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The Roles of Zinc and Metallothionein in Hepatocyte Antioxidant Defense

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PAGE 1

THE ROLES OF ZINC AND METALLOTHIONEIN IN HEPATOCYTE ANTIOXIDANT DEFENSE By LOUIS LICHTEN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA

PAGE 2

Copyright 2004 by Louis Lichten

PAGE 3

This thesis is dedicated to my father, a nd my grandparents. Without their love and support, this would never have been possible.

PAGE 4

ACKNOWLEDGMENTS I would like to thank Kelly Clayton for always encouraging me to do my best. I would also like to thank my lab mates for all their help. I thank Ray Blanchard for his wisdom in answering all my questions, and Jeff Bobo for always being willing to help me with my experiments. I thank Cal Green for his help with solving experimental problems. I would also like to thank Juan Liuzzi for helping when I needed it. I greatly appreciate Steve Daviss help from the very beginning of this project. I would also like to thank Dr. Gregory, and Dr. Kilberg for their help and participation on my thesis committee. Finally, I would like to thank Dr. Cousins for giving me the opportunity to work on this project. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT.......................................................................................................................ix CHAPTER 1 INTRODUCTION........................................................................................................1 2 LITERATURE REVIEW.............................................................................................4 Zinc...............................................................................................................................4 Interrelations of Zinc and Metallothionein...................................................................4 Zinc and Metallothionein as Cellular Antioxidants......................................................6 Metallothionein, Nitric Oxide, and Oxidative Stress....................................................7 3 MATERIALS AND METHODS...............................................................................10 Animals.......................................................................................................................10 Hepatocyte Isolation...................................................................................................10 Culture Period.............................................................................................................11 Treatment Period........................................................................................................12 Cytotoxicity Assays....................................................................................................12 Data Analysis..............................................................................................................15 4 RESULTS...................................................................................................................16 Lactate Dehydrogenase Assay....................................................................................16 Metallothionein Genotype Differences.......................................................................17 Zinc Supplementation.................................................................................................19 Altered Genotype and Zinc Supplementation.............................................................20 5 DISCUSSION.............................................................................................................27 APPENDIX CYTOTOXICITY EXPERIMENTS...........................................................32 v

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LIST OF REFERENCES...................................................................................................33 BIOGRAPHICAL SKETCH.............................................................................................37 vi

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LIST OF TABLES Table page 4-1 LD50 responses due to altered metallothionein genotype.......................................19 4-2 LD 50 responses to toxicants of hepatocytes from MT KO, CK, CT, and MT Tg mice as a result of zinc status.......................................................................24 4-3 Summary of LD 50 responses between the metallothionein genotypes after pretreatment by zinc.........................................................................................26 vii

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LIST OF FIGURES Figure page 4-1 Influence of SNAP on LDH activity........................................................................16 4-2 Viability due to altered metallothionein genotype...................................................18 4-3 Effect of zinc status on subsequent toxicant induced viability changes in control knockout and metallothionein knockout hepatocyes...................................22 4-4 Effect of zinc status on subsequent toxicant induced viability changes in control transgenic and metallothionein transgenic hepatocyes................................23 4-5 Differences in hepatocyte viability due to altered MT genotypes and zinc pretreatment..............................................................................................................25 A-1 Differences in CT and Tg viability due to altered MT genotypes and zinc pretreatment..............................................................................................................32 viii

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science THE ROLES OF ZINC AND METALLOTHIONEIN IN HEPATOCYTE ANTIOXIDANT DEFENSE By Louis Lichten August 2004 Chair: Robert Cousins Major Department: Food Science and Human Nutrition Zinc and metallothionein are involved in cellular antioxidant defense, but the basic mechanisms are not known. Zinc is a biologically redox inert metal, that has been associated with protection from various oxidant producing species. However, zinc induces the cysteine-rich, metal-binding protein, metallothionein, through interaction with the transcription factor MTF-1. Metallothionein may have a role in preventing oxidative stress, by quenching free radicals and other oxidants. Therefore, it is hard to distinguish between protection due to zinc, and protection due to induction of metallothionein. Hence, metallothionein null (KO) mice and metallothionein transgenic (Tg) mice provide a unique opportunity to study altered states of metallothionein expression. These mouse models were used to determine the effects of zinc supplementation and/ or metallothionein gene expression on the susceptibility to oxidative and nitrosative stress. Metallothionein KO mice were significantly less viable than control (CK) mice after ix

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tertiary-butyl hydroperoxide (tBH) and s-nitroso n-acetyl penicillamine (SNAP) treatments. Zinc supplementation (40 M) had no effect on viability of KO hepatocytes. The presence of metallothionein in CK hepatocytes demonstrated protection at baseline and after zinc supplementation. However, overexpression of metallothionein provided no further protection against tBH or SNAP. These results are in contrast to a direct antioxidant role for metallothionein in murine hepatocytes. x

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CHAPTER 1 INTRODUCTION Zinc is an essential mineral nutrient, possessing various biological functions including catalytic and structural roles in metalloenzymes and metalloproteins, as well as regulatory roles in synaptic signaling and gene expression (Cousins 1996). If zinc is absent from a biological system, an organism becomes more susceptible to oxidative insult. Zinc-deprived animals have demonstrated either increased free-radical production or enhanced injury from exposure to oxidative stress (reviewed in Powell 2000). The antioxidant mechanism of zinc protection is uncertain, but may relate to (1) zinc as a structural factor for copper-zinc superoxide dismutase, (2) the ability of zinc to protect sulfhydryl groups from oxidation, and (3) the ability of zinc to antagonize the catalytic properties of redox-active transition metals (reviewed in Powell 2000). A final possible antioxidant mechanism may be the ability of zinc to induce metallothionein (MT). Metallothionein is a cysteine-rich protein induced by zinc through the metal responsive transcription factor-1 (MTF-1), and interaction with metal response elements in the metallothionein promoter (Andrews 2000). Interestingly, metallothionein is also induced by oxidants through antioxidant response elements and/ or MTF-1 (Andrews 2000). At the protein level, metallothionein may be protective against oxidants by scavenging free radicals. This antioxidant potential was first realized by the quenching of the hydroxyl radical by metallothionein (Thornalley and Vasak 1985). Further, many studies have shown metallothionein to be protective against a variety of oxidative insults: radiation exposure (Matsubara 1987), anticancer drugs (Satoh et al. 1988), carbon 1

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2 tetrachloride (Schroeder and Cousins, 1991), S-nitroso N-acetyl penicillamine (SNAP) (Schwartz et al. 1995), peroxynitrite (Cai et al. 2000), and ethanol toxicity (Zhou et al. 2002). The mechanism of protection by metallothionein may be the result of interaction of an oxidant with the redox-active zinc-sulfur ligands of metallothionein, causing reduction of the oxidant and subsequent release of zinc (Jacob et al 1998). This mechanism allows metallothionein to be considered as a possible link between cellular zinc and the redox state of the cell (reviewed in Maret 2000). To look at the efficacy of metallothionein protection, metallothionein gene knock-out (KO), and metallothionein transgenic (TG) mice can be used. In a study by Davis et al. (2001), expression of metallothionein protected against carbon tetrachloride-induced hepatotoxicity, but metallothionein overexpression did not provide further protection. These results are inconsistent with direct antioxidant activity of metallothionein. Therefore, experiments were designed to more directly assess the antioxidant activity of metallothionein by treatment of primary hepatocyte cultures (from these genotypes) with tertiary-butyl hydroperoxide (tBH), and a nitric oxide donor (SNAP). Current studies utilizing the same study design and a redox-insensitive method to detect cell viability demonstrate the protection provided by metallothionein against oxidative and nitrosative stress. Hypothesis 1: Basal increase of metallothionein expression in the transgenic mice will provide isolated hepatocytes increased protection from nitric oxide and/or tBH, while the ablation of metallothionein in the knock-out hepatocytes will be deleterious. Hypothesis 2: Supplementation of hepatocyte cultures with zinc prior to nitric oxide or tBH exposure will further enhance the viability of transgenic cells, through induction of metallothionein.

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3 The specific aim of these experiments is to determine whether the presence of metallothioenein will protect primary hepatocytes from tBH or nitric oxide-induced oxidative stress, as measured by cell viability.

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CHAPTER 2 LITERATURE REVIEW Zinc Zinc is an essential mineral nutrient that is required for many biological roles including catalytic, structural, and regulatory roles (Cousins 1996). Zinc functions catalytically in more than 60 enzymes, such as metallopeptidases (Turner et al. 1996). Metalloenzymes are considered to be zinc-dependent when removal of zinc causes reduction in activity, and replacement of zinc restores activity. Structurally, greater than 1% of the genome codes for proteins containing zinc finger or similar domains, where zinc serves a structural role (Chesters 1992). Typically, zinc acts to stabilize the tertiary structure of metalloproteins (Vallee et al. 1991). Zinc is typically coordinated by four amino acid side chains: usually the sulfur atom of cysteine, or sometimes histidine. The regulatory role of zinc includes the binding of the metal to specific transcription factors, allowing binding of the transcription factor to metal responsive elements in the promoters of metal responsive genes (reviewed in Andrews 2000). Zinc binding may also have a regulatory role, through activation of signal transduction pathways (reviewed in Maret 2000). Interrelations of Zinc and Metallothionein In vivo, zinc metabolism is controlled homeostatically, through mechanisms related to absorption and excretion (via zinc transporters), and intracellular proteins such as metallothionein. Metallothioneins are small (6-7 kDa), cysteine-rich heavy-metal-binding proteins, and can bind up to seven zinc atoms (reviewed in Cousins 4

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5 1996). Approximately 5 to 10% of the total cellular zinc is found in complex with metallothionein under normal physiological conditions (Andrews 2001). Metallothioneins are the most abundant heavy-metal-binding proteins in the body (reviewed in Andrews 2000). Metallothioneins are thought to function in the homeostasis of zinc through involvement in zinc absorption, tissue distribution, and protection against acute stress (Schroeder and Cousins 1991b). During periods of acute stress, there is a decrease in plasma zinc levels, along with an increase in tissue zinc, particularly in the liver (reviewed in Cousins 1996). This reduction in plasma zinc is directly related to the changes in kinetics of zinc metabolism, which lead to increased binding of the metal to metallothionein (Dunn and Cousins 1991). In support of a link among zinc, metallothionein, and regulation of zinc metabolism, results of experiments with endotoxin-challenged metallothionein null mice (MT KO) show no decrease in plasma zinc (Philcox et al. 1995). Therefore, metallothionein plays a crucial role in plasma-zinc clearance and tissue-zinc uptake. Metallothionein KO mice lack functional expression of metallothionein, and allow zinc metabolism to be studied in the absence of metallothionein (Davis et al. 1998). Transgenic mice have been developed that have approximately 55 additional copies of the metallothionein-I gene in their genome (Palmiter et al. 1993). Metallothionein transgenic mice provide a model for studying the effect of exaggerated metallothionein levels on zinc metabolism. Metallothionein is transcriptionally regulated by zinc, through metal responsive elements in the promoter region of the gene (Andrews 2000). However, the metallothionein gene is also transcriptionally regulated by glucocorticoid hormones and

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6 specific cytokines. This allows for the possibility that these metalloproteins plays a role in inflammatory and stress-related responses (reviewed in Davis and Cousins 2000). Zinc and Metallothionein as Cellular Antioxidants The oxidation of many different cellular constituents is involved in the pathogenesis of an array of diseases (Gutteridge and Halliwell 2000). Cellular oxidative stress occurs when the antioxidant defense system in the cell becomes overwhelmed. There are many different forms of oxidants, including reactive oxygen species (H 2 O 2 O 2 and OH), reactive nitrogen species (NO and ONOO ), and carbon centered radicals (e.g., CCl 3 ). These oxidative species can damage lipids, proteins, and nucleic acids (Farber 1994). When oxidative stressors are introduced into cells, specific cellular antioxidant defense mechanisms are present to quench or reduce the radical mediated damage that may occur. These defense mechanisms involve antioxidant nutrients such as tocopherols and ascorbate, as well as the cellular antioxidants glutathione and superoxide dismutase (Yu 1994). If damage does occur, there are ways for the cell to repair itself. For instance, oxidized bases are removed from DNA to prevent further damage, oxidized lipid membranes are reduced by the action of glutathione peroxidase, and damaged proteins are committed to a proteasome degradation pathway. If these cellular defenses are overcome, and oxidative stress occurs in great amounts, the cell or cells may not survive. Therefore, establishing a way to reduce oxidative stress is important. Supplemental zinc provides additional protection against certain radicals (Blain et al. 1998). Zinc is a potent inducer of metallothionein expression. Consequently, the antioxidant protection attributed to zinc may be due to metallothionein induction. Metallothionein expression is also induced in response to oxidative stress (reviewed in

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7 Davis and Cousins 2000). Therefore, there is a great degree of correlation between increased metallothionein synthesis and protection from oxidative stress. However, this protection is not always observed. In studies using metallothionein knock out and metallothionein transgenic mice, carbon tetrachloride induced hepatotoxicity was reduced initially by the presence of metallothionein in control compared to knock out mice (Davis et al. 2001). Later time points showed no difference between genotypes in response to carbon tetrachloride. Also, metallothionein transgenic and transgenic control mice did not differ in the levels of hepatotoxicity, despite a large degree of difference in hepatic metallothionein and zinc content. Further examination of zinc and metallothionein as cellular antioxidants in cells from this mouse model led to differing results. In two independent studies, overexpresssion of metallothionein was found to be ineffective at increasing viability, and may actually be deleterious (Davis et al. unpublished data). These results are a remarkable contrast from the apparent protective effects of metallothionein observed with carbon tetrachloride treated rat hepatocytes (Schroeder and Cousins, 1990), and numerous other reports describing a protective effect of this protein. While many experiments have shown metallothionein and zinc to have cytoprotective roles, some have not. This disparity in protection must be investigated further utilizing a different oxidative stressor, such as nitric oxide. Metallothionein, Nitric Oxide, and Oxidative Stress During hepatic inflammation or endotoxemia, hepatocytes can respond to cytokine or bacterial LPS stimulation by activating the inducible form of nitric oxide synthase, which can generate a large amount of nitric oxide from arginine (Nssler et al. 1993). This production of NO has been shown to be both hepatoprotective and cytotoxic

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8 (Wink and Mitchell 1998). The paradoxical effects of nitric oxide may relate to its ability to interact with O 2 to form peroxynitrite, which is highly reactive and may account for part of nitric oxide-induced cytotoxicity (Fu et al. 2001). The susceptibility of cells to peroxynitrite and nitric oxide is primarily dependent upon their reducing capacity (thiol content) (Kim et al. 1999). Metallothionein has been shown, in vitro, to react directly with peroxynitrite to prevent lipoprotein and DNA damage caused by this reactive nitrogen species (Cai et al. 2000). However, the protection by metallothionein in this study was not dose dependent. Increasing levels of the protein did not provide further protection against DNA damage, or LDL oxidation. Nonetheless, metallothionein over expression has been documented to be protective against SNAP (NO-donor) induced killing of, and DNA single-strand breaks in NIH 3T3 cells (Schwarz et al. 1995). Further in vitro studies have elucidated a link between the cellular redox state and metal ion homeostasis (Jacob et al. 1998, reviewed in Maret 2000). The metal thiolate clusters of metallothionein possess the unique ability to function as a redox unit, therefore it has the potential to be involved in a redox sensitive signaling pathway (Pearce et al. 2000). Indeed, nitric oxide has been shown to S-nitrosylate metallothionein and release zinc from cultured pulmonary artery endothelial cells as confirmed by fluorescence protein modified metallothionein that undergoes conformational changes in the presence of nitric oxide (Pearce et al., 2000). These alterations in cellular zinc homeostasis may lead to the protective effects of metallothionein against nitric oxide toxicity (Schwarz et al. 1995). Overall, the data on the protective effects provided by zinc and metallothionein against oxidative stress is not definitive. Therefore, further characterization of the

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9 interaction of zinc and/or metallothionein to oxidative insult, is necessary. The pathogenesis of many liver diseases may begin with oxidative injury (Gutteridge and Halliwell, 2000). For that reason, hepatocytes provide a good model to understand the possible role of zinc as an antioxidant. Consequently, the goal of this study is to elucidate the role of zinc and metallothionein as hepatic cytoprotective agents, utilizing the murine model of altered metallothionein gene expression (i.e. MT-KO and MT-TG mice).

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CHAPTER 3 MATERIALS AND METHODS Animals All breeding of MT-KO and MT-Tg mice was done in-house from founder mice purchased from the Jackson Laboratory, Bar Harbor, ME. The C57BL/6 mice were crossed with SJL mice, possessing multiple copies of the murine metallothionein-1 gene, to create the line of metallothionein overexpressing mice (Palmiter et al. 1993). The control transgenic mice were created by backcrossing against the C57BL/6 strain. The MT-KO genotype was developed by crossing 129/SvCPJ mice MT (-/-), with C57BL/6 mice (Masters 1997). The control KO mice are 129S3/SvImJ mice. Sixto eight-week old male mice were used in all experiments. Mice were fed a modified commercial rodent diet (Harland TEKLAD, TEKLAD Rodent Diet (W) 8604) and given tap water. Hepatocyte Isolation Liver perfusions were alternated among all four genotypes. Mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg/kg). The isolation of liver cells involves many steps. First, the liver was infused with a calcium-free solution containing 0.142 M NaCl, 0.0067 M KCl, and 0.01 M HEPES buffer (pH 7.3), via the subhepatic inferior vena cava (modification of Schroeder and Cousins 1991a). Initial flow rate for the calcium-free solution was set at 2 mL/min. As the liver began to blanche, an incision was made in the portal vein, allowing perfusate to freely flow without recirculation. After opening the rib cage, a hemostat was then placed on the inferior vena cava superior to the diaphragm. The flow rate of calcium-free solution was 10

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11 then increased to 8 mL/min for approximately 10-12 min. The subsequent step involved perfusion of a solution containing 0.0667 M NaCl, 0.0067 M KCl, 0.00476 M CaCl 2 0.10 M HEPES buffer (pH 7.3), and 0.04% collagenase (Sigma type IV collagenase, C-5138) at a flow rate of 7 mL/min for 14 min. Upon completion of digestion, the liver was excised rapidly from the body cavity, and transferred to a sterile glass culture dish containing approximately 15 mL of the collagenase solution. The culture dish was then transferred to a sterile cell culture hood. Prior to liberation of hepatocytes, the gall bladder was carefully removed. Incisions were then made in each of the liver lobes to allow easy access to the hepatocytes. Finally, using a cell scraper, cells were released from the liver lobes gently, and as quickly as possible. Culture Period After liberation, the liver cells were passed through a 100 um cell strainer to remove debris and large cell aggregates. The hepatocytes were then suspended in a buffered wash medium (Williams Medium E + 10 mM HEPES pH 7.3), and collected by centrifugation at approximately 50 x g for 4 min. Cells were then washed in the same buffer two more times to improve cell purity. The final cell pellet was resuspended in attachment medium (WME supplemented with 10% FBS, 100 nM insulin, 100 nM Dexamethasone, 100 U/mL penicillin, 100 mg/mL streptomycin). An aliquot of the final cell suspension was then removed, and placed into solution with Trypan Blue (Sigma T8154) and PBS for determination of cell viability. Viability of the hepatocytes was then assessed by Trypan Blue Exclusion as measured with a hemacytometer. Only suspensions with > 90% viability were used in experiments. If viability was satisfactory, cells were seeded at 2.5 x 10 5 cells/ well in 12-well, collagen coated culture plates (6-10

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12 g collagen/ well). After plating, the cells are allowed to attach for 3 h (37C, 5% CO 2 ). Following selective attachment of parenchymal cells, medium in each well was exchanged for fresh medium only, or fresh medium containing 40 M zinc. These culture conditions did not change for 18-22 h, when oxidant treatment began. Treatment Period Upon completion of the culture period, all media was removed and replaced with medium containing 0-3 mM SNAP for 2 to 14 h, or 0-75 M tBH for 2 h. Cytotoxicity Assays After the treatment period had ended, medium from each well was removed and set aside. Cell monolayers were then washed twice with PBS, and cells were harvested in 1% Triton X-100 and disrupted by repeated passage through a 200 L pipette tip. The medium as well as cell extracts were then centrifuged (130 00 x g for 20s) to remove cellular debris, stored at -20 C for up to 48 h prior to enzyme analysis. Lactate dehydrogenase activity was measured spectrophotometrically, for cell viability determination, by three different methods. The first method involved incubating aliquots of medium or cell extracts with a solution containing phosphate buffer (0.1 M, pH 7.4), sodium pyruvate (9.1 mM), and -NADH (3.3 mM). LDH in the sample catalyzes the reaction of pyruvate to lactate, resulting in oxidation of NADH to NAD. Absorbance at 340 nm is used to measure the rate of disappearance of NADH, and is directly proportional to LDH in sample (Henry et al. 1960). The second method utilized the lactate dehydrogenase (LD-L) assay kit (Sigma procedure 228-UV). This assay procedure combined the same method of medium acquisition and cell harvesting as above. However, the extracts were then incubated in a lactate dehydrogenase reagent (50mM lactate, 7 mM NAD, 0.05% NaN 3, and buffer, pH 8.9), and the reduction rate of

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13 NAD was read immediately spectrophotometrically at 340 nm. The final method used to analyze LDH activity was the Sigma TOX-7 assay. This assay involved reduction of NAD to NADH, which was then used in a second coupled stoichiometric conversion of a tetrazolium dye to a colored compound. The production of the colored compound was then assessed spectrophotometrically at 590 nm. In all three methods a calculation of the cell viability via percent LDH activity was made, by determining the LDH activity of medium, and LDH activity in the cell extract (medium activity/ (medium activity + cell activity)) (Jauregui et al. 1981). Cell viability was also determined using cell proliferation reagent WST-1 (Roche Cat. No. 1 644 807). The assay is based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenase through production of NADH in viable cells. Briefly, hepatocytes were cultured in microtiter plates (tissue culture grade, 96 well, flat bottom plate). After the treatment period, each well was brought to a final volume of 100 l in a humidified atmosphere (37 C and 5% CO 2 ). The WST-1 reagent was then added (10L/well). The plate was then incubated for 2 h in a humidified atmosphere (37 C and 5% CO 2 ). Before assaying, the plate was placed on a shaker for 5 min to ensure thorough mixing. The absorbance at 450 nm was then measured spectrophotometrically with 690 nm as the reference wavelength. Culture medium served as the blank. Absorbance of samples directly correlates to the viability of the cells. The crystal violet assay was also used to determine cytotoxicity (Kim et al 1999). Upon conclusion of the treatment period, medium was removed from each well, and the cells were washed several times with PBS. The PBS was then removed and subsequently replaced with a dye solution containing 0.1% v/v crystal violet, 4% v/v formaldehyde,

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14 and 30% v/v ethanol diluted in PBS. The cells were then incubated in this solution for approximately 15 min. After incubation, the free dye solution was removed, and the cells were washed with PBS five times. The crystal violet dye within the cells was then extracted using 0.1% wt/v SDS diluted in PBS. The resultant extract was centrifuged (13,000xg for 20s) to remove cellular debris. Instead of simply diluting samples for each assay, a standard procedure was used to bring the absorbance of control extracts to an A 590 between 0.650 and 0.750. First, the initial A 590 of a control sample was read on the Molecular Devices spectra max 340PC 384 plate reader, with 0.1% wt/v SDS as the blank. Then, a series of 4, 1: 10 dilutions were made, and a plot of volume percent crystal violet versus absorbance was made. A linear least squares line of the absorbance at these dilutions was then used to determine the molar absorptivity of crystal violet in the control sample. Next, the volume percent crystal violet required to give an A 590 of 0.6 for the (untreated) control samples was determined. This absorbance was then used to represent 100% viability for a given group of hepatocytes. Finally, the same dilution used for control samples was carried out for all the samples. The analytical absorbance directly correlated to viable cell number. Trypan blue exclusion was used as a final means of determining cell viability. After treatment, cells were incubated for five min with dilute trypan blue (0.1 %). Dye was then removed, and cells were washed five times with PBS. Cells were then visualized on a Zeiss Axiovert S100 microscope (Carl Zeiss, Thornwood, NY) fitted with a SPOT digital CCD camera (Diagnostics Instruments, Sterling Heights, MI). Digital pictures were taken, and viability was determined by a simple picture grid.

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15 Data Analysis Data were analyzed by ANOVA for a three-way factorial design (2x2x2) to determine significant main effects using genotype, zinc status, and oxidant treatment as independent variables. Fishers LSD was used to determine significant differences (designated by p< 0.05, ** p< 0.01, and *** p<0.001).

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CHAPTER 4 RESULTS Lactate Dehydrogenase Assay Lactate dehydrogenase (LDH) enzyme leakage by hepatocytes was the first method used as a measure of cytotoxicity. However, this assay was eliminated from further use after analysis of the LDH assay with treatments of SNAP revealed significant interaction of treatment and the analyte being measured (i.e., NADH). This interaction led to suppression of NADH absorption (Fig. 4-1). Similar results were obtained with the WST-1 assay which also depends on production of NADH in a coupled reaction (data not shown). To correct this error in viability assessment, the crystal violet inclusion assay was employed as a non redox sensitive measure of viability. 01020304050Control10 mM SNAPTreatmentsLDH Activity (OD x min -1 ) *** Figure 4-1. Influence of SNAP on LDH activity. Samples containing fetal bovine serum (source of LDH) were untreated (Control) or treated with 10 mM SNAP, and LDH activity (in this case, production rate of NADH) was measured in a coupled reaction resulting in production of a tetrazolium dye (A540). Data are represented as the mean SD (n = 10 cultures). Treatment with SNAP for 12 h led to significant (p < 0.001) suppression of LDH activity, which was directly related to oxidation of NADH. 16

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17 Metallothionein Genotype Differences Hepatocyte viability due to genotype variation alone was investigated in hepatocyte cultures from all four genotypes by the crystal violet assay. The consequences of tBH and SNAP exposure on metallothionein absence or presence were determined by utilization of KO and CK hepatocytes (Fig. 4-2A and Fig 4-2B). After 2 h tBH exposure viability was drastically different between CK and KO cultures. At all concentrations between 15 M and 65 M tBH CK hepatocytes were significantly more viable than KO hepatocytes (p < 0.001). At concentrations of 0, 5, 10, and 75 M tBH viability was not significantly different between CK and KO hepatocytes. Viability was significantly reduced at all but the lowest SNAP concentration. Effects of metallothionein overexpression using CT and Tg genotypes are shown in Figures 4-2C and 4-2D. Treatment with tBH led to no significant differences in viability between CT and Tg genotypes at any concentraiton level, while treatment with SNAP at 0.5 mM was the only significantly different concentration (p < 0.05). Examination of the concentrations of the toxicants required to kill 50% of the hepatocytes revealed that, there is no benefit for mice that overexpress metallothionein (Table 4-1). However, when KO mice are compared to CK mice there is a large difference in LD 50 (Table 4-1). The CK hepatocytes required 56% more tBH, and 73% more SNAP, to reach the same level of lethality as the KO hepatocytes. Therefore, CK cultures are more viable than KO cultures under the same treatment conditions.

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18 0 10 20 30 40 50 60 70 8 0 0 10 20 30 40 50 60 70 80 90 100 110************************CK ZnN KO ZnN tBH Concentration ( M ) Viability (% Control) 0 0.1 0.5 1.0 2.0 2.5 3.0 0 10 20 30 40 50 60 70 80 90 100 110*CT ZnN TG ZnN SNAP Concentration ( mM ) Viability (% Control) 0 10 20 30 40 50 60 70 8 0 0 10 20 30 40 50 60 70 80 90 100 110CT ZnN Tg ZnN tBH Concentration ( M ) Viability (% Control)ABCD 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 10 20 30 40 50 60 70 80 90 100 110CK ZnN KO ZnN SNAP Concentration ( mM ) Viability (% Control)************** 0 10 20 30 40 50 60 70 8 0 0 10 20 30 40 50 60 70 80 90 100 110************************CK ZnN KO ZnN tBH Concentration ( M ) Viability (% Control) 0 0.1 0.5 1.0 2.0 2.5 3.0 0 10 20 30 40 50 60 70 80 90 100 110*CT ZnN TG ZnN SNAP Concentration ( mM ) Viability (% Control) 0 10 20 30 40 50 60 70 8 0 0 10 20 30 40 50 60 70 80 90 100 110CT ZnN Tg ZnN tBH Concentration ( M ) Viability (% Control)ABCD 0 10 20 30 40 50 60 70 8 0 0 10 20 30 40 50 60 70 80 90 100 110************************CK ZnN KO ZnN tBH Concentration ( M ) Viability (% Control) 0 0.1 0.5 1.0 2.0 2.5 3.0 0 10 20 30 40 50 60 70 80 90 100 110*CT ZnN TG ZnN SNAP Concentration ( mM ) Viability (% Control) 0 10 20 30 40 50 60 70 8 0 0 10 20 30 40 50 60 70 80 90 100 110CT ZnN Tg ZnN tBH Concentration ( M ) Viability (% Control)ABCD 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 10 20 30 40 50 60 70 80 90 100 110CK ZnN KO ZnN SNAP Concentration ( mM ) Viability (% Control)************** Figure 4-2. Viability due to altered metallothionein genotype. Hepatocyte cultures from KO and CK (left panels), and CT and Tg (right panels) mice were treated with 0-75 M tBH for 2 h (A and C), or 0-3 mM SNAP for 12 h (B and D). Viability was assessed by the crystal violet assay. Data are represented as the mean SD (n = 3 cultures from a representative experiment). Statistically significant differences (designated by for p < 0.05 and *** for p < 0.001) were determined by ANOVA and Fishers LSD.

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19 Table 4-1. LD50 responses due to altered metallothionein genotype. MT Genotype tBH LD 50 SNAP LD 50 KO 20.0 3 M 0.75 0.02 mM CK 45.0 2 M*** 2.75 0.13 mM*** CT 35.0 2 M 2.5 0.3 mM TG 37.0 4 M, ns 2.5 0.2 mM, ns Statistically significant differences (designated by *** for p < 0.001 and ns for not significant) were determined by ANOVA and Fishers LSD. Zinc Supplementation The effect of zinc supplementation on the individual metallothionein genotypes was investigated using a dose of 40 M zinc. Hepatocytes form KO, CK, CT, and Tg mice were treated with 40 M zinc for 18-22 h prior to addition of toxicants. Treatment with either 0-75 M tBH, or 0-3 mM SNAP for designatated amounts of time followed. As expected, without the presence of metallothionein in the KO cells, there was no effect produced by zinc on viability of tBH (Fig. 4-3A) or SNAP treated hepatocytes (Fig. 4-3B). On the contrary, zinc supplementation of CK hepatocytes prior to toxicant exposure protected both tBH and SNAP treated cultures (Fig. 4-3C and Fig. 4-3D). The LD 50 response of CK hepatocytes supplemented with zinc to tBH and SNAP treatment was 25% and 29% higher respectively, than zinc normal cultures (Table 4-2). Control transgenic hepatocytes supplemented with 40 M zinc were resistant to tBH and SNAP over the majority of concentrations used (Fig. 4-4A and Fig. 4-4B), while the un-supplemented hepatocytes were not. The tBH concentration required to kill 50 percent of the zinc supplemented CT hepatocytes was 18% higher than the ZnN cells, and the LD 50 for SNAP was 17% higher (Table 4-2). Zinc supplementation of

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20 metallothionein transgenic hepatocytes led to no overall difference in viability (compared to ZnN cultures) when tBH was used as a stressor (Fig. 4-4C). This was an unexpected result considering the large difference in viabilities obtained when SNAP was used as a stressor (Fig. 4-4D). The overall trend for SNAP treated Tg cultures was an increase in viability with zinc treatment when compared to zinc normal cultures. Zinc treated cultures from 1-3 mM SNAP were significantly different than the untreated counterparts. The dose of SNAP required to kill 50% of the ZnN hepatocytes was 2.5 mM, while the zinc supplemented hepatocytes required 3.5 mM (Fig. A-1). This represented a 29% increase in protection of Tg hepatocytes due to zinc supplementation alone. Thus, zinc increased viability within each genotype except for the KO group. Altered Genotype and Zinc Supplementation Zinc supplementation caused the greatest difference on subsequent toxicological injury between the metallothionein genotypes. Pretreatment of CK hepatocytes with zinc led to a large significant difference (p < 0.001) in viability from 10-75 M tBH, when compared to KO hepatocytes pretreated with zinc (Fig. 4-5A). The tBH concentration required to kill 50% of CK hepatocytes was 58% higher than KO hepatocytes (Table 4-3). The greatest difference in protection was realized by SNAP treated CK versus KO hepatocytes. Pretreatment of CK hepatocytes with zinc led to 79% more protection against SNAP (Table 4-3). Only one concentration of tBH (50 M) caused a significant difference between Tg and CT cells, but the overall trend in viability shows no difference between the two genotypes (Fig. 4-5C). In cells treated with 0.5, 1.0, and 2.5 mM SNAP, zinc pretreatment caused a significant increase in the viability of TG hepatocytes (Fig. 4-5D). The highest increase in viability occurred at 2.5 mM SNAP (40% increase in viability due to TG genotype), but declined to no difference at 3.0 mM SNAP. The dose

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21 of SNAP required to kill 50 percent of CT and Tg hepatocytes was the same (Fig. A-1). As with tBH treatment, the trend in viability was the same for both CT and Tg hepatocytes. Metallothionein KO hepatocytes provided a means to determining the effect of the presence of metallothionein. CK hepatocytes were significantly more viable than their KO counterparts for both SNAP and tBH treatments. This difference was exaggerated when zinc was supplemented in the medium (Fig. 4-5). Zinc caused an increase in metallothionein expression which was directly related to increased CK hepatocyte viability (Table 4-3). Hepatocytes of the Tg genotype gained no advantage in viability under these treatment conditions (Table 4-1 and Table 4-3).

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22 0 0.1 0.5 1.0 2.0 2.5 3.0 0 10 20 30 40 50 60 70 80 90 100 110KO Zn+ KO ZnN SNAP Concentration ( mM ) Viability (% Control) 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 90 100 110KO Zn+ KO ZnN tBH Concentration ( M ) Viability (% Control)ABC 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 10 20 30 40 50 60 70 80 90 100 110CK ZnN CK Zn+ ********* SNAP Concentration ( mM ) Viability (% Control) 0 10 20 30 40 50 60 70 8 0 0 10 20 30 40 50 60 70 80 90 100 110********************CK Zn+ CK ZnN tBH Concentration (M)Viability (% Control)D 0 0.1 0.5 1.0 2.0 2.5 3.0 0 10 20 30 40 50 60 70 80 90 100 110KO Zn+ KO ZnN SNAP Concentration ( mM ) Viability (% Control) 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 90 100 110KO Zn+ KO ZnN tBH Concentration ( M ) Viability (% Control)ABC 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 10 20 30 40 50 60 70 80 90 100 110CK ZnN CK Zn+ ********* SNAP Concentration ( mM ) Viability (% Control) 0 10 20 30 40 50 60 70 8 0 0 10 20 30 40 50 60 70 80 90 100 110********************CK Zn+ CK ZnN tBH Concentration (M)Viability (% Control)D Figure 4-3. Effect of zinc status on subsequent toxicant induced viability changes in control knockout and metallothionein knockout hepatocyes. Primary hepatocyte cultures from KO (A and B), and CK (C and D) mice were pretreated with zero (designated ZnN) or 40 M zinc (designated Zn+), followed by 0-75 M tBH for 2 h, or 0 3mM SNAP for 12 h. Viability was assessed by crystal violet inclusion. Data are represented as the mean SD (n = 3 cultures from a representative experiment). Statistically significant differences (designated by for p < 0.05, ** for p < 0.01, and *** for p < 0.001) were determined by ANOVA and Fishers LSD.

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23 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 80 90 100 110*********************CT ZnN CT Zn+ tBH Concentration ( M ) Viability (% Control) 0 10 20 30 40 50 60 70 8 0 0 10 20 30 40 50 60 70 80 90 100 110***Tg ZnN Tg Zn+ tBH Concentration ( M ) Viability (% Control) 0 0.1 0.5 1.0 2.0 2.5 3.0 0 10 20 30 40 50 60 70 80 90 100 110Tg Zn+ Tg ZnN ***********SNAP Concentration (mM)Viability (% Control)AB CD 0 0.1 0.5 1.0 2.0 2.5 3.0 0 10 20 30 40 50 60 70 80 90 100 110**********CT ZnN CT Zn+ SNAP Concentration ( mM ) Viability (% Control) 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 80 90 100 110*********************CT ZnN CT Zn+ tBH Concentration ( M ) Viability (% Control) 0 10 20 30 40 50 60 70 8 0 0 10 20 30 40 50 60 70 80 90 100 110***Tg ZnN Tg Zn+ tBH Concentration ( M ) Viability (% Control) 0 0.1 0.5 1.0 2.0 2.5 3.0 0 10 20 30 40 50 60 70 80 90 100 110Tg Zn+ Tg ZnN ***********SNAP Concentration (mM)Viability (% Control)AB CD 0 0.1 0.5 1.0 2.0 2.5 3.0 0 10 20 30 40 50 60 70 80 90 100 110**********CT ZnN CT Zn+ SNAP Concentration ( mM ) Viability (% Control) Figure 4-4. Effect of zinc status on subsequent toxicant induced viability changes in control transgenic and metallothionein transgenic hepatocyes. Primary hepatocyte cultures from CT (A and B), and Tg (C and D) mice, were pretreated with 0 (designated ZnN) or 40 M zinc (designated Zn+), followed by 0-75 M tBH for 2 h, or 0 3mM SNAP for 12 h. Viability was assessed by crystal violet inclusion. Data are represented as the mean SD (n = 3 cultures from a representative experiment). Statistically significant differences (designated by for p < 0.05, ** for p < 0.01, and *** for p < 0.001) were determined by ANOVA and Fishers LSD.

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24 Table 4-2. LD 50 responses to toxicants of hepatocytes from MT KO, CK, CT, and MT Tg mice as a result of zinc status. Genotype Toxicant Zinc status LD 50 ZnN 25.0 2 M tBH Zn+ 25.0 3 M, ns ZnN 0.75 0.2 mM KO SNAP Zn+ 0.75 0.15 mM, ns ZnN 45.0 2 M tBH Zn+ 60.0 4 M*** ZnN 2.5 0.3 mM CK SNAP Zn+ 3.5 0.2 mM ZnN 45 3 M tBH Zn+ 55 1 M*** ZnN 2.5 0.4 mM CT SNAP Zn+ 3.0 0.15 mM** ZnN 45 5 M tBH Zn+ 47 3 M, ns ZnN 2.5 0.25 mM TG SNAP Zn+ 4.0 0.11 mM*** Cultures supplemented with 40 M zinc are labeled Zn+, unsupplemented cultures are labeled ZnN. Statistically significant differences (designated by for p < 0.05, ** for p < 0.01, *** for p < 0.001, and ns for not significant) were determined by ANOVA and Fishers LSD.

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25 A B 0 10 20 30 40 50 60 70 8 0 0 10 20 30 40 50 60 70 80 90 100 110******************************CK Zn+ KO Zn+ tBH Concentration ( M ) Viability (% Control) 0 10 20 30 40 50 60 70 8 0 0 10 20 30 40 50 60 70 80 90 100 110***CT Zn+ Tg Zn+ tBH Concentration ( M ) Viability (% Control) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 10 20 30 40 50 60 70 80 90 100 110CK Zn+ KO Zn+ SNAP Concentration (mM)Viability (% Control)****************** 0 0.1 0.5 1.0 2.0 2.5 3.0 0 10 20 30 40 50 60 70 80 90 100 110 ********CT Zn+ TG Zn+ SNAP Concentration ( mM ) Viability (% Control) C D Figure 4-5. Differences in hepatocyte viability due to altered MT genotypes and zinc pretreatment. Hepatocytes from mice of differing metallothionein genotypes were compared to their respective controls. KO versus CK mice (left panel), and CT versus Tg (right panel) mice were pretreated with 40 M zinc for 18-22h, followed by addition of 0-75 M tBH for 2 h (A and C), or 0-3 mM SNAP for 12 h (B and D). Viability was assessed by the crystal violet assay. Data are represented as the mean SD (n = 3 cultures from a representative experiment). Statistically significant differences (designated by ** for p < 0.01, and *** for p < 0.001) were determined by ANOVA and Fishers LSD.

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26 Table 4-3. Summary of LD 50 responses between the metallothionein genotypes after pretreatment by zinc. MT Genotype tBH LD 50 SNAP LD 50 KO 25.0 3 M 0.75 0.15 mM CK 60.0 4 M*** 3.5 0.2 mM*** CT 55.0 1 M 4.0 0.25 mM TG 50.0 4 M, ns 4.0 0.33 mM, ns Values obtained from representative experiments. Statistically significant differences (designated by for p < 0.05, ** for p < 0.01, *** for p < 0.001, and ns for not significant) were determined by ANOVA and Fishers LSD.

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CHAPTER 5 DISCUSSION One common method for assessing cell viability is the lactate dehydrogenase enzyme leakage assay. When perforation of the cellular plasma membrane occurs during cell death, the cytosolic enzyme lactate dehydrogenase (among other enzymes) leaks out. This leakage can be quantified by using the enzymatic conversion of lactate to pyruvate, with subsequent formation of the analyte NADH. The highly reactive nature of NADH (E = -320 mV) suggests that in a solution with oxidative compounds, it will be oxidized. As a consequence, an experiment was conducted to determine whether treatments to be used in subsequent experiments would interact with (oxidize) NADH. As shown in figure 4-1, addition of SNAP to the LDH assay led to suppression of absorbance from NADH. Presumably, this was caused by NO production with concomitant oxidation of NADH. Therefore, a redox-insensitive measure of viability was used for all subsequent experiments. Oxidative damage to cellular constituents plays a prominent role in the pathogenesis of many diseases (Gutteridge and Halliwell 2000). Results of this study demonstrate the importance of metallothionein in protection against cellular oxidants. The absence of metallothionein in the knock-out hepatocytes proved to be deleterious when cultures of CK and KO hepatocytes were treated with tertiary-butyl hydroperoxide or S-nitroso n-acetyl penicillamine for 2 h, and 12 h respectively. The presence of metallothionein in CK cultures provided more protection against toxicant injury than did overexpression of metallothionein in transgenic cultures. However, because baseline 27

PAGE 38

28 cellular metallothionein protein content of CT and Tg cultures was found to be similar (Davis et al. unpublished data), it was not surprising to find a lack of difference between the viabilities of CT and Tg cultures based on genotype difference alone. Therefore, addition of 40 M zinc prior to toxicant treatment was used to increase cellular metallothionein levels (Cao et al. 2001). As a result, hepatocytes that were able to increase metallothionein expression did so (data not shown). Supplemental zinc increased viability within each genotype, except for the knock out hepatocytes. The increase in viability of CK, CT, and Tg cells may thus be attributed directly to zinc induced metallothionein expression. Even though zinc was able to increase viability of CK, CT, and Tg cells when the genotypes were examined individually, only the CK cells were more viable than the KO cultures when the genotypes were compared to one another. Consequently, the presence of metallothionein and its induction by zinc provided CK hepatocytes protection against oxidative insult, while ablation of metallothionein in KO hepatocytes was deleterious (Table 4-5). Contrary to the hypothesis, overexpression of metallothionein in transgenic cultures provided no increased protection. These results are in agreement with previous findings from this lab (Davis et al. 2001). Carbon tetrachloride had been used in the past to examine the role of metallothionein in protection against hepatotoxicity. In those studies, no combination of metallothionein gene expression (i.e., Tg, CT, CK, KO) and either adequate or supplemental zinc provided hepatic protection. Even though, the mice had drastically different levels of liver zinc and liver metallothionein. Many studies have brought to light the role of metallothionein as an antioxidant. Metallothionein has demonstrated the ability to quench reactive oxygen and reactive

PAGE 39

29 nitrogen species at an even higher efficiency than the established cellular antioxidant glutathione (Miura et al. 1997 and Quesada et al. 1996). The extensive network of zinc-sulfur clusters in metallothionein provide the chemical basis for its antioxidant capacity (reviewed in Maret 2000). The reduction potential of the zinc-sulfur clusters are extremely low (E < -340 mV) and thus provide a means to reduce a variety of cellular oxidants. Even though zinc bound to metallothionein is extremely stable (pK d = 12.9), zinc in metallothionein can readily exchange with free zinc in solution (Steinebach et al. 1992). Thus, metallothionein may play a critical role in redox sensing and zinc distribution in the cell. In fact, a variety of oxidative and nitrosative stressors have also been shown to interact with zinc-sulfer clusters of zinc-finger proteins, and lead to concomitant loss of zinc (Kroncke et al. 2002). One such nitrosative stressor is nitric oxide. NO has the ability to release zinc from zinc dependant transcription factors, as well as metallothionein (Schapiro et al. 2003, St. Croix et al. 2002). In vitro studies utilizing a nitric oxide donor (DEA/ NO) have demonstrated that NO has the ability to liberate zinc specifically from the domain of metallothionein (Zangger et al 2001). More recent studies in aortic endothelial cells have shown that iNOS derived NO has the ability to increase labile zinc through interaction with metallothioenin (Spahl et al. 2003). The current study utilized SNAP as a model for NO induced stress. The availability of metallothionein to interact with nitric oxide in CK hepatocytes provided protection that was not seen in KO hepatocytes. This protection was augmented when zinc increased metallothionein expression in CK cells, but not in Tg hepatocytes (compared to CT cells). A possible explanation for protection by the presence and induction of metallothionein by

PAGE 40

30 zinc, but not the over-expression of MT, could be the release of zinc from MT and subsequent signaling events such as induction of zinc dependent transcription factors or zinc dependent proteins. In these cells, exposure to NO after zinc pretreatment caused increased induction of metallothionein mRNA, with the largest increase in Tg hepatocytes (Lichten et al. unpublished observations). NO mediated release of zinc from metallothionein may be a primary means of increasing free zinc in the cell. Consequently, the ratio of thionein (apo-metallothionein) to metallothionein may be one mechanism with which the cell governs the levels of intracellular zinc (St. Croix et al. 2002). However, when metallothionein expression is altered, a shift in the MT/T ratio may occur, causing increased production of thionein. Therefore, over-expression of metallothionein may lead to production of large amounts of thionein that may sequester zinc before it can be utilized by other apoproteins. In these experiments, oxidative stress to the hepatocytes was produced by tBH. The mitochondria are the primary target of this oxidative stressor (Mehendale et al. 1994). It is presumed that tBH causes mitochondrial permeability transition (MPT) followed by mitochondrial depolarization, uncoupling of oxidative phosphorylation, ATP depletion, and finally cell death. If metallothionein has the capacity to act as an antioxidant, then MPT may be inhibited, and cell death (either necrosis or apoptosis) may be prevented. Once again, the presence of metallothionein was beneficial to CK cultures, but over expression of metallothionein in Tg cells was not (Table 4-3). However, the magnitude of viability differences seen in the zinc supplemented CK and KO cultures were not as great. This may be due to the difference between oxidative and nitrosative stress towards the zinc-sulfur clusters of metallothionein. Zinc finger proteins have been

PAGE 41

31 shown to react differently towards these stressors (Kroncke et al 2002). NO may be able to release zinc from zinc-sulfur clusters without destroying them, while oxidative stressors such as tBH could destroy the clusters. Thus, as with zinc finger proteins, a reversible exchange of zinc may occur through NO interaction that does not occur when an oxidant is applied. Therefore, zinc may be released from metallothionein in greater amounts after interaction with tBH causing decreases in viability not only due to free radical formation, but in the case of metallothionein transgenics, zinc toxicity. In summary, the absence of metallothionein in MT KO hepatocytes led to significant loss of viability after NO or tBH treatment. The presence, and induction of metallothionein by zinc, increased the viability of CK hepatocytes, but not transgenic hepatocytes. Therefore, there is a limit to the protection provided by metallothionein, which directly questions the role of metallothionein as a cellular antioxidant. However, the primary role of metallothionein may not be to act as an antioxidant, but to be a transducer of zinc signaling.

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APPENDIX CYTOTOXICITY EXPERIMENTS 020406080100120012345SNAP Concentration (mM)Viability (% control ) Tg Zn+ CT Zn+ Figure A-1. Differences in CT and Tg viability due to altered MT genotypes and zinc pretreatment CT versus Tg (right panel) mice were pretreated with 40 M zinc for 18-22h, followed by addition 0-4 mM SNAP for 12 h Viability was assessed by the crystal violet assay 32

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LIST OF REFERENCES Andrews, G.K. (2000) Regulation of metallothionein gene expression by oxidative stressand metal ions. Biochem. Pharmacol. 59:95-104. Andrews, G.K. (2001) Cellular zinc sensors: MTF-1 regulation of gene expression.Biometals. 14:223-37 Blain, D., Kubow, S., Chan, H.M. (1998) Zinc pretreatment inhibits isotretinoin teratogenicity and induces embryonic metallothionein in CD-1 mice. J. Nutr.128:1239-46. Cai, L., Klein, J.B., Kang, Y.J. (2000) Metallothionein inhibits peroxynitrite-induced DNA and lipoprotein damage. J. Biol. Chem. 275:38957-60. Cao, J., Bobo, J.A., Liuzzi, J.P., Cousins, R.J. (2001) Effects of intracellular zinc depletion on metallothionein and ZIP2 transporter expression and apoptosis. J. Leukoc. Biol. 70:559-66. Chesters, J.K. (1992) Trace element gene interactions. Nutr. Rev. 50:21723. Cousins, R.J. (1996) Zinc. In Present Knowledge in Nutrition, 7 th ed., pp.293-306 (Filer, L.J., & Ziegler, E.E., editors). Washington, D.C.: International Life Sciences Institute-Nutrition Foundation. Davis, S.R., Cousins, R.J. (2000) Metallothionein expression in animals: a physiological perspective on function. J. Nutr. 130:1085-8. Davis, S.R., Samuelson, D.A., Cousins, R.J. (2001) Metallothionein expression protects against carbon tetrachloride-induced hepatotoxicity, but overexpression and dietary zinc supplementation provide no further protection in metallothionein transgenic and knockout mice. J. Nutr.131:215-22. Dunn, M.A., Cousins, R.J. (1989) Kinetics of zinc metabolism in the rat: effect of dibutyryl cAMP. Am. J. Physiol. 256:420-30. Farber, J.L. (1994) Mechanisms of cell injury by activated oxygen species. Environ. Health. Perspect. 102:17-24. Fu, Y., Porres, J.M., Lei, X.G. (2001) Comparative impacts of glutathione peroxidase-1 gene knockout on oxidative stress induced by reactive oxygen and nitrogen species in mouse hepatocytes. Biochem. J. 359:687-95. 33

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34 Geller, D.A., Di Silvio, M., Nussler, A.K., Wang, S.C., Shapiro, R.A., Simmons, R.L., Billiar, T.R. (1993) Nitric oxide synthase expression is induced in hepatocytes in vivo during hepatic inflammation. J. Surg. Res. 55:427-32. Gutteridge, J.M., Halliwell, B. (2000) Free radicals and antioxidants in the year 2000. A historical look to the future. Ann. N. Y. Acad. Sci. 899:136-47. Hyde, R.J., Hooper, N.M., Turner, A.J. (1996) Molecular cloning and expression in COS1 cells of pig kidney aminopeptidase P. Biochem. J.319:197-201. Jacob, C., Maret, W., Vallee, B.L. (1998) Control of zinc transfer between thionein, metallothionein, and zinc proteins. Proc. Natl. Acad. Sci. 31:3489-94. Jauregui, H.O., Hayner, N.T., Driscoll, J.L., Williams-Holland, R., Lipsky, M.H. & Galleti, P.M. (1981) Trypan blue uptake and lactate dehydrogenase in adult rat hepatocytes freshly isolated cells, cell suspensions, and primary monolayer cultures. In Vitro. 17:1100-1110. Kim, Y.M., Bombeck, C.A., Billiar, T.R. (1999) Nitric oxide as a bifunctional regulator of apoptosis. Circ. Res. 84:253-6. Kroncke, K.D., Klotz, L.O., Suschek, C.V., Sies, H. (2002) Comparing nitrosative versus oxidative stress toward zinc finger-dependent transcription. Unique role for NO. J.Biol. Chem. 277:13294-301. Lichtlen, P., Schaffner, W. (2001) The "metal transcription factor" MTF-1: biological facts and medical implications. Swiss Med. Wkly. 131:647-52. Maret, W. (2000) The function of zinc metallothionein: a link between cellular zinc and redox state. J. Nutr. 130:1455-1458. Masters, B.A., Kelly, E.J., Quaife, C.J., Brinster, R.L. & Palmiter RD. (1994) Targeted disruption of metallothionein I and II genes increases sensitivity to cadmium. Proc. Natl. Acad. Sci. U S A. ;91:584-8. Matsubara, J. (1987) Alteration of radiosensitivity in metallothionein induced mice and a possible role of Zn-Cu-thionein in GSH-peroxidase system. Experientia. Suppl.;52:603-12. Matsubara, J., Tajima, Y., Karasawa, M. (1987) Metallothionein induction as a potentmeans of radiation protection in mice. Radiat. Res. 111:267-75. Miura, T., Muraoka, S., Ogiso, T. (1997) Antioxidant activity of metallothionein compared with reduced glutathione. Life Sci. 60:301-9. Miura, T., Satoh, T., Takeuchi, H. (1988) Role of metal-ligand coordination in the folding pathway of zinc finger peptides. Biochim. Biophys. Acta. 1384:171-9.

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35 Nussler, A.K., Geller, D.A., Sweetland, M.A., Di Silvio, M., Billiar, T.R., Madariaga, J.B., Simmons, R.L., Lancaster, J.R. (1993) Induction of nitric oxide synthesis and its reactions in cultured human and rat hepatocytes stimulated with cytokines plus LPS. Biochem. Biophys. Res. Commun. 194:826-35. Palmiter, R.D., Sandgren, E.P., Koeller, D.M. & Brinster, R.L. (1993) Distal regulatoryelements from the mouse metallothionein locus stimulate gene expression intransgenic mice. Mol. Cell Biol. ;13:5266-75. Pearce, L.L., Gandley, R.E., Han, W., Wasserloos, K., Stitt, M., Kanai, A.J., McLaughlin,M.K., Pitt, B.R., Levitan, E.S. (2000) Role of metallothionein in nitric oxidesignaling as revealed by a green fluorescent fusion protein. Proc. Natl. Acad. Sci.4;97:477-82. Pearce, L.L., Wasserloos, K., St. Croix, C.M., Gandley, R., Levitan, E.S., Pitt, B.R. (2000) Metallothionein, nitric oxide and zinc homeostasis in vascular endothelialcells. J. Nutr. 130:1467-70. Philcox, J.C., Coyle, P., Michalska, A., Choo, A.K.H. & Rofe, A.M. (1995) Endotoxin-induced inflammation does not cause hepatic zinc accumulation in mice lacking metallothionein gene expression. Biochem. J. 308:543-546. Powell, S.R. (2000) The antioxidant properties of zinc. J. Nutr. 130:1447-54. Quesada, A.R., Byrnes, R.W., Krezoski, S.O., Petering, D.H. (1996) Direct reaction ofH2O2 with sulfhydryl groups in HL-60 cells: zinc-metallothionein and other sites. Arch. Biochem. Biophys. 334:241-50. St. Croix, C.M., Wasserloos, K.J., Dineley, K.E., Reynolds, I.J., Levitan, E.S., Pitt, B.R.(2002) Nitric oxide-induced changes in intracellular zinc homeostasis are mediated by metallothionein/thionein. Am. J. Physiol. Lung Cell Mol. Physiol. 282:185-92. Satoh, M., Naganuma, A., Imura, N. (1988) Involvement of cardiac metallothionein inprevention of adriamycin induced lipid peroxidation in the heart. Toxicology.53:231-7. Schapiro, J.M., Libby, S.J., Fang, F.C. (2003) Inhibition of bacterial DNA replication by zinc mobilization during nitrosative stress. Proc. Natl. Acad. Sci. U. S. A. 100:8496-501. Schroeder, J.J., Cousins, R.J. (1990) Interleukin 6 regulates metallothionein geneexpression and zinc metabolism in hepatocyte monolayer cultures. Proc. Natl.Acad. Sci. U. S. A. 87:3137-41. Schroeder, J.J., Cousins, R.J. (1991a) Maintenance of zinc-dependent hepatic functions in rat hepatocytes cultured in medium without added zinc. J. Nutr. 121:844-53.

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36 Schroeder, J.J., Cousins, R.J. (1991b) Metallothionein and zinc metabolism in hepatocytes. Methods. Enzymol. 205:575-84. Schwarz, M.A., Lazo, J.S., Yalowich, J.C., Allen, W.P., Whitmore, M., Bergonia, H.A., Spahl, D.U., Berendji-Grun, D., Suschek, C.V., Kolb-Bachofen, V., Kroncke, K.D. (2003) Regulation of zinc homeostasis by inducible NO synthase-derived NO: nuclear metallothionein translocation and intranuclear Zn2+ release. Proc. Natl. Acad. Sci. U. S. A. 100:13952-7. Steinebach, O.M., Wolterbeek, B.T. (1992) Metallothionein biodegradation in rathepatoma cells: a compartmental analysis aided 35S-radiotracer study. Biochim. Biophys. Acta. 1116:155-65. Thornalley, P.J. & Vasak, M. (1985) Possible role for metallothionein in protectionagainst radiation-induced oxidative stress: Kinetics and mechanism of its reactionwith superoxide and hydroxyl radicals. Biochem. Biophys. Acta. 827:36-44. Turner, J.S., Glands, P.D., Samson, A.C., Robinson, N.J. (1996) Zn2+-sensing by the cyanobacterial metallothionein repressor SmtB: different motifs mediate metal-induced protein-DNA dissociation. Nucleic Acids Res. 24:3714-21. Tzeng, E., Billiar, T.R., Robbins, P.D., Lancaster, jr, J.R., & Pitt, B.R. (1995)Metallothionein Protects Against the Cytotoxic and DNA-Damaging Effects ofNitric Oxide. Proc. Natl. Acad. Sci. 92: 4452-4456. Vallee, B.L., Coleman, J.E., Auld, D.S. (1991) Zinc fingers, zinc clusters, and zinc twists in DNA-binding protein domains. Proc. Natl. Acad. Sci. U. S. A. 88:999-1003. Wink, D.A., Mitchell, J.B. (1998) Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic. Biol. Med. 25:434-56. Yu, B.P. (1994) Cellular damage from reactive oxygen species. Physiol. Rev. 74:139-162. Zago, M.P., Oteiza, P.I. (2001) The antioxidant properties of zinc: interactions with ironand antioxidants. Free Radic. Biol. Med. 31:266-74. Zangger, K., Oz, G., Haslinger, E., Kunert, O., Armitage, I.M. (2001) Nitric oxideselectively releases metals from the amino-terminal domain of metallothioneins: potential role at inflammatory sites. FASEB J. 15:1303-5. Zhou, Z., Sun, X., James, Kang, Y. (2002) Metallothionein protection against alcoholicliver injury through inhibition of oxidative stress. Exp. Biol. Med. 227:214-22.

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BIOGRAPHICAL SKETCH Louis Lichten was born in Miami, Florida on April 24, 1979. He received his Bachelor of Science degree from the University of Florida in 2002, and plans to continue on in the doctorate program for nutritional sciences. 37


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THE ROLES OF ZINC AND METALLOTHIONEIN IN HEPATOCYTE
ANTIOXIDANT DEFENSE















By

LOUIS LICHTEN


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE


UNIVERSITY OF FLORIDA



























Copyright 2004

by

Louis Lichten
































This thesis is dedicated to my father, and my grandparents. Without their love and
support, this would never have been possible.















ACKNOWLEDGMENTS

I would like to thank Kelly Clayton for always encouraging me to do my best. I

would also like to thank my lab mates for all their help. I thank Ray Blanchard for his

wisdom in answering all my questions, and Jeff Bobo for always being willing to help me

with my experiments. I thank Cal Green for his help with solving experimental

problems. I would also like to thank Juan Liuzzi for helping when I needed it. I greatly

appreciate Steve Davis's help from the very beginning of this project. I would also like

to thank Dr. Gregory, and Dr. Kilberg for their help and participation on my thesis

committee. Finally, I would like to thank Dr. Cousins for giving me the opportunity to

work on this project.
















TABLE OF CONTENTS



A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES ................................................... vii

LIST OF FIGURES ................................ .. ......... ............................ viii

A B S T R A C T ........................................................................................................ ........... ix

CHAPTER

1 IN T R O D U C T IO N ................................................. .............................................. .

2 LITER A TU R E R EV IEW .................................................................... ...............4...

Z in c ............... ... .. ......................... ..... ...................................................... ........ .. 4
Interrelations of Zinc and Metallothionein.............................................................4...
Zinc and Metallothionein as Cellular Antioxidants ................................................6...
Metallothionein, Nitric Oxide, and Oxidative Stress..............................................7...

3 MATERIALS AND METHODS .......................................................................... 10

A n im als ....................................................................................................... ......... 10
Hepatocyte Isolation .............................. .... ........ ...... ............... 10
C u ltu re P erio d ............................................................................................................. 1 1
T reatm ent P erio d ....................................................................................................... 12
Cytotoxicity Assays ........................................ .......................................... 12
D ata A n aly sis .............................................................................................................. 1 5

4 R E S U L T S ................................................................................................................. .. 1 6

L actate D ehydrogenase A ssay ...................................... ...................... ............... 16
Metallothionein Genotype Differences................................................................. 17
Z inc Supplem entation .............................. ....... ................... .... ...... .. ............... 19
Altered Genotype and Zinc Supplementation........................................................20

5 DISCUSSION ........................ ......... ............... 27

APPENDIX CYTOTOXICITY EXPERIMENTS ......................................................32


v









L IST O F R E FE R E N C E S ... ......................................................................... ................ 33

BIOGRAPHICAL SKETCH ......................................................................................37
















LIST OF TABLES

Table page

4-1 LD50 responses due to altered metallothionein genotype. ................................... 19

4-2 LD50 responses to toxicants of hepatocytes from MT KO, CK, CT, and
M T Tg m ice as a result of zinc status.................................................. ................ 24

4-3 Summary of LD5o responses between the metallothionein genotypes
after pretreatm ent by zinc ........................................ ........................ ................ 26
















LIST OF FIGURES


Figure page

4-1 Influence of SNAP on LDH activity................................................. 16

4-2 Viability due to altered metallothionein genotype.. ............................ ................ 18

4-3 Effect of zinc status on subsequent toxicant induced viability changes in
control knockout and metallothionein knockout hepatocyes ............................... 22

4-4 Effect of zinc status on subsequent toxicant induced viability changes in
control transgenic and metallothionein transgenic hepatocyes................................23

4-5 Differences in hepatocyte viability due to altered MT genotypes and zinc
p retire atm en t.............................................................................................................. 2 5

A-i Differences in CT and Tg viability due to altered MT genotypes and zinc
p retire atm en t.............................................................................................................. 3 2















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

THE ROLES OF ZINC AND METALLOTHIONEIN IN HEPATOCYTE
ANTIOXIDANT DEFENSE

By

Louis Lichten

August 2004

Chair: Robert Cousins
Major Department: Food Science and Human Nutrition

Zinc and metallothionein are involved in cellular antioxidant defense, but the

basic mechanisms are not known. Zinc is a biologically redox inert metal, that has been

associated with protection from various oxidant producing species. However, zinc

induces the cysteine-rich, metal-binding protein, metallothionein, through interaction

with the transcription factor MTF-1. Metallothionein may have a role in preventing

oxidative stress, by quenching free radicals and other oxidants. Therefore, it is hard to

distinguish between protection due to zinc, and protection due to induction of

metallothionein.

Hence, metallothionein null (KO) mice and metallothionein transgenic (Tg) mice

provide a unique opportunity to study altered states of metallothionein expression. These

mouse models were used to determine the effects of zinc supplementation and/ or

metallothionein gene expression on the susceptibility to oxidative and nitrosative stress.

Metallothionein KO mice were significantly less viable than control (CK) mice after









tertiary-butyl hydroperoxide (tBH) and s-nitroso n-acetyl penicillamine (SNAP)

treatments. Zinc supplementation (40 pM) had no effect on viability of KO hepatocytes.

The presence of metallothionein in CK hepatocytes demonstrated protection at

baseline and after zinc supplementation. However, overexpression of metallothionein

provided no further protection against tBH or SNAP. These results are in contrast to a

direct antioxidant role for metallothionein in murine hepatocytes.














CHAPTER 1
INTRODUCTION

Zinc is an essential mineral nutrient, possessing various biological functions

including catalytic and structural roles in metalloenzymes and metalloproteins, as well as

regulatory roles in synaptic signaling and gene expression (Cousins 1996). If zinc is

absent from a biological system, an organism becomes more susceptible to oxidative

insult. Zinc-deprived animals have demonstrated either increased free-radical production

or enhanced injury from exposure to oxidative stress (reviewed in Powell 2000). The

antioxidant mechanism of zinc protection is uncertain, but may relate to (1) zinc as a

structural factor for copper-zinc superoxide dismutase, (2) the ability of zinc to protect

sulfhydryl groups from oxidation, and (3) the ability of zinc to antagonize the catalytic

properties of redox-active transition metals (reviewed in Powell 2000). A final possible

antioxidant mechanism may be the ability of zinc to induce metallothionein (MT).

Metallothionein is a cysteine-rich protein induced by zinc through the metal

responsive transcription factor-1 (MTF-1), and interaction with metal response elements

in the metallothionein promoter (Andrews 2000). Interestingly, metallothionein is also

induced by oxidants through antioxidant response elements and/ or MTF-1 (Andrews

2000). At the protein level, metallothionein may be protective against oxidants by

scavenging free radicals. This antioxidant potential was first realized by the quenching of

the hydroxyl radical by metallothionein (Thornalley and Vasak 1985). Further, many

studies have shown metallothionein to be protective against a variety of oxidative insults:

radiation exposure (Matsubara 1987), anticancer drugs (Satoh et al. 1988), carbon









tetrachloride (Schroeder and Cousins, 1991), S-nitroso N-acetyl penicillamine (SNAP)

(Schwartz et al. 1995), peroxynitrite (Cai et al. 2000), and ethanol toxicity (Zhou et al.

2002). The mechanism of protection by metallothionein may be the result of interaction

of an oxidant with the redox-active zinc-sulfur ligands of metallothionein, causing

reduction of the oxidant and subsequent release of zinc (Jacob et al 1998). This

mechanism allows metallothionein to be considered as a possible link between cellular

zinc and the redox state of the cell (reviewed in Maret 2000).

To look at the efficacy of metallothionein protection, metallothionein gene

knock-out (KO), and metallothionein transgenic (TG) mice can be used. In a study by

Davis et al. (2001), expression of metallothionein protected against carbon

tetrachloride-induced hepatotoxicity, but metallothionein overexpression did not provide

further protection. These results are inconsistent with direct antioxidant activity of

metallothionein. Therefore, experiments were designed to more directly assess the

antioxidant activity of metallothionein by treatment of primary hepatocyte cultures (from

these genotypes) with tertiary-butyl hydroperoxide (tBH), and a nitric oxide donor

(SNAP). Current studies utilizing the same study design and a redox-insensitive method

to detect cell viability demonstrate the protection provided by metallothionein against

oxidative and nitrosative stress.

* Hypothesis 1: Basal increase of metallothionein expression in the transgenic mice
will provide isolated hepatocytes increased protection from nitric oxide and/or tBH,
while the ablation of metallothionein in the knock-out hepatocytes will be
deleterious.

* Hypothesis 2: Supplementation of hepatocyte cultures with zinc prior to nitric
oxide or tBH exposure will further enhance the viability of transgenic cells, through
induction of metallothionein.









The specific aim of these experiments is to determine whether the presence of

metallothioenein will protect primary hepatocytes from tBH or nitric oxide-induced

oxidative stress, as measured by cell viability.














CHAPTER 2
LITERATURE REVIEW

Zinc

Zinc is an essential mineral nutrient that is required for many biological roles

including catalytic, structural, and regulatory roles (Cousins 1996). Zinc functions

catalytically in more than 60 enzymes, such as metallopeptidases (Turner et al. 1996).

Metalloenzymes are considered to be zinc-dependent when removal of zinc causes

reduction in activity, and replacement of zinc restores activity. Structurally, greater than

1% of the genome codes for proteins containing zinc finger or similar domains, where

zinc serves a structural role (Chesters 1992). Typically, zinc acts to stabilize the tertiary

structure of metalloproteins (Vallee et al. 1991). Zinc is typically coordinated by four

amino acid side chains: usually the sulfur atom of cysteine, or sometimes histidine. The

regulatory role of zinc includes the binding of the metal to specific transcription factors,

allowing binding of the transcription factor to metal responsive elements in the promoters

of metal responsive genes (reviewed in Andrews 2000). Zinc binding may also have a

regulatory role, through activation of signal transduction pathways (reviewed in Maret

2000).

Interrelations of Zinc and Metallothionein

In vivo, zinc metabolism is controlled homeostatically, through mechanisms

related to absorption and excretion (via zinc transporters), and intracellular proteins such

as metallothionein. Metallothioneins are small (6-7 kDa), cysteine-rich

heavy-metal-binding proteins, and can bind up to seven zinc atoms (reviewed in Cousins









1996). Approximately 5 to 10% of the total cellular zinc is found in complex with

metallothionein under normal physiological conditions (Andrews 2001).

Metallothioneins are the most abundant heavy-metal-binding proteins in the body

(reviewed in Andrews 2000). Metallothioneins are thought to function in the

homeostasis of zinc through involvement in zinc absorption, tissue distribution, and

protection against acute stress (Schroeder and Cousins 1991b). During periods of acute

stress, there is a decrease in plasma zinc levels, along with an increase in tissue zinc,

particularly in the liver (reviewed in Cousins 1996). This reduction in plasma zinc is

directly related to the changes in kinetics of zinc metabolism, which lead to increased

binding of the metal to metallothionein (Dunn and Cousins 1991). In support of a link

among zinc, metallothionein, and regulation of zinc metabolism, results of experiments

with endotoxin-challenged metallothionein null mice (MT KO) show no decrease in

plasma zinc (Philcox et al. 1995). Therefore, metallothionein plays a crucial role in

plasma-zinc clearance and tissue-zinc uptake.

Metallothionein KO mice lack functional expression of metallothionein, and

allow zinc metabolism to be studied in the absence of metallothionein (Davis et al. 1998).

Transgenic mice have been developed that have approximately 55 additional copies of

the metallothionein-I gene in their genome (Palmiter et al. 1993). Metallothionein

transgenic mice provide a model for studying the effect of exaggerated metallothionein

levels on zinc metabolism.

Metallothionein is transcriptionally regulated by zinc, through metal responsive

elements in the promoter region of the gene (Andrews 2000). However, the

metallothionein gene is also transcriptionally regulated by glucocorticoid hormones and









specific cytokines. This allows for the possibility that these metalloproteins plays a role

in inflammatory and stress-related responses (reviewed in Davis and Cousins 2000).

Zinc and Metallothionein as Cellular Antioxidants

The oxidation of many different cellular constituents is involved in the

pathogenesis of an array of diseases (Gutteridge and Halliwell 2000). Cellular oxidative

stress occurs when the antioxidant defense system in the cell becomes overwhelmed.

There are many different forms of oxidants, including reactive oxygen species (H202, 02-,

and OH), reactive nitrogen species (NO and ONOO-), and carbon centered radicals

(e.g., 'CC13). These oxidative species can damage lipids, proteins, and nucleic acids

(Farber 1994). When oxidative stressors are introduced into cells, specific cellular

antioxidant defense mechanisms are present to quench or reduce the radical mediated

damage that may occur. These defense mechanisms involve antioxidant nutrients such as

tocopherols and ascorbate, as well as the cellular antioxidants glutathione and superoxide

dismutase (Yu 1994). If damage does occur, there are ways for the cell to repair itself.

For instance, oxidized bases are removed from DNA to prevent further damage, oxidized

lipid membranes are reduced by the action of glutathione peroxidase, and damaged

proteins are committed to a proteasome degradation pathway. If these cellular defenses

are overcome, and oxidative stress occurs in great amounts, the cell or cells may not

survive. Therefore, establishing a way to reduce oxidative stress is important.

Supplemental zinc provides additional protection against certain radicals (Blain et

al. 1998). Zinc is a potent inducer of metallothionein expression. Consequently, the

antioxidant protection attributed to zinc may be due to metallothionein induction.

Metallothionein expression is also induced in response to oxidative stress (reviewed in









Davis and Cousins 2000). Therefore, there is a great degree of correlation between

increased metallothionein synthesis and protection from oxidative stress. However, this

protection is not always observed. In studies using metallothionein knock out and

metallothionein transgenic mice, carbon tetrachloride induced hepatotoxicity was reduced

initially by the presence of metallothionein in control compared to knock out mice (Davis

et al. 2001). Later time points showed no difference between genotypes in response to

carbon tetrachloride. Also, metallothionein transgenic and transgenic control mice did

not differ in the levels of hepatotoxicity, despite a large degree of difference in hepatic

metallothionein and zinc content. Further examination of zinc and metallothionein as

cellular antioxidants in cells from this mouse model led to differing results. In two

independent studies, overexpresssion of metallothionein was found to be ineffective at

increasing viability, and may actually be deleterious (Davis et al. unpublished data).

These results are a remarkable contrast from the apparent protective effects of

metallothionein observed with carbon tetrachloride treated rat hepatocytes (Schroeder

and Cousins, 1990), and numerous other reports describing a protective effect of this

protein.

While many experiments have shown metallothionein and zinc to have

cytoprotective roles, some have not. This disparity in protection must be investigated

further utilizing a different oxidative stressor, such as nitric oxide.

Metallothionein, Nitric Oxide, and Oxidative Stress

During hepatic inflammation or endotoxemia, hepatocytes can respond to

cytokine or bacterial LPS stimulation by activating the inducible form of nitric oxide

synthase, which can generate a large amount of nitric oxide from arginine (Nussler et al.

1993). This production of NO has been shown to be both hepatoprotective and cytotoxic









(Wink and Mitchell 1998). The paradoxical effects of nitric oxide may relate to its ability

to interact with 02- to form peroxynitrite, which is highly reactive and may account for

part of nitric oxide-induced cytotoxicity (Fu et al. 2001). The susceptibility of cells to

peroxynitrite and nitric oxide is primarily dependent upon their reducing capacity (thiol

content) (Kim et al. 1999). Metallothionein has been shown, in vitro, to react directly

with peroxynitrite to prevent lipoprotein and DNA damage caused by this reactive

nitrogen species (Cai et al. 2000). However, the protection by metallothionein in this

study was not dose dependent. Increasing levels of the protein did not provide further

protection against DNA damage, or LDL oxidation. Nonetheless, metallothionein over

expression has been documented to be protective against SNAP (NO-donor) induced

killing of, and DNA single-strand breaks in NIH 3T3 cells (Schwarz et al. 1995).

Further in vitro studies have elucidated a link between the cellular redox state and

metal ion homeostasis (Jacob et al. 1998, reviewed in Maret 2000). The metal thiolate

clusters of metallothionein possess the unique ability to function as a redox unit, therefore

it has the potential to be involved in a redox sensitive signaling pathway (Pearce et al.

2000). Indeed, nitric oxide has been shown to S-nitrosylate metallothionein and release

zinc from cultured pulmonary artery endothelial cells as confirmed by fluorescence

protein modified metallothionein that undergoes conformational changes in the presence

of nitric oxide (Pearce et al., 2000). These alterations in cellular zinc homeostasis may

lead to the protective effects of metallothionein against nitric oxide toxicity (Schwarz et

al. 1995).

Overall, the data on the protective effects provided by zinc and metallothionein

against oxidative stress is not definitive. Therefore, further characterization of the






9


interaction of zinc and/or metallothionein to oxidative insult, is necessary. The

pathogenesis of many liver diseases may begin with oxidative injury (Gutteridge and

Halliwell, 2000). For that reason, hepatocytes provide a good model to understand the

possible role of zinc as an antioxidant. Consequently, the goal of this study is to

elucidate the role of zinc and metallothionein as hepatic cytoprotective agents, utilizing

the murine model of altered metallothionein gene expression (i.e. MT-KO and MT-TG

mice).














CHAPTER 3
MATERIALS AND METHODS

Animals

All breeding of MT-KO and MT-Tg mice was done in-house from founder mice

purchased from the Jackson Laboratory, Bar Harbor, ME. The C57BL/6 mice were

crossed with SJL mice, possessing multiple copies of the murine metallothionein-1 gene,

to create the line of metallothionein overexpressing mice (Palmiter et al. 1993). The

control transgenic mice were created by backcrossing against the C57BL/6 strain. The

MT-KO genotype was developed by crossing 129/SvCPJ mice MT (-/-), with C57BL/6

mice (Masters 1997). The control KO mice are 129S3/SvImJ mice. Six- to eight-week

old male mice were used in all experiments. Mice were fed a modified commercial

rodent diet (Harland TEKLAD, TEKLAD Rodent Diet (W) 8604) and given tap water.

Hepatocyte Isolation

Liver perfusions were alternated among all four genotypes. Mice were anesthetized

with an intraperitoneal injection of sodium pentobarbital (60 mg/kg). The isolation of

liver cells involves many steps. First, the liver was infused with a calcium-free solution

containing 0.142 M NaCl, 0.0067 M KC1, and 0.01 M HEPES buffer (pH 7.3), via the

subhepatic inferior vena cava (modification of Schroeder and Cousins 1991 a). Initial

flow rate for the calcium-free solution was set at 2 mL/min. As the liver began to

blanche, an incision was made in the portal vein, allowing perfusate to freely flow

without recirculation. After opening the rib cage, a hemostat was then placed on the

inferior vena cava superior to the diaphragm. The flow rate of calcium-free solution was









then increased to 8 mL/min for approximately 10-12 min. The subsequent step involved

perfusion of a solution containing 0.0667 M NaCl, 0.0067 M KC1, 0.00476 M CaC12,

0.10 M HEPES buffer (pH 7.3), and 0.04% collagenase (Sigma type IV collagenase, C-

5138) at a flow rate of 7 mL/min for 14 min. Upon completion of digestion, the liver was

excised rapidly from the body cavity, and transferred to a sterile glass culture dish

containing approximately 15 mL of the collagenase solution. The culture dish was then

transferred to a sterile cell culture hood.

Prior to liberation of hepatocytes, the gall bladder was carefully removed.

Incisions were then made in each of the liver lobes to allow easy access to the

hepatocytes. Finally, using a cell scraper, cells were released from the liver lobes gently,

and as quickly as possible.

Culture Period

After liberation, the liver cells were passed through a 100 um cell strainer to

remove debris and large cell aggregates. The hepatocytes were then suspended in a

buffered wash medium (William's Medium E + 10 mM HEPES pH 7.3), and collected by

centrifugation at approximately 50 x g for 4 min. Cells were then washed in the same

buffer two more times to improve cell purity. The final cell pellet was resuspended in

attachment medium (WME supplemented with 10% FBS, 100 nM insulin, 100 nM

Dexamethasone, 100 U/mL penicillin, 100 mg/mL streptomycin). An aliquot of the final

cell suspension was then removed, and placed into solution with Trypan Blue (Sigma

T8154) and PBS for determination of cell viability. Viability of the hepatocytes was then

assessed by Trypan Blue Exclusion as measured with a hemacytometer. Only

suspensions with > 90% viability were used in experiments. If viability was satisfactory,

cells were seeded at 2.5 x 105 cells/ well in 12-well, collagen coated culture plates (6-10









[tg collagen/ well). After plating, the cells are allowed to attach for 3 h (370C, 5% CO2).

Following selective attachment of parenchymal cells, medium in each well was

exchanged for fresh medium only, or fresh medium containing 40 [iM zinc. These

culture conditions did not change for 18-22 h, when oxidant treatment began.

Treatment Period

Upon completion of the culture period, all media was removed and replaced with

medium containing 0-3 mM SNAP for 2 to 14 h, or 0-75 [LM tBH for 2 h.

Cytotoxicity Assays

After the treatment period had ended, medium from each well was removed and set

aside. Cell monolayers were then washed twice with PBS, and cells were harvested in

1% Triton X-100 and disrupted by repeated passage through a 200 pL pipette tip. The

medium as well as cell extracts were then centrifuged (130 00 x g for 20s) to remove

cellular debris, stored at -20 C for up to 48 h prior to enzyme analysis.

Lactate dehydrogenase activity was measured spectrophotometrically, for cell

viability determination, by three different methods. The first method involved incubating

aliquots of medium or cell extracts with a solution containing phosphate buffer (0.1 M,

pH 7.4), sodium pyruvate (9.1 mM), and 3-NADH (3.3 mM). LDH in the sample

catalyzes the reaction of pyruvate to lactate, resulting in oxidation of NADH to NAD.

Absorbance at 340 nm is used to measure the rate of disappearance of NADH, and is

directly proportional to LDH in sample (Henry et al. 1960). The second method utilized

the lactate dehydrogenase (LD-L) assay kit (Sigma procedure 228-UV). This assay

procedure combined the same method of medium acquisition and cell harvesting as

above. However, the extracts were then incubated in a lactate dehydrogenase reagent

(50mM lactate, 7 mM NAD, 0.05% NaN3, and buffer, pH 8.9), and the reduction rate of









NAD was read immediately spectrophotometrically at 340 nm. The final method used to

analyze LDH activity was the Sigma TOX-7 assay. This assay involved reduction of

NAD to NADH, which was then used in a second coupled stoichiometric conversion of a

tetrazolium dye to a colored compound. The production of the colored compound was

then assessed spectrophotometrically at 590 nm. In all three methods a calculation of the

cell viability via percent LDH activity was made, by determining the LDH activity of

medium, and LDH activity in the cell extract (medium activity/ (medium activity + cell

activity)) (Jauregui et al. 1981).

Cell viability was also determined using cell proliferation reagent WST-1 (Roche

Cat. No. 1 644 807). The assay is based on the cleavage of the tetrazolium salt WST-1

by mitochondrial dehydrogenase through production of NADH in viable cells. Briefly,

hepatocytes were cultured in microtiter plates (tissue culture grade, 96 well, flat bottom

plate). After the treatment period, each well was brought to a final volume of 100 pl in a

humidified atmosphere (37 C and 5% C02). The WST-1 reagent was then added

(10lO/well). The plate was then incubated for 2 h in a humidified atmosphere (37 C and

5% C02). Before assaying, the plate was placed on a shaker for 5 min to ensure thorough

mixing. The absorbance at 450 nm was then measured spectrophotometrically with 690

nm as the reference wavelength. Culture medium served as the blank. Absorbance of

samples directly correlates to the viability of the cells.

The crystal violet assay was also used to determine cytotoxicity (Kim et al 1999).

Upon conclusion of the treatment period, medium was removed from each well, and the

cells were washed several times with PBS. The PBS was then removed and subsequently

replaced with a dye solution containing 0.1% v/v crystal violet, 4% v/v formaldehyde,









and 30% v/v ethanol diluted in PBS. The cells were then incubated in this solution for

approximately 15 min. After incubation, the free dye solution was removed, and the cells

were washed with PBS five times. The crystal violet dye within the cells was then

extracted using 0.1% wt/v SDS diluted in PBS. The resultant extract was centrifuged

(13,000xg for 20s) to remove cellular debris. Instead of simply diluting samples for

each assay, a standard procedure was used to bring the absorbance of control extracts to

an A590 between 0.650 and 0.750. First, the initial A590 of a control sample was read on

the Molecular Devices spectra max 340PC384 plate reader, with 0.1% wt/v SDS as the

blank. Then, a series of 4, 1: 10 dilutions were made, and a plot of volume percent crystal

violet versus absorbance was made. A linear least squares line of the absorbance at these

dilutions was then used to determine the molar absorptivity of crystal violet in the control

sample. Next, the volume percent crystal violet required to give an A590 of 0.6 for the

(untreated) control samples was determined. This absorbance was then used to represent

100% viability for a given group of hepatocytes. Finally, the same dilution used for

control samples was carried out for all the samples. The analytical absorbance directly

correlated to viable cell number.

Trypan blue exclusion was used as a final means of determining cell viability.

After treatment, cells were incubated for five min with dilute trypan blue (0.1 %). Dye

was then removed, and cells were washed five times with PBS. Cells were then

visualized on a Zeiss Axiovert S100 microscope (Carl Zeiss, Thornwood, NY) fitted with

a SPOT digital CCD camera (Diagnostics Instruments, Sterling Heights, MI). Digital

pictures were taken, and viability was determined by a simple picture grid.






15


Data Analysis

Data were analyzed by ANOVA for a three-way factorial design (2x2x2) to

determine significant main effects using genotype, zinc status, and oxidant treatment as

independent variables. Fisher's LSD was used to determine significant differences

(designated by p< 0.05, ** p< 0.01, and *** p<0.001).














CHAPTER 4
RESULTS

Lactate Dehydrogenase Assay

Lactate dehydrogenase (LDH) enzyme leakage by hepatocytes was the first

method used as a measure of cytotoxicity. However, this assay was eliminated from

further use after analysis of the LDH assay with treatments of SNAP revealed significant

interaction of treatment and the analyte being measured (i.e., NADH). This interaction

led to suppression of NADH absorption (Fig. 4-1). Similar results were obtained with the

WST-1 assay which also depends on production of NADH in a coupled reaction (data not

shown). To correct this error in viability assessment, the crystal violet inclusion assay

was employed as a non redox sensitive measure of viability.


50 -
50







Control 10 mM SNAP
Treatments


Figure 4-1. Influence of SNAP on LDH activity. Samples containing fetal bovine serum
(source of LDH) were untreated (Control) or treated with 10 mM SNAP, and LDH
activity (in this case, production rate of NADH) was measured in a coupled reaction
resulting in production of a tetrazolium dye (A540). Data are represented as the mean +
SD (n = 10 cultures). Treatment with SNAP for 12 h led to significant (p < 0.001)
suppression of LDH activity, which was directly related to oxidation of NADH.









Metallothionein Genotype Differences

Hepatocyte viability due to genotype variation alone was investigated in hepatocyte

cultures from all four genotypes by the crystal violet assay. The consequences of tBH

and SNAP exposure on metallothionein absence or presence were determined by

utilization of KO and CK hepatocytes (Fig. 4-2A and Fig 4-2B). After 2 h tBH exposure

viability was drastically different between CK and KO cultures. At all concentrations

between 15 gM and 65 tM tBH CK hepatocytes were significantly more viable than KO

hepatocytes (p < 0.001). At concentrations of 0, 5, 10, and 75 tM tBH viability was not

significantly different between CK and KO hepatocytes. Viability was significantly

reduced at all but the lowest SNAP concentration. Effects of metallothionein

overexpression using CT and Tg genotypes are shown in Figures 4-2C and 4-2D.

Treatment with tBH led to no significant differences in viability between CT and Tg

genotypes at any concentraiton level, while treatment with SNAP at 0.5 mM was the only

significantly different concentration (p < 0.05). Examination of the concentrations of the

toxicants required to kill 50% of the hepatocytes revealed that, there is no benefit for

mice that overexpress metallothionein (Table 4-1). However, when KO mice are

compared to CK mice there is a large difference in LD50 (Table 4-1). The CK

hepatocytes required 56% more tBH, and 73% more SNAP, to reach the same level of

lethality as the KO hepatocytes. Therefore, CK cultures are more viable than KO

cultures under the same treatment conditions.














go0

go a i s o A 6 TT lo
tNiitAP ononirtlon (mM) HSNAP ons ntration (mM)











KG and CK (left panels), and CT and Tg (right panels) mice were treated with 0-75 OT
0.05 and *** for AP 0.001) were determi) ned by onANOVntratA andn (Fisher's LSM)

Figure 4-2. Viability due to altered metallothionein genotype. Hepatocyte cultures from
KO and CK (left panels), and CT and Tg (right panels) mice were treated with 0-75 PM
tBH for 2 h (A and C), or 0-3 mM SNAP for 12 h (B and D). Viability was assessed by
the crystal violet assay. Data are represented as the mean SD (n = 3 cultures from a
representative experiment). Statistically significant differences (designated by for p <
0.05 and *** for p < 0.001) were determined by ANOVA and Fisher's LSD.









Table 4-1 LD50 response e


Statistically significant differences (designated by *** for p < 0.001 and ns for not
significant) were determined by ANOVA and Fisher's LSD.


Zinc Supplementation

The effect of zinc supplementation on the individual metallothionein genotypes

was investigated using a dose of 40 [tM zinc. Hepatocytes form KO, CK, CT, and Tg

mice were treated with 40 [tM zinc for 18-22 h prior to addition of toxicants. Treatment

with either 0-75 [tM tBH, or 0-3 mM SNAP for designatated amounts of time followed.

As expected, without the presence of metallothionein in the KO cells, there was no effect

produced by zinc on viability of tBH (Fig. 4-3A) or SNAP treated hepatocytes (Fig.

4-3B). On the contrary, zinc supplementation of CK hepatocytes prior to toxicant

exposure protected both tBH and SNAP treated cultures (Fig. 4-3C and Fig. 4-3D). The

LD50 response of CK hepatocytes supplemented with zinc to tBH and SNAP treatment

was 25% and 29% higher respectively, than zinc normal cultures (Table 4-2).

Control transgenic hepatocytes supplemented with 40 tM zinc were resistant to

tBH and SNAP over the majority of concentrations used (Fig. 4-4A and Fig. 4-4B), while

the un-supplemented hepatocytes were not. The tBH concentration required to kill 50

percent of the zinc supplemented CT hepatocytes was 18% higher than the ZnN cells, and

the LD50 for SNAP was 17% higher (Table 4-2). Zinc supplementation of


MT Genotype tBH LD50 SNAP LD50

KO 20.0 3 atM 0.75 0.02 mM

CK 45.0 2 aM*** 2.75 0.13 mM***

CT 35.0 2 aM 2.5 0.3 mM

TG 37.0 4 [M, ns 2.5 0.2 mM, ns









metallothionein transgenic hepatocytes led to no overall difference in viability (compared

to ZnN cultures) when tBH was used as a stressor (Fig. 4-4C). This was an unexpected

result considering the large difference in viabilities obtained when SNAP was used as a

stressor (Fig. 4-4D). The overall trend for SNAP treated Tg cultures was an increase in

viability with zinc treatment when compared to zinc normal cultures. Zinc treated

cultures from 1-3 mM SNAP were significantly different than the untreated counterparts.

The dose of SNAP required to kill 50% of the ZnN hepatocytes was 2.5 mM, while the

zinc supplemented hepatocytes required 3.5 mM (Fig. A-1). This represented a 29%

increase in protection of Tg hepatocytes due to zinc supplementation alone. Thus, zinc

increased viability within each genotype except for the KO group.

Altered Genotype and Zinc Supplementation

Zinc supplementation caused the greatest difference on subsequent toxicological

injury between the metallothionein genotypes. Pretreatment of CK hepatocytes with zinc

led to a large significant difference (p < 0.001) in viability from 10-75 gM tBH, when

compared to KO hepatocytes pretreated with zinc (Fig. 4-5A). The tBH concentration

required to kill 50% of CK hepatocytes was 58% higher than KO hepatocytes (Table 4-

3). The greatest difference in protection was realized by SNAP treated CK versus KO

hepatocytes. Pretreatment of CK hepatocytes with zinc led to 79% more protection

against SNAP (Table 4-3). Only one concentration of tBH (50 [M) caused a significant

difference between Tg and CT cells, but the overall trend in viability shows no difference

between the two genotypes (Fig. 4-5C). In cells treated with 0.5, 1.0, and 2.5 mM SNAP,

zinc pretreatment caused a significant increase in the viability of TG hepatocytes (Fig.

4-5D). The highest increase in viability occurred at 2.5 mM SNAP (40% increase in

viability due to TG genotype), but declined to no difference at 3.0 mM SNAP. The dose









of SNAP required to kill 50 percent of CT and Tg hepatocytes was the same (Fig. A-1).

As with tBH treatment, the trend in viability was the same for both CT and Tg

hepatocytes.

Metallothionein KO hepatocytes provided a means to determining the effect of

the presence of metallothionein. CK hepatocytes were significantly more viable than

their KO counterparts for both SNAP and tBH treatments. This difference was

exaggerated when zinc was supplemented in the medium (Fig. 4-5). Zinc caused an

increase in metallothionein expression which was directly related to increased CK

hepatocyte viability (Table 4-3). Hepatocytes of the Tg genotype gained no advantage in

viability under these treatment conditions (Table 4-1 and Table 4-3).














a40
200
1k40o
I 8
0 ,


IB 2o 30 4 o 010 7
tBH Concentration (14M)


8 0 10 20 o 0 40 Sio ~M
tBH Concentration (^M)


6 0:1 0:5 1.0 2: 0 2.5 3:0 0.0 0.8 1.0 1.8 2.0 2.8 3.0
SNAP Concentration (mM) SNAP Conoentration (mM)
Figure 4-3. Effect of zinc status on subsequent toxicant induced viability changes in
control knockout and metallothionein knockout hepatocyes. Primary hepatocyte cultures
from KO (A and B), and CK (C and D) mice were pretreated with zero (designated ZnN)
or 40 [M zinc (designated Zn+), followed by 0-75 [M tBH for 2 h, or 0 3mM SNAP
for 12 h. Viability was assessed by crystal violet inclusion. Data are represented as the
mean SD (n = 3 cultures from a representative experiment). Statistically significant
differences (designated by for p < 0.05, ** for p < 0.01, and *** for p < 0.001) were
determined by ANOVA and Fisher's LSD.


70 10


10,
10,
i0,
40, "< >
iB0,^^
190,^
IB0,^&
!B ^1^


























S 10 20 30 40 50 60 70
tBH Concentration (MM)


-- CT ZnN


0 0.1 0.5 1.0 2.0 2.5 3.0
SNAP Concentration (mM)


110o
100
90'
I 70
:50
40
o30
S 20
10B
0


B


Figure 4-4. Effect of zinc status on subsequent toxicant induced viability changes in
control transgenic and metallothionein transgenic hepatocyes. Primary hepatocyte
cultures from CT (A and B), and Tg (C and D) mice, were pretreated with 0 (designated
ZnN) or 40 pM zinc (designated Zn+), followed by 0-75 PM tBH for 2 h, or 0 3mM
SNAP for 12 h. Viability was assessed by crystal violet inclusion. Data are represented
as the mean SD (n = 3 cultures from a representative experiment). Statistically
significant differences (designated by for p < 0.05, ** for p < 0.01, and *** for p <
0.001) were determined by ANOVA and Fisher's LSD.


10-
0-
80

30
10-
0.
0 10 20 30 40 50 60 70 80
tBH Concentration (pM)


-m-Tg ZnN
* 100 -.-Tg Zn
90 ***

70.
50.
40.
30,
20,
10.
6 1 085 0 21 0 2Y 5 3A 0
SNAP Concentration (mrM)












Table 4-2. LD50 responses to toxicants of hepatocytes from MT KO, CK, CT, and MT
Tg mice as a result of zinc status.
Genotype Toxicant Zinc status LD50
KO tBH ZnN 25.0 + 2 [M
Zn+ 25.0 3 [M, ns
SNAP ZnN 0.75 + 0.2 mM
Zn+ 0.75 + 0.15 mM, ns
CK tBH ZnN 45.0 + 2 [M
Zn+ 60.0 + 4 [M***
SNAP ZnN 2.5 + 0.3 mM
Zn+ 3.5 + 0.2 mM
CT tBH ZnN 45 + 3 kM
Zn+ 55 + 1 [M***
SNAP ZnN 2.5 + 0.4 mM
Zn+ 3.0 + 0.15 mM**
TG tBH ZnN 45 + 5 [pM
Zn+ 47 + 3 [M, ns
SNAP ZnN 2.5 + 0.25 mM
Zn+ 4.0 + 0.11 mM***
Cultures supplemented with 40 [M zinc are labeled Zn+, unsupplemented cultures are
labeled ZnN. Statistically significant differences (designated by for p < 0.05, ** for p <
0.01, *** for p < 0.001, and ns for not significant) were determined by ANOVA and
Fisher's LSD.














A B



80 080 20 30 40 50 60 70 80 10 20 30 40 50 60 70 80
0 *** 70

40 ** 4100-
9300 30:
20, 20-



S10. 710-
0 10 20 30 40 50 60 70 8 10 20 3 0 04,0 '0 6 70 8;0
C tBH Concentration (VI) D tBH1 Concentration (2M)
110F *** Ds -- CK Zn+ 110-
copae0t0v KO Zn+ KG vs C
80- *** 80-
6 60- 70-
50- 60-
40- 50o
30- 40o
.2 20- o30
( 10- 20-
0- I I I I I 10-
0.0 0.5 1.0 1.5 2.0 2.5 3.0 o
SNAP Concentration (mM) o 0.1 0.5 1.0 2.0 2.5 3.0
SNAP Concentration (mM)

Figure 4-5. Differences in hepatocyte viability due to altered MT genotypes and zinc
pretreatment. Hepatocytes from mice of differing metallothionein genotypes were
compared to their respective controls. KO versus CK mice (left panel), and CT versus Tg
(right panel) mice were pretreated with 40 pM zinc for 18-22h, followed by addition of 0-
75 [tM tBH for 2 h (A and C), or 0-3 mM SNAP for 12 h (B and D). Viability was
assessed by the crystal violet assay. Data are represented as the mean + SD (n = 3
cultures from a representative experiment). Statistically significant differences
(designated by ** for p < 0.01, and *** for p < 0.001) were determined by ANOVA and
Fisher's LSD.









Table 4-3. Summary of LD5o responses between the metallothionein genotypes after
pretreatment by zinc.
MT Genotype tBH LDso SNAP LDso

KO 25.0 3 [M 0.75 0.15 mM

CK 60.0 4 pM*** 3.5 + 0.2 mM***

CT 55.0 1 [M 4.0 + 0.25 mM

TG 50.0 4 [M, ns 4.0 + 0.33 mM, ns

Values obtained from representative experiments. Statistically significant differences
(designated by for p < 0.05, ** for p < 0.01, *** for p < 0.001, and ns for not
significant) were determined by ANOVA and Fisher's LSD.














CHAPTER 5
DISCUSSION

One common method for assessing cell viability is the lactate dehydrogenase

enzyme leakage assay. When perforation of the cellular plasma membrane occurs during

cell death, the cytosolic enzyme lactate dehydrogenase (among other enzymes) leaks out.

This leakage can be quantified by using the enzymatic conversion of lactate to pyruvate,

with subsequent formation of the analyte NADH. The highly reactive nature of NADH

(E = -320 mV) suggests that in a solution with oxidative compounds, it will be oxidized.

As a consequence, an experiment was conducted to determine whether treatments to be

used in subsequent experiments would interact with (oxidize) NADH. As shown in

figure 4-1, addition of SNAP to the LDH assay led to suppression of absorbance from

NADH. Presumably, this was caused by NO production with concomitant oxidation of

NADH. Therefore, a redox-insensitive measure of viability was used for all subsequent

experiments.

Oxidative damage to cellular constituents plays a prominent role in the

pathogenesis of many diseases (Gutteridge and Halliwell 2000). Results of this study

demonstrate the importance of metallothionein in protection against cellular oxidants.

The absence of metallothionein in the knock-out hepatocytes proved to be deleterious

when cultures of CK and KO hepatocytes were treated with tertiary-butyl hydroperoxide

or S-nitroso n-acetyl penicillamine for 2 h, and 12 h respectively. The presence of

metallothionein in CK cultures provided more protection against toxicant injury than did

overexpression of metallothionein in transgenic cultures. However, because baseline









cellular metallothionein protein content of CT and Tg cultures was found to be similar

(Davis et al. unpublished data), it was not surprising to find a lack of difference between

the viabilities of CT and Tg cultures based on genotype difference alone. Therefore,

addition of 40 tM zinc prior to toxicant treatment was used to increase cellular

metallothionein levels (Cao et al. 2001). As a result, hepatocytes that were able to

increase metallothionein expression did so (data not shown).

Supplemental zinc increased viability within each genotype, except for the knock

out hepatocytes. The increase in viability of CK, CT, and Tg cells may thus be attributed

directly to zinc induced metallothionein expression. Even though zinc was able to

increase viability of CK, CT, and Tg cells when the genotypes were examined

individually, only the CK cells were more viable than the KO cultures when the

genotypes were compared to one another. Consequently, the presence of metallothionein

and its induction by zinc provided CK hepatocytes protection against oxidative insult,

while ablation of metallothionein in KO hepatocytes was deleterious (Table 4-5).

Contrary to the hypothesis, overexpression of metallothionein in transgenic cultures

provided no increased protection. These results are in agreement with previous findings

from this lab (Davis et al. 2001). Carbon tetrachloride had been used in the past to

examine the role of metallothionein in protection against hepatotoxicity. In those studies,

no combination of metallothionein gene expression (i.e., Tg, CT, CK, KO) and either

adequate or supplemental zinc provided hepatic protection. Even though, the mice had

drastically different levels of liver zinc and liver metallothionein.

Many studies have brought to light the role of metallothionein as an antioxidant.

Metallothionein has demonstrated the ability to quench reactive oxygen and reactive









nitrogen species at an even higher efficiency than the established cellular antioxidant

glutathione (Miura et al. 1997 and Quesada et al. 1996). The extensive network of zinc-

sulfur clusters in metallothionein provide the chemical basis for it's antioxidant capacity

(reviewed in Maret 2000). The reduction potential of the zinc-sulfur clusters are

extremely low (E < -340 mV) and thus provide a means to reduce a variety of cellular

oxidants. Even though zinc bound to metallothionein is extremely stable (pKd = 12.9),

zinc in metallothionein can readily exchange with free zinc in solution (Steinebach et al.

1992). Thus, metallothionein may play a critical role in redox sensing and zinc

distribution in the cell.

In fact, a variety of oxidative and nitrosative stressors have also been shown to

interact with zinc-sulfer clusters of zinc-finger proteins, and lead to concomitant loss of

zinc (Kroncke et al. 2002). One such nitrosative stressor is nitric oxide. NO has the

ability to release zinc from zinc dependant transcription factors, as well as

metallothionein (Schapiro et al. 2003, St. Croix et al. 2002). In vitro studies utilizing a

nitric oxide donor (DEA/ NO) have demonstrated that NO has the ability to liberate zinc

specifically from the 0 domain of metallothionein (Zangger et al 2001). More recent

studies in aortic endothelial cells have shown that iNOS derived NO has the ability to

increase labile zinc through interaction with metallothioenin (Spahl et al. 2003). The

current study utilized SNAP as a model for NO induced stress. The availability of

metallothionein to interact with nitric oxide in CK hepatocytes provided protection that

was not seen in KO hepatocytes. This protection was augmented when zinc increased

metallothionein expression in CK cells, but not in Tg hepatocytes (compared to CT cells).

A possible explanation for protection by the presence and induction of metallothionein by









zinc, but not the over-expression of MT, could be the release of zinc from MT and

subsequent signaling events such as induction of zinc dependent transcription factors or

zinc dependent proteins. In these cells, exposure to NO after zinc pretreatment caused

increased induction of metallothionein mRNA, with the largest increase in Tg

hepatocytes (Lichten et al. unpublished observations). NO mediated release of zinc from

metallothionein may be a primary means of increasing free zinc in the cell.

Consequently, the ratio of thionein (apo-metallothionein) to metallothionein may be one

mechanism with which the cell governs the levels of intracellular zinc (St. Croix et al.

2002). However, when metallothionein expression is altered, a shift in the MT/T ratio

may occur, causing increased production of thionein. Therefore, over-expression of

metallothionein may lead to production of large amounts of thionein that may sequester

zinc before it can be utilized by other apoproteins.

In these experiments, oxidative stress to the hepatocytes was produced by tBH.

The mitochondria are the primary target of this oxidative stressor (Mehendale et al.

1994). It is presumed that tBH causes mitochondrial permeability transition (MPT)

followed by mitochondrial depolarization, uncoupling of oxidative phosphorylation, ATP

depletion, and finally cell death. If metallothionein has the capacity to act as an

antioxidant, then MPT may be inhibited, and cell death (either necrosis or apoptosis) may

be prevented. Once again, the presence of metallothionein was beneficial to CK cultures,

but over expression of metallothionein in Tg cells was not (Table 4-3). However, the

magnitude of viability differences seen in the zinc supplemented CK and KO cultures

were not as great. This may be due to the difference between oxidative and nitrosative

stress towards the zinc-sulfur clusters of metallothionein. Zinc finger proteins have been









shown to react differently towards these stressors (Kroncke et al 2002). NO may be able

to release zinc from zinc-sulfur clusters without destroying them, while oxidative

stressors such as tBH could destroy the clusters. Thus, as with zinc finger proteins, a

reversible exchange of zinc may occur through NO interaction that does not occur when

an oxidant is applied. Therefore, zinc may be released from metallothionein in greater

amounts after interaction with tBH causing decreases in viability not only due to free

radical formation, but in the case of metallothionein transgenics, zinc toxicity.

In summary, the absence of metallothionein in MT KO hepatocytes led to

significant loss of viability after NO or tBH treatment. The presence, and induction of

metallothionein by zinc, increased the viability of CK hepatocytes, but not transgenic

hepatocytes. Therefore, there is a limit to the protection provided by metallothionein,

which directly questions the role of metallothionein as a cellular antioxidant. However,

the primary role of metallothionein may not be to act as an antioxidant, but to be a

transducer of zinc signaling.















APPENDIX
CYTOTOXICITY EXPERIMENTS



--- Tg Zn+
S-A- CT Zn+


SNAP Concentration (mM)
Figure A-1. Differences in CT and Tg viability due to altered MT genotypes and zinc
pretreatment CT versus Tg (right panel) mice were pretreated with 40 tM zinc
for 18-22h, followed by addition 0-4 mM SNAP for 12 h. Viability was
assessed by the crystal violet assay















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BIOGRAPHICAL SKETCH

Louis Lichten was born in Miami, Florida on April 24, 1979. He received his

Bachelor of Science degree from the University of Florida in 2002, and plans to continue

on in the doctorate program for nutritional sciences.