells exposed to 1000 uM lead (top) and control hippocampal cells (bottom) at 48 hrs in culture. 43


rot cortical cells {2% fcs} 250 ,....._ E 3 200 .c a. 150 c .!! c 0 100 )( 0 c 0 50 Cll E ,....._ E 3 200 .c a. c 150 .!! c 100 0 c g 50 E 1 o-o 1 o- 1 o- 1 o-7 1 o- 1 o-' 1 o- 1 o-3 rat cortical cells ( 10% fcs) PbCI2 Concentration {M) rot hippocampal cells {2% fcs) 250 200 150 100 50 rot hippocampal cells (10% fcs} 200 150 100 50 1o-o 1o- 1o- 10-7 10- 1o-' 10- 1o-3 PbCI2 Concentration (M} Fig. 5. The effects of lead on mean axon length. Asterisks indicate statistical significance (p< .05). The dashed line represents the control level. Data points are means.. s.e.m. 44


FCS showed no effects of lead on axon length (Fig. 5, right). The cortical neurons in 10% FCS medium exposed to lead showed no effects of lead on axon length, similar to what was observed in 2% FCS medium (Fig. 5, bottom). Number of Branches Per Axon In the 2% FCS medium, high PbC12 concentrations (100-1000 uM) caused an increase in the number of axon branches present in both cortical and hippocampal neurons (Fig. 6, top). Additionally, cortical neurons showed an increase in branching at 100 nM PbC12 concentration. The 10% FCS medium eliminated the effects of lead on axon branching (seen with the 2% FCS medium) for both cell types (Fig. 6, bottom). The increase in FCS did not significantly affect axon branching in the control neurons of either cell type. Number of Dendrites Per Cell In 2% FCS medium, control cortical neurons produced significantly more dendrites than the hippocampal control neurons (Fig. 7, top; p< .001). Lead did not significantly affect the number of dendrites in either cell type at either concentration of FCS (Fig. 7). In 10% FCS medium, control hippocampal neurons produced significantly more dendrites than the control hippocampal neurons in 2% 45


J c 0 )( 0 --..... 2 "' QJ .I:. u c 0 .... .a "'I=: c J 0 )( 0 --..... "' 2 QJ .I:. u c 0 .... .a "'I=: rat cortical cells (2% fcs) rat cortical cells (10% fcs) 1 o-o 10-8 1 o- 1 o-7 1 o-e 1 o-' 10- 10-J PbCI2 Concentration (M) rat hippocampal cells (2% fcs) 2 rat hippocampal cells ( 10% fcs) 2 0 1o-o 10-8 1o- 10-7 10- 10-' 1o- 10-J PbCI2 Concentration (M) Fig. 6. The effects of lead on the number of branches per axon. Asterisks indicate statistical significance (p < .05). The dashed line represents the control level. Data points are means s.e.m. 46


rat cortical cells (2% fcs) rat hippocampal cells (2% fcs) 4 Q; u "-3 Ul 'i: "C 2 c:: cu "C """ rat cortical cells ( 10% fcs) rat hippocampal cells ( 10% fcs) 4 4 2 _.___....._ ...... _.___....._ ...... 1 o-10 1 o- 1 o-1 1 o-7 1 o- 1 o-' 1 o- 1 o-3 1o-10 1o- 10-1 10-7 1o- 10-' 1o- 10-3 PbCI2 Concentration (M) PbCI2 Concentration (M) Fig. 7. The effects of lead on the number of dendrites per cell. Asterisks indicate statistical significance (p < .05). The dashed line represents the control level. Data points are means s.e.m. 47


FCS ( 1.51 to 3.05, p< .001). The cortical neuron controls, however, showed no significant increase in dendrite numbers in response to the increase in FCS (Fig. 7). Mean Dendrite Length In the 2% FCS medium, the rat hippocampal cells displayed a significant increse in dendrite lengths when exposed to 500 uM and l 000 uM concentrations (Fig. 8, top). The cortical neuron dendrite lengths were not affected by lead at any concentration tested (Fig. 8, top). In 10% FCS, both cell type controls showed significant increases in dendrite lengths compared to 2% FCS medium control cells (hippocampal cells: 28 urn up to 51 urn, cortical cells: 40 urn up to 51 urn, p < .01). In 10% FCS medium, hippocampal cells no longer showed any significant effects of lead on dendrite length (Fig. 8, bottom, compare with top). Other Parameters Other parameters were measured, but not depicted graphically because there were no significant effects observed. Dendritic branching. The number of dendrite branches is not reported because in this cell culture system, after 2 days of growth, dendritic branching is too infrequent (usually a mean of much less than 1 branch per dendrite) to make any meaningful comparisons. 48


rat cortical cells (2% fcs) E 160 3 140 .r:: 0> 120 c Oil ....J 100 ;::: "U c Oil 0 c 0 Oil ::! .r:: 80 60 40 20 0> 120 c Oil ....J 100 Oil ;::: 80 "U 60 0 c 40 0 Oil ::! 20 rat cortical cells ( 10% fcs) 0 PbCI2 Concentration (M) rat hippocampal cells (2% fcs) rat hippocampal cells ( 10% fcs) PbCI2 Concentration (M) Fig. 8. The effects of lead on mean dendrite length. Asterisks indicate statistical significance (p < .05). The dashed line represents the control level. Data points are means. s.e.m. 49


Discussion Table 1 provides a summary of the effects of lead on several parameters of growth and differentiation for hippocampal cells and cortical cells, as well as a comparison between 2% and 10% FCS supplemented medium. Together, Table 1 and Figures 1-8 reveal several interesting similarities and differences among cell types. 2% Versus 10% FCS Medium Comparison The interactions among lead, cell type and FCS concentrations are complex. It is possible that the increase in FCS provides a more optimal growth environment for the cells, making them generally more resistant to the toxic effects of lead. In general, the data indicate that an increase in FCS did attenuate the cells' response to lead, making it less toxic. All figures, except Fig. 2 (% initiation), show that 10% FCS reduced lead's effects for both cell types, in some cases obliterating the effects (See Figs. 1, 5, 6, and 8). The hypothesis that 10% FCS medium provides for optimal growth can be substantiated by the observation that the 10% FCS medium produced a significant increase in the inititiation of cortical neurons, axon length in both hippocampal and cortical cells, number of dendrites for hippocampal controls and dendrite lengths for both hippocampal and cortical cells (Refer to Figs. 2, 5, 7, and 8). Initiation 50


U'l Table 11 Lead Effects on Hippocampal and Cortical Neurons Hippocampal Neurons Cortical Neurons I % FCS I 2% I 10% II 2% I 10% Levels' I PbCI, I Ww I Med I Hi I Ww I Med I Hi II Ww I Med I Hi I Ww I Med I Survival 0 -+ 0 0 0 0 0 0 0 Initia-0 -0 -0 --tion Mean Axon 0 0 + 0 0 0 0 0 0 0 Length Branches/ 0 0 + 0 0 0 + 0 + 0 Axon Dendrites per 0 0 0 0 0 0 0 0 0 0 Cell Dendrite 0 0 + 0 0 0 0 0 0 0 Length 1 a II+ II or a "-" denotes at least one value significantly different (increased or decreased) from the control value within the concentration range 2 low = 0.0001 0.5 uM; Medium = 1.0-10 uM; High = 100-1000 uM 0 0 0 0 0 0 I Hi I 0 0 0 -0 I 0 __ I


rate of hippocampal neurons was not affected (incr.eased or decreased) by the increase in FCS, indicating that at least for this parameter, hippocampal cells did not benefit from the increase in FCS. For both hippocampal and cortical cells, survival was decreased upon the increase in FCS. It appears that there may be a somewhat inverse relationship between initiation rate and survival rate, as will be discussed below. Other in vitro studies of cultured rat cortical neurons have shown that the cells are sensitive to the amount and type of serum supplement, requiring at least 5-10% serum supplement for optimum growth (Dichter, 1978). Also, the dependence of cortical neurons on 10% FCS for optimum growth may have artificially made them appear more sensitive to lead in 2% FCS. For example, in 2% FCS meduim, the numbers of axon branches were increased at 10 nM lead in the cortical neurons, but not the hippocampal neurons, making the cortical neurons appear to be more sensitive to lead (Fig. 6). It should be noted that an increase in a growth parameter may be just as harmful to the development of proper synaptic connections as a decrease. Overall, in 2% FCS medium with lead, the hippocampal cells were more sensitive to lead than the cortical cells (Figs. 1, 5, and 8). Any other instances where the cortical cells appeared to be more sensitive, could have resulted from the cortical neuron's need for an increase in FCS for better growth and a more vigorous response to the 52


stimulatory or inhibitory effects of lead (growth was compromised in the 2% FCS medium). So it possible that hippocampal neurons may actually be more sensitive to lead's effects than the cortical neurons. However, the 10% FCS did promote better growth in hippocampal cells for certain parameters such as axon and dendrite lengths and the number of dendrites. Another factor that may play a role in the reduction of lead's effects in response to 10% FCS is that FCS contains proteins and other organic molecules that might bind lead. For instance, lead can bind with the nitrogenous and sulfhydryl groups of many proteins and amino acids (Simons, in press). When the FCS concentration is increased from 2% to 10%, there is a potential for more lead to be bound to substances in the FCS. Audesirk (unpublished data) observed that in 10% FCS medium with nanomolar to micromolar total lead concentrations, the free lead concentration is approximately 20% of that in the 2% FCS supplemented medium. Also, Audesirk et al. (1989) reported that the free lead concentration in Eagle's Minimum Essential Medium with 10% FCS was to 3 orders of magnitude lower than the total PbC12 added. Therefore, in 10% FCS medium, more lead is bound and perhaps less accessible (or less toxic) to the cells than in its free form. For example, a higher total lead concentration would be needed to block calcium channels, since free lead is responsible for the blockage (Audesirk, 1990). If not enough free lead is 53


present, calcium channels will not be inhibited, hence no effects on neurite growth attributed to VSCC blockage will be produced. This phenomenon could account for part of the reduction of lead's effects in 10% FCS medium at the higher total lead concentrations (i.e., dendrite length increase in hippocampal cells). However, the increase in FCS did not totally suppress the effects of lead on cell growth and development. Inititiation for both cell types in 10% FCS medium was still affected in a multimodal manner with high and low lead concentrations inhibiting initiation. It is possible that bound lead may enter the cell, perhaps by endocytosis, and therefore exert intracellular effects on growth (see discussion below). As indicated by other studies, it is probable that both bound and unbound lead have the potential to affect the functioning of the cells (Scott and Lew, 1986). The observation that FCS lowers the amount of free lead is relevant when comparing actual concentrations of total lead found in humans. For example, the total lead concentrations examined in this study are similar to or lower than those found in the cerebrospinal fluid of non-lead exposed adults (25 nM total lead; Cavalieri et al., 1984). Furthermore, cerebrospinal fluid has 0.5% as much protein as FCS so that cerebrospinal fluid contains fewer lead binding proteins than FCS. 54


In summary, the interactions among lead, cell type and FCS are complex. However, lead does affect the development of neurons in virro at concentrations similar to or lower than those reported for cerebrospinal fluid of humans (Cavalieri et al., 1984). Comparison of Survival and Initiation The 10% FCS medium significantly lowered the survival rates for both control cell types, but enhanced the percentage of cells initiating neurites in cortical cells (Figs. 1 and 2). Also, in 2% FCS medium, the higher lead concentrations enhanced survival but decreased initiation. This supports an earlier observation (Audesirk et al., 1991) that neuron survival and initiation seem to have an inverse relationship. This could be interpreted to mean that the cells that have initiated tend to be more suseptible to death than the non-initiated cells. Overall, the growth parameter of initiation appeared to be the most sensitive indicator of lead neurotoxicity in this culture system. Lead significantly inhibited neurite initiation in both hippocampal and cortical neurons at high and low concentrations for both 2 and 10% FCS (Fig. 2). It has been proposed that the mechanism of lead's actions at the higher doses ( 100-1000 uM) might be due to L-type calcium channel blockage (Audesirk et al., 1991). This is supported by a previous study in which 55


Audesirk et al. (1990) found that neurite initiation in cultured chick neurons and NlE-115 neuroblastomas was reduced by blocking L-type calcium channels. Further, lead partially blocks L-type channels in hippocampal neurons (Audesirk and Audesirk, in press). Also, Rogers and Hendry ( 1990) showed that L-type channel blockage resulted in inhibition of neurite initiation in sympathetic neurons. Therefore, it is likely that higher doses of lead block L-type calcium channels and in turn this may cause a inhibition of neurite initiation. The disruption of intracellular calcium regulation may be a cause of the effects of lead on initiation. The process of neurite growth has 3 main steps; initiation of neurite from cell body, neuronal growth cone motility and neurite elongation (Mattson and Kater, 1987). The growth cone is an actin-based structure located at the tip of a growing neurite. It is a motile structure thought to be involved in initiation and the pathfinding of neurites (Smith, 1988). It is hypothesized that certain growth processes (such as initiation and neurite elongation) have specific optimal concentrations of calcium, which is termed the calcium hypothesis (Mattson and Kater, 1987). If the optimum [Ca2+]; is increased or decreased then neurite initiation and elongation may be disrupted. For example, in Helisoma neurons, low concentrations of calcium channel blockers inhibited growth cone motility but stimulated neurite elongation, indicating that the growth 56


cone has a higher optimum calcium concentration than the process of elongation (Mattson and Kater, 1987). However, it should be noted that the optimum calcium concentration for growth cone movement and neurite elongation may vary among cell types (Kater et al., 1988). Connor (1986) also showed that the growth cones of actively extending neurites (of cultured rat brain cells isolated from the diencephalon) had a higher [Ca2+]; than the rest of the cell. Therefore, if lead blocks calcium channels and decreases [Ca2+]i at the growth cone then inhibition of growth cone motility may occur and hence initiation may be disrupted (or decreased). As intimated above, inhibition of initiation at low lead doses is probably not a result of calcium channel blockage, since the free lead concentration is too low to block the channels (Audesirk eta/., 1991). Therefore, lead is probably exerting its effects either by mimicking calcium or disrupting calcium regulated processes inside the cell (Audesirk, 1990; Bressler and Goldstein, 1991; Pounds, 1984). As explained in the introduction, lead can stimulate PKC in picomolar concentrations (Markovac and Goldstein, 1988). Also, it may interfere with calcium regulation by increasing intracellular calcium (Schanne et a/., 1989a, b). This disruption in intracellular calcium concentration or calcium regulated processes could in turn have detrimental effects on development including initiation. For example, if the [Ca2+]i at the growth cone is too high 57


(above optimum), then the growth cone may be inhibited and neurite initiation may be decreased. Cellular Mechanisms That Regulate Initiation. Dendrite Production. and Axonal Branching As stated previously, initiation in both cortical and hippocampal neurons was inhibited by lead. However, the number of dendrites per cell was not affected by lead at any concentration (Fig. 7). It might be expected that if initiation was reduced, so too should be the numbers of dendrites. Audesirk et al. (1991) have proposed, based on the responses of hippocampal cells, N1E-115 neuroblastoma cell and B-50 neuroblastoma cells to lead, that the processes of dendrite production, neurite initiation, and axon branching may be controlled by different developmental mechanisms. Even though initiation was affected by lead, dendrite numbers were not affected, supporting the hypothesis that the two processes are controlled separately. These results are consistent with a (previous) study involving cultured chick embryos (Audesirk et al., 1989) in which lead inhibited neurite initiation, but not the number of neurites produced by those cells that initiated. The number of axon branches per cell increased at high lead concentrations for hippocampal cells and high and low lead concentrations for cortical neurons (Fig. 6). Again, this suggests that initiation and 58


branching are processes controlled by different mechanisms. There is not very much known about the mechanism of neurite branching. It has been postulated that the mechanism of branching is due to the advancing growth cone dividing, and that this process is regulated by an optimum calcium concentration (Mattson and Kater, 1987). As mentioned earlier, growth cones show an elevated calcium concentration when they are motile. If active growth cones have higher [Ca2+l, and high concentrations of lead block VSCC, then it would be expected that growth cone motility and the process of branching would be inhibited. As stated previously, axon branching was increased in hippocampal and cortical cells in high lead concentrations. Therefore, some other mechanism besides calcium channel blockage by lead must be responsible for lead increasing the number of axon branches, or perhaps axon branching in cortical and hippocampal cells requires a different calcium concentration (optimum) than initiation. In conclusion, lead may interfere with neurite development through actions on [Ca2+]; by altering calcium influx through calcium channels or by interfering with intracellular calcium regulation. Also, lead appears to affect initiation, dendrite numbers, and axonal branching in different ways, suggesting that different mechanisms operate to control these processes. 59


Axon and Dendrite Elongation High concentrations ( 100 uM-1000 uM) of lead stimulated axon and dendrite elongation in hippocampal neurons, but not cortical neurons (Figs. 5 and 8). The results for cortical neurons (in 2 and 10% FCS) and hippocampal cells (in 10% FCS) are consistent with a previous study of chick embryo neurons, where neurite elongation was not affected by 1000 uM total lead in 10% FCS medium (Audesirk et al., 1989). Neurite extension occurs when tubu1in monomers are polymerized into stable microtubules (via microtubule-associated proteins or MAPS) which comprise part of the cytoskeleton (Matus et al., 1986; Meininger and Binet, 1989). As mentioned previously, Mattson and Kater (1987) investigated the role of [Ca2+]; on the process of elongation in Helisoma neurons. They showed that a somewhat reduced level of calcium stimulated neurite extension. This observation and lead's ability to block calcium channels at high concentrations, suggests a possible mechanism for lead's stimulatory effect on axon and dendrite elongation in hippocampal neurons. However, preliminary results from our laboratory, indicate that L-channel blockers do not cause increased axon or dendrite elongation in cultured hippocampal neurons (unpublished data). For example, cadmium did not affect axon or dendrite length at any concentration tested (1-50 uM). Initiation of hippocampal cells, was decreased by 10 and 50 uM 60


cadmium concentrations. Hippocampal cells exposed to nicardipine (L type calcium channel blocker) at 10-50 uM concentrations also showed a decrease in initiation. However, cells exposed to 25-50 uM nicardapine showed a decrease in axon and dendrite length. This contradicts both the lead and cadmium studies, and suggests that the various calcium-channel blockers may be affecting the cells through different mechanisms (intracellular mechanisms as well as calcium channel blocking). In 10% FCS medium, control axon and dendrite lengths were increased, while in 2% FCS medium lead's stimulatory effects were reduced. Since the maximum mean axon lengths were similar under the two conditions in lead, higher FCS levels may have brought the control axon elongation rate closer to its maximum, masking the effects of lead (Fig. 5). Cortical neuron controls had significantly longer axons and dendrites than the hippocampal cells, and were less influenced by the increase in FCS and by lead. These results suggest that lead affected axon elongation and dendrite elongation in a similar manner. It has been assumed that the actual mechanisms of dendrite and axon growth are similar, even though the two differ structurally and functionally (Meininger and Binet, 1989). These results support the hypothesis of a similar elongation mechanism in both axons and dendrites. 61


Overall Cell Type Comparison Overall, there appear to be some similarities between the cell types with respect to lead's toxic effects. Both cortical and hippocampal cells displayed a multimodal effect of lead, particularly with respect to initiation. Bimodal responses to a range of lead concentrations have been reported in vivo for hippocampal development. For example, Slomianka er al. (1989) found that neonatal rats exposed to low doses of lead had a decrease in thickness of the hippocampus, whereas rats exposed to higher doses of lead showed no effects on the thickness of the hippocampus. Also, biphasic responses to lead have been reported in other assays such as mitochondrial calcium uptake, where low lead levels inhibited uptake, but high lead levels increased calcium uptake (Suszkiw et al., 1984). Therefore a bimodal response to lead may not be uncommon. However, this complicates interpretations of results. One explanation is that lead may affect several developmental processes, each at a different dose. Some of these effects may be excitatory and some inhibitory to a given morphological end-point, such as initiation. Although many similarities were seen between cortical and hippocampal cells, there were some important differences. Generally, it appeared that cortical cells were less affected by lead than the hippocampal cells. Lead did not affect survival and dendrite lengths of cortical neurons 62


as was observed with the hippocampal cells. There may be several explanations for the cortical neurons lack of response to lead. One possibility is that cortical neurons are inherently more resistant to lead's effects, and the hippocampal cells are more sensitive. Also, as stated in the introduction, the hippocampus appears to accumulate lead more readily, perhaps lending to further sensitivity in vivo (Grand jean, 1978). Our results are in vitro so whether there is more in lead accumulation in hippocampal cells is not known. Alternately, the differences in lead toxicity between the hippocampal and cortical cells could be due to differences in the composition of cell types found in the hippocampal and cortical regions. Banker and Cowan (1977) state that the majority of cultured E18 rat hippocampal neurons are pyramidal cells. Kriegstein and Dichter (1983) found a greater variety of cell types (pyramidal, fusiform, and multipolar) in cultured El5 rat cortical cells. However, this is may not be directly comparable to our system, which employs the use of E18 cortical cells. To avoid some of the ambiguity, measurements of axons and dendrite growth parameters were made on pyramidal-type cells only. However, initiation and survival assessments were made on all cell types present, which could account for some of the differences seen in survival rates between the hippocampal and cortical cells. Also, in this case, the motor cortex region (as opposed to 63


the entire cerebral cortex) was examined so that a more uniform cell distribution could be obtained. However, it cannot be ruled out that upon dissection of the motor cortex, other surrounding areas were taken as well and there is no guarantee that the motor cortex region does not contain the same cell distribution as the rest of the cerebral cortex. Overall, differential sensitivity of the different brain regions, combined with the different exposure levels to lead in vivo, compound the complexity of lead's toxic effects on the developing nervous system. Future Experiments In the 2 day hippocampal and cortical cell cultures, several parameters of growth were adversely affected. However, it was not possible to assess parameters such as dendritic branching, because at 2 days in culture, the cells are not fully matured and therefore do not display extensive branching. One possible future experiment would be to assess the effects of lead on older, cultured cells (5-7 days). By examining older cultures, long term lead exposure effects could be examined. There are several ways in which to promote cell growth for longer than 2 days. One way is to use what is called a sandwich technique where glial cells are placed on top of cultured cells (or vice versa). The glial cells promote growth by secreting beneficial substances such as growth factors 64


(Banker, 1980). The drawback to this method is that glial cells may impart a protective effect on the cells or absorb lead (Holtzman et al., 1987). However, this situation would be comparable to what occurs in vivo. Another way to promote long term cell growth is through the use of a highly enriched, defined medium (Brewer and Cotman, 1989). This medium is now available commercially (Gibco). Preliminary results from our laboratory suggest that this medium promotes growth and survival for 2 weeks or more. Also, an advantage of using this medium is that it is serum free and lacks proteins that may bind lead which may cause complications in interpreting results. Long term cell growth may present some problems for morphological measurements. As the cells mature, they tend to aggregate and the neurites become somewhat unidentifiable. To avoid this problem, it may be feasible to use immunostaining techniques to stain specifically dendrites and axons. Several studies have successfully employed fluorescent or peroxidase conjugated antibodies to MAPs (e.g., MAP2 and tau). MAP2 localizes to dendrites, whereas tau localizes in axons (Caceres et al., 1986; Kosik and Finch, 1987). Therefore, by using antibody immunostaining techniques, it may be feasible to measure long term lead effects. 65


CHAPTER 3 FURA/TPEN EXPERIMENTS Fura Introduction Since evidence thus far indicates that lead interferes with calcium related processes, it is desirable to actually measure [Ca2+]; in the presence of lead. Fura-2 is a fluorescent dye that can be used to measure [Ca2+]; (Grynkiewicz eta/., 1985; Tsien, 1989). Fura-2 is similar in structure to the chelator EGTA and binds calcium with a high affinity (K11=2.24 x w-7 M, recall from introduction that is a dissociation constant and a lower dissociation concentration equates to a higher binding affinity) (Grynkiewicz eta/., 1985; Tsien, 1989). Fura-2 acetoxymethyl ester (AM) works by permeating the cell membrane and is hydrolyzed (de-esterified by intracellular esterases) to its free acid form. Consequently, the fura free acid is trapped inside the cell (no longer has esters to enable it to permeate the membrane) and is free to bind with calcium in a 1:1 ratio (Tsien, 1989). It is then possible to measure the [Ca2+]; by examining the fluorescence of fura-2 excited by wavelengths at 340 nm (bound furalcalcium complex) and 380 nm (unbound fura)_ The bound fura-2/calcium complex fluoresces at 340 nm.


and the unbound fura-2 fluoresces at 380 nm. Therefore, as [Ca2+]; increases, excitation is greater at the 340 nm versus the 380 nm wavelength (Tsien and Poenie, 1986). The [Ca2+]; can be calculated by the ratioed images obtained at 340 and 380 nm wavelengths. In order to obtain the ratioed images, we employ the Quantex QX-7 imaging system and Nikon inverted microscope. The setup consists of a microscope with a filter wheel attachment and a xenon light source that provides the proper fura excitation wavelengths. The filter wheel is controlled by a computer that changes the filters so that the cells are exposed to 340 nm excitation and then 380 nm excitation wavelengths. The actual imaging of the cells is possible through the use of an intensified charge-coupled device (CCD) camera attached to the microscope. The CCD camera takes the images (at 340 nm and 380 nm excitation wavelengths) and relays them to a video processor which converts the images to numbers (ratios). A video monitor is used to view the images as they are being taken. The computer stores the information obtained by the processer and a program is used to calculate the [Ca2+]; upon acquisition and analysis of data. The following formula is used by the computer program to calculate [Ca2+]; from the images: R l [Cal = Ko _mn (F0/F,). mu R (Grynkiewicz er al., 1985) 67


Where Kd = the dissociation constant for binding of fura to calcium (224 nM), R= intensity of ratioed image (340/380 nm) obtained from a cell, fluorescence ratio (340/380 nm) of fura-2 free acid in the absence of calcium (EGTA solution), Rnax=the fluorescence ratio (340/380 nm) of fura-2 free acid in a saturated calcium solution, and FofF, = fluorescence of EGTA solution at 380 nmlfluorescence of saturated calcium solution at 380 nm. The calibration numbers used for the equation are the default numbers of the computer program or actual calibration numbers (approximately the same as the computer default numbers) obtained when images are taken of a saturated calcium or EGTA solution. The Use of TPEN as a Tool to Measure fCa2+k It would be ideal to measure [Ca2+]; using fura-2 in the presence of lead. However, lead also binds with fura-2 x 1Q12M compared to fura/calcium Kd=2.24 X l0"7M, the lead Kd is 5 X 104 times lower, hence higher binding affinity) and gives off a similar spectrum (shifted slightly to longer wavelengths) as the fura-2/calcium complex (Tomsig and Suszkiw, 1990). To circumvent the problem of lead's interference with the fura2/calcium signal, it may be feasible to use N,N,N' ,N'-tetrakis (2pyridylmethyl ethylenediamine) or TPEN. TPEN is a membrane-permeant 68


heavy metal chelator similar in structure to EGTA (Arslan, eta/., 1985). TPEN has high affinity for heavy metals and low affinity for calcium (Arslan et al., 1985). The affinity for lead to bind with TPEN has not been reported. However, Arslan et al. (1985) found that with quin-2, an indicator dye similar to fura-2, TPEN had similar or higher affinities for the heavy metals than did quin-2. Therefore, TPEN is effective in binding the heavy metals that are interfering with the quin-2/calcium signal. Also, it has been shown that fura-2 has a lower affinity for divalent heavy metals and higher affinity for calcium than quin-2 (Grynkiewicz et al., 1985). Therefore, it follows that TPEN will be able to bind heavy metals with a greater affinity than fura-2. This is an important factor when considering the use of TPEN to measure [Ca2+]; in the presence of lead. Another consideration is TPEN's toxicity. Arslan et al. ( 1985) found that TPEN is relatively nontoxic to cells for short durations (no longer than 4 hours at a 10 uM concentration). If TPEN were toxic, it may cause a false increase in [Ca2+]; by causing cell death, which is characterized by a rise in intracellular calcium (Arslan et al., 1985). Therefore, it may be feasible to use TPEN in order to measure [Ca2+]; in cells exposed to lead. Others have successfully used TPEN for chelation of endogenous heavy metals that interfere with [Ca2+]; measurements of 69


fura-2 and quin-2 indicators (Komulainen and Bondy, 1987; Verhage er al., 1988). Purpose of the Experiment The objective of the second part of my thesis research was to examine the feasibility of using TPEN in conjunction with lead exposed cells in order to measure [Ca2+]; using the fura-2 indicator and Quantex QX-7 imaging system. The reasoning behind this objective is that lead interferes with the calcium/fura-2 signal by binding with fura and fluorescing at similar excitation wavelengths. Therefore, preliminary experiments were designed to avoid the problem of lead interfering with the calcium signal. The procedure involved applying TPEN, then applying a high dose of lead to block calcium channels. The purpose of this was to allow TPEN to enter the cell first. Then, upon exposure to lead, the TPEN would bind any lead that entered the cell making the lead inaccessible to bind with the fura. Theoretically, what would be measured were only the effects of lead on calcium channels. The limitation of using TPEN is that it binds lead and therefore, may interfere with any effects lead may have inside the cell. The objective of these preliminary experiments was to determine whether it is feasible to use TPEN in conjunction with lead in order to measure [Ca2+]; in future experiments. 70


Fura Materials and Methods First, an experiment was done to assess whether TPEN was actually binding lead and preventing it from interacting with fura-2. This experiment was repeated twice and consisted of using preparations without cells. Images were taken under the following conditions: 1) Dish #1 consisted of 500 uL of 25 mM HEPES buffer with 10 uL fura-2 (pentapotassium salt, the calcium sensitive de-esterified form of fura-2). The HEPES was necessary to buffer the calcium and lead. 2) Dish # 2 consisted of 500 uL of 25 mM HEPES containing 1 uM PbC12 and 10 uL fura-2 pentapotassium salt. 3) Dish #3 contained 500 uL of 25 mM HEPES containing 1 uM PbC12 50 uM TPEN and 10 uL fura pentapotassium salt. Fura-2 Loading of Cells Hippocampal cells were cultured onto special 35 mm plastic dishes. Each dish was made by removing the center and replacing it with a glass coverslip attached with Sylgard 184 (Dow Corning). For cells to attach properly, a high molecular weight (M.W. approximately 500,000; 1 mg/ml 01 H20) poly-0-lysine was applied to dishes for 20-30 minutes then rinsed. 71


For fura-2 loading, the hippocampal cells were thawed and plated at a density of 200,000 cells/dish in 300 uL of culture medium (see cell culture methods) containing 2ug/ml of fura-2AM (in DMSO, Molecular Probes). Usually, two dishes of cells were set up in case one dish of cells did not load with fura-2 properly. Next, the cells were incubated in a 5% C02 humidified incubator at 37 oc for 1/2 hour, which allowed the fura-2AM to enter the cells and become de-esterified by intracellular esterases. Then an additional 2 ml of control medium was gently placed in the dishes. The media allowed for excess non-hydrolyzed fura-2AM to be washed from the cells. The cells were then ready for the perfusion experiment with TPEN and PbC12 TPEN Experimental Procedure For the perfusion experiments using PbC12 and TPEN, four protocols were employed. Each experiment's duration was 1 hour and 32 minutes. Also, each experiment was designed so that during the perfusion periods (i.e., 15 minutes, 12 minutes, 5 minutes and 1 hour) images were obtained at standard time intervals during the perfusion periods. For example, for the 15 and 12 minute perfusions, images were taken at 3 minute intervals for a total of 6 and 4 images, respectively. Then an image was taken at the end of the 5 minute control perfusion. Images were taken every minute 72


for 10 minutes, then every 2 minutes for the remaining 50 minutes to total 1 hour. All images taken during each perfusion period were averaged together. The following are descriptions of the protocols: 1) The first experiment was designed to examine the effects of TPEN and lead on the cells. It consisted of control media perfusion for 15 minutes (same media as used for cell culturing) in order to get baseline data of [Ca2+];. Then 50 uM TPEN (made from a 25 mM stock in ethanol and diluted in the medium) was perfused for 12 minutes, to allow TPEN to diffuse into the cells. Next, the cells were rinsed with control media for 5 minutes, to remove extracellular TPEN. Then 500 uM PbC12 (diluted in cell culture media, except containing 50 mM KCl) was perfused over cells for 1 hour. The high potassium medium was used to depolarize cells and to open calcium channels. 2) The second experiment was designed to examine the effects of lead in a high KCl medium (HKCl, 50 mM) on the cells. It consisted of perfusing cells with control media for 32 minutes (parallel to # 1 above; 15 minutes of control and 12 minutes of control instead of TPEN and a 5 minute control rinse), then perfusion with 500 uM PbCl2 in a high KCl medium for 1 hour. 3) The third experiment was designed to examine the effects of TPEN alone. Therefore, control media was perfused for 15 minutes, followed by 73


12 minutes of 50 uM TPEN perfusion. Then a 5 minute control rinse was performed. Finally, control media with 50 mM KCl was perfused for 1 hour. 4) Experiment number four was designed to examine the effects of the 50 mM KCl medium alone on [Ca2+];. It consisted of control media perfusion for 32 minutes (parallel to #1, a 15 minute control perfusion then 12 minutes of control and finally 5 minutes of control) followed by control media with high KCl perfusion for 1 hour. Imaging System Operation Once cells were loaded with fura, and the above media preparations were made, the actual perfusion experiments were conducted using the Quantex QX-7 calcium imaging system. Calibration of the system was not performed for each experiment because extensive calibrations showed that the computer program default numbers were the correct calibration numbers. However, the xenon bulb was checked for proper alignment before each use. This was done by using a HBSS solution (500 uL) with 10 uL of fura-2 pentapotassium salt. An image was taken, and analyzed using the system's default calibration numbers. If the image was uniform (i.e., 3 areas within the field were within 10% calcium concentration of each other) then the experiment was 74


begun. If there was not an even distribution, the bulb was centered and not adjusted thereafter. Next, for the actual perfusion experiment, the fura loaded cells were placed on a stage warmer (37 C) on the inverted microscope using the lOOX objective and a Nikon #2 neutral density filter. A peristaltic pump was used to perfuse the media at a rate of 200 uL/minute. Usually, a field of 8-10 cells was located. The CCD camera was set to a sensitivity level of 200, and intensifier was set on 250. The images were taken through 340 and 380 nm filters at 0.54 second exposures and averaged for 16 frames. The perfusion experiment was then conducted (see above procedure for perfusion set-up). Data Analysis of Images Images were obtained, stored in the computer, and later analyzed to obtain [Ca2+];. Each exposure was viewed and a background region (dark region in dish) and halo region (region next to cell) was subtracted Then, using the default calibration numbers, the [Ca2+;] was calculated by the computer using the following equation: [Cal = Ko l R (F0 / F,). LRmu R (Grynkiewicz et a/., 1985) 75


Where, Kd=224 nM, Rnax= 13.25, Rnin=0.48, FjF.= 10. For definitions of this equation refer to the introduction to this chapter. Fura-2 Imaging Statistical Analysis Each experiment was performed twice to obtain a total of 10-15 cells, for each data time point. For the experiment to be considered valid, the control cells' average [Ca2+]; value had to be within 20-150 nM [Ca2+];. Statistical analysis on the TPEN/lead binding assay was performed using a two-tailed Student's t-test for independent measures with a significance level of p <0.05. For the perfusion experiments, a two-tailed Student's ttest for repeated measures was used at a significance level of p < 0.05. Fura Results Assessment of TPEN/Lead Binding Table 2 displays the results (mean of 2 replicates) of the various preparations of TPEN and lead without cells. The sample with 25 mM HEPES displayed a [Ca2+] of 74 nM + 13 s.e.m. Adding 1 uM PbC12 elevated the apparent [Ca2+] to 174 nM 9 s.e.m. (significantly increased from control, p < 0.05). Since no extra calcium was added this indicated that lead was binding with the fura to give a false increase in [Ca2+] 76


Table 2. TPEN/Lead Binding Assessment I Sample I Treatment I [Ca2+]i I #1 500uL of 25 mM 74 nM + 13 SEM Hepes + 10 ul fura (control) #2 500 uL of 25 mM *174 nM + 9 SEM Hepes + 1 uM PbCl2 + 10 uL fura #3 500 uL of 25 mM 83 nM + 1 SEM Hepes + 1 uM PbCl2 +50 uM TPEN + 10 uL fura Denotes values significantly different from control (p < .05). measurement. The solution with both 1 uM PbC12 and 50 uM TPEN showed a [Ca2+] of 83 nM + 1 s.e.m., which was not significant from the HEPES control; p < 0.05). It is reasonable to assume that TPEN (50 uM) is an effective lead chelator that essentially eliminates any effect of lead on the tluorescence of fura-2. 77


TPEN/Lead Perfusion Experiments High potassium chloride medium. The high (50 mM) potassium control (HKCl) perfusion experiment displayed no significant differences in [Ca2+]; between the control (low KCl) and the experimental (HKCl) cells (Fig. 9). However, at the end of the perfusion experiment, the [Ca2+l rose from 90 nM to 136 nM calcium, although this was not statistically significant. TPEN/HKCl medium. The TPEN control perfusion experiment showed no significant differences in the [Ca2+]; between the experimental (TPEN) or control (no TPEN) exposed hippocampal cells (Fig. 10). Lead in HKCl medium. There were no significant effects of lead on [Ca2+]; observed (i.e., no significant increase or decrease from control line) (see Fig. 11). TPEN!lead in HKCl medium. The TPEN!lead exposed cells showed a significant decrease in [Ca2+]; beginning at 27 minutes (TPEN application) and extending to 62 minutes (part of lead exposure period) (Fig. 12). At 1 hour 32 minutes (duration of lead exposure), there was a slight increase in [Ca2+];, although it was not significant. 78


HKCI Medium 200 ..--.. :::::!! 180 c c 160 0 140 :.:::; 0 '-120 ..... c Q) u 100 c 80 0 u 60 E 40 :I u 20 0 u 0 0 10 20 30 40 50 60 70 80 90 100 Time (Minutes) Fig. 9. Perfusion of hippocampal cells with HKCl medium. Cells were perfused with regular control medium for 32 minutes, the HKCl for 60 minutes. Data points are means s.e.m. (10-15 cells) during the indicated time interval and are expressed as percent of control. Asterisks indicate statistical significance (p < .05). TPEN Medium 200 ..--.. :::::!! 180 c COHTIIOl 160 c 0 140 ..... 0 120 '-..... c 100 Q) u c 80 0 u 60 E 40 :I u 20 0 u 0 0 10 20 30 40 50 60 70 80 90 100 Time (Minutes) Fig. IO. Perfusion of hippocampal cells with TPEN medium. Cells were exposed to control medium for 15 minutes, 50 uM TPEN for 12 minutes, control rinse for 5 minutes, then HKCI medium for 60 minutes. Data points are means. s.e.m. (I0-15 cells) during the indicated time interval and are expressed as percent of control. Asterisks indicate statistical significance (p < .05). 79


Lead Medium 200 r"'. 180 c 160 c 0 :;; 140 c 120 L.. .... c 100 cu u c 80 0 u 60 E 40 ::J u 20 c u 0 0 10 20 30 40 50 60 70 80 90 100 Time (Minutes) Fig. 11. Perfusion of hippocampal cells with lead medium. Cells were perfused with control medium for 32 minutes. Then 500 uM in HKCI was applied for 60 minutes. Data points are means s.e.m. (10-15 cells) for the indicated time interval and are expressed as percent of control. Asterisks indicate statistical significance (p < .05). Lead and TPEN 200 r"'. 180 CONTROl c -r1lr-........, 160 c 0 140 :;; c 120 L.. .... c 100 cu u c 80 0 u 60 E 40 ::J u 20 c u 0 0 10 20 30 40 50 60 70 80 90 100 Time (Minutes) Fig. 12. Perfusion of hippocampal cells with TPEN/Iead medium Cells were perfused with control medium for 15 minutes 50 uM TPEN for 12 minutes control rinse for 5 minutes then 500 uM in HKCI for 60 minutes Data points are means s.e.m. (10-15 cells) for the indicated time interval and are expressed as percent of control. Asterisks indicate statistical significance (p< .05). 80


Fura Discussion Fura-2 studies were performed in order to assess the feasibility of using TPEN in measurements of [Ca2+]i in lead exposed cells, particularly in relation to the ability of lead to block calcium channels. TPEN/Lead Bindine Assay The experiment of TPEN/lead binding was done in order to verify that TPEN could bind with lead in our system, thereby making lead inaccessible to bind with fura-2. Results from Table 2 indicate that 50 uM TPEN can bind 1 uM lead, thereby making lead inaccessible to compete with calcium in binding with fura-2. For example, the HEPES buffer with fura had a calcium concentration of 74 nM, while the HEPES with fura and lead had 174 nM calcium concentration. It should be noted that the 174 nM calcium concentration was not a true calcium concentration since no extra calcium was added. Therefore, it can be reasoned that lead was binding to the fura causing an increase in the apparent calcium concentration reading. Upon addition of TPEN to the solution, the calcium concentration was similar to control level, indicating that TPEN was binding with the lead and making the lead unavailable to bind with the fura. The concentration of lead ( 1 uM) is probably much higher than what is found inside the cell. Schanne 81


eta/. (1989 a,b) found that micromolar exposure to lead equated to picomolar concentrations of lead inside of osteoblast and neuroblastoma cells. So, it was assumed in the subsequent perfusion experiments that TPEN entered the cell and was able to bind with any lead that entered the cell. Of course, it would be ideal to examine the binding affinity of TPEN for lead, and compare this with the binding affinity of fura-2 for lead. These experiments were beyond the scope of this research. TPEN and Lead Perfusion Experiments The hypothesis that one of lead's actions is to disrupt [Ca2+]; by blocking calcium channels was examined by using TPEN, lead and various controls with hippocampal cells. High potassium chloride (HKCI) control. The purpose of the HKCl control was to examine whether an increase from 20 mM KCL (normal medium) to 50 mM KCl (HKCl) had any effects on [Ca2+];. Theoretically, when the cell is depolarized by the increase in potassium, calcium channels open, thereby allowing for calcium influx. Therefore, it would be expected that an increase in potassium would cause an increase in [Ca2+];. For example, Tomsig and Suszkiw ( 1991) observed a transient rise in [Ca2+]; in bovine chromaffin cells immediately after 1-2 minutes exposure to a high KCl medium. However, my results indicate a slowly rising 82


increase in calcium (not signifcant from controls) over a 1 hour 32 minute period (Fig. 9). Perhaps the reason that a significant rise in calcium was not observed was that the increase from 20 mM to 50 mM KCl was not enough to cause a dramatic increase in depolarization. The experiment by Tomsig and Suszkiw (1991) used a high KCl medium containing 140 mM KCl, a substantially higher concentration than what was used in this experiment. It is also possible that the 1-2 minute time point measured by Tomsig and Suszkiw (1991) was missed in this study. TPEN Medium. The TPEN experiment was designed to see if TPEN alone had any effects on [Ca2+]i. Generally, TPEN is not toxic to the cells for short periods of time (no longer than 4 hours) and does not bind any appreciable amounts of calcium (Arslan et al., 1985). Indeed, in this experiment, TPEN showed no significant increase or decrease in [Ca2+]i (Fig. 10). If TPEN was not affecting [Ca2+]j, then the graphs of HKCl medium and TPEN medium should be similar. The graphs are similar, except upon TPEN application (between 15-27 minutes), the [Ca2+]i was decreased to some extent, more so than the cells in HKCl medium. The difference between the TPEN and HKCL medium could be a result of the small sample sizes (i.e., 10-15 cells). Also, the decrease in [Ca2+l seen with the TPEN medium could be explained by TPEN binding with other endogenous heavy metals (e.g., zinc, copper and iron) that interfere or 83


bind with fura-2 and give a false increase in calcium measurement. Similar results were observed by Komulainen and Bondy (1987) using synaptosomal preparations, where addition of TPEN caused a 10% decrease in [Ca2+t after 1-3 minutes. It was postulated that TPEN bound with interfering heavy metals, so that only the [Ca2+]; was being measured following TPEN appplication. Lead medium. The cells in 500 uM PbC12 medium showed no significant effects of lead alone on [Ca2+]; (Fig. 11). However, a slight decrease was observed at 32 minutes which corresponded to lead application. The data could be interpreted to mean that the decrease in [Ca2+t was due to lead blocking calcium channels. By the end of the perfusion experiment the [Ca2+]; began to rise. If calcium channels were being blocked by lead, an increase in calcium influx would not be expected. However, it could be speculated that the increase in [Ca2+]; might be due to the cell compensating for the decreased [Ca2+];, by increasing release of calcium from intracellular stores, or by calcium channel up-regulation. However, calcium channel up-regulation probably would not be observed in l hour 32 minutes. It is also possible that lead may have entered the cell and became bound with fura-2, or could have directly caused an increase in calcium by interfering with intracellular calcium regulation (Schanne et al., 1989a,b). Therefore, the calcium 84


channel blockage could have been masked by lead entering the cell and interfering with the calcium/fura-2 signal or by directly affecting calcium regulation. TPEN!lead calcium measurements. Finally, the TPEN/lead experimental cells displayed a significant decrease in [Ca2+]; from 27-62 minutes (TPEN applied at 27 minutes, then PbC12 applied at 37 minutes, Fig. 12). These results are in agreement with the hypothesis that lead may enter the cell, but is bound by the TPEN, so it is not able to bind with fura-2, or cause an increase in calcium by interfering with calcium regulation. Therefore, it is possible that what is being measured is lead blocking the calcium channels. Indeed, a significant decrease in [Ca2+]; would indicate that lead is blocking calcium channels, because although TPEN alone does cause a slight decrease in [Ca2+]; (Fig. 10), this is neither as large nor long-lasting as the decrease observed with the TPEN/lead exposed cells (Fig. 12). However, with the TPEN!lead exposure, TPEN alone did cause a significant decrease in [Ca2+]; at 27 minutes (see Fig. 12). The discrepancy between results with TPEN alone and TPEN/lead medium may be due to the small sample sizes, and further repeat experiments may provide more information. Perhaps, the slight increase in [Ca2+]; in the TPEN!lead exposed cells after 62 minutes was due to the cell compensating for the calcium 85


decrease, by increasing [Ca2+]i via release from intracellular stores. Alternately, the slight increase in calcium observed with the TPEN/lead samples, may have been due to lead entering the cell and binding with fura-2 or lead causing an increase in intracellular calcium. TPEN should be binding any lead that enters the cell. However, it is also plausible that TPEN diffused out of cell, in which case lead may be binding with the fura. Also, the TPEN concentration may be too small to bind with the lead that enters the cell. However, this is unlikely since 50 uM TPEN can bind with 1 uM lead. Although, the exact concentration of lead that would enter hippocampal cells exposed to 500 uM total lead has not been measured, it has been suggested by Schanne et al. (1989a,b) that picomolar concentrations of lead are found inside osteoblast and neuroblastoma cells upon extracellular micromolar exposure of lead in 5% FCS medium. It can be extrapolated that upon 500 uM lead exposure, 500 pM or 0.5 nM is the approximate concentration inside the hippocampal cells. Therefore, 50 uM TPEN should be binding this concentration of lead, and what is being observed is the blockage of VSCC by lead, which is in agreement with many voltage clamp studies (see introduction; Audesirk, in press). Alternatively, the decrease in [Ca2+]i could be due to the TPEN application prior to the lead perfusion (Fig. 12). However, the TPEN alone (TPEN medium, Fig. 10) did not significantly decrease [Ca2+];. 86


Future Experiments The effects that were observed for the fura-2 experiments occurred towards the end of the perfusion time (1-hour, 32 minutes). The logical next step would be to extend this time frame to 3-4 hours and observe the effects of lead since other studies did not see effects of lead until 2-3 112 hours (Schanne et al., 1989a,b). There are also other variations on the experiments that were performed. For example, a higher lead concentration (1000 uM) might be utilized. Also, perhaps a higher KCl and low KCl comparison could be performed. The high KCl promotes cell depolarization and hence calcium channel opening. In this experiment, a 50 mM KCl was used (normal media contains 20 mM). Perhaps a higher increase in KCl in conjunction with an increase in lead, would display more dramatic effects of lead on channel blockage. Also, it may be preferable to use a lower TPEN concentration (10-25 uM) because TPEN is potentially toxic to the cell. It may be best to use the minimum amount of TPEN that will bind lead. Lastly, is TPEN really necessary? Evidence suggests that upon micromolar lead exposure, picomolar concentrations were measured in the cell over a 2-3 l/2 hour exposure time (Schanne et al., l989a,b). However, it is probably more accurate to use TPEN, because the exact concentration of lead that enters hippocampal cells is not known. 87

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Another possible experiment, would be to apply lead first, then subsequently expose cells to TPEN. This would allow lead to enter the cells and exert effects such as increasing intracellular calcium. However, it would be difficult to determine intracellular effects of lead because it could either be lead or calcium that is binding with fura-2. Whether this experiment would reveal any effects is uncertain. These are just a few possible experiments for using TPEN to measure lead's effects on [Ca2+];. 88

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CHAPTER 4 FINAL SUMMARY/CONCLUSIONS Lead affected cortical and hippocampal neurons in a similar multimodal manner, affecting neurite initiation and axon branching in particular. Hippocampal neurons were additionally affected by lead in the parameters of survival, axon length and dendrite length, suggesting a higher sensitivity of the cells to lead. Also, the increase in FCS from 2% to 10% tended to reduce the deleterious effects of lead. Therefore, it appears that lead interferes with multiple regulatory processes of cell growth. The mechanisms of lead toxicity are not well understood, but it is believed that lead interferes with calcium regulation (Audesirk, 1985; Bressler and Goldstein, 1991; Pounds, 1984). In an attempt to verify lead's effects on calcium regulation, the feasibility of using TPEN with the fura-2 indicator and calcium imaging system were examined. Initial results indicate that TPEN may be a useful tool in future experiments designed to elucidate lead's interference with normal intracellular calcium regulation. In summary, lead's multiple, concentration dependent effects and its binding with proteins and interactions with fura-2 make it a challenging toxin to study in vitro. However, as exemplified by the studies mentioned above, we have made some progress in studying the complex effects and

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interactions of lead. In the future, further studies will undoubtedly reveal more information regarding lead neurotoxicity. 90

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