Roles of interleukin 6, zinc, and metallothionein in cytoprotection

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Title:
Roles of interleukin 6, zinc, and metallothionein in cytoprotection
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Schroeder, Joseph J., 1960-
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Thesis (Ph. D.)--University of Florida, 1990.
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Includes bibliographical references (leaves 72-81).
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by Joseph J. Schroeder.
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Full Text










ROLES OF INTERLEUKIN 6, ZINC, AND METALLOTHIONEIN
IN CYTOPROTECTION














By

JOSEPH J. SCHROEDER
7


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

1990

UNIVERSITY OF FLORIDA LIBRARIES






























I dedicate this dissertation to my wife Stephanie in sincere

appreciation of her love, patience, and encouragement.












ACKNOWLEDGEMENTS

I express special thanks to my committee chairman and advisor Dr.

Robert Cousins not only for his support and advice but for providing me

with a dynamic environment in which to work. I am grateful to the

members of my doctoral committee Dr. Jesse Gregory, Dr. David

Richardson, Dr. Gary Rodrick, and Dr. Rachel Shireman, for their

thoughtful criticisms and helpful suggestions. Also, I thank Dr. Claude

McGowan who served on my committee during part of my doctoral study.

I recognize the contributions of my laboratory coworkers Ellen

Barber, Kirsten Huber, Linda Lee-Ambrose, Annette Leinart, and Drs. Lynn

Blalock, Daphne Coppen, Michael Dunn, Arthur Grider, Jim Hempe, and Jim

Hoadley. Without their research developments and discoveries, much of

what I accomplished would not have been possible.

I also extend my gratitude to the U.S.D.A. fellowship committee for

providing me with financial support, to Ann Coutu and Virginia Mauldin

for their secretarial expertise, to Walter Jones for helping to prepare

figures, to William Dougall and Dr. Harry Nick for measuring manganous

superoxide dismutase gene expression, and to Dr. William Buhi for

providing technical assistance regarding 2D gel electrophoresis.

Finally, I extend my sincere appreciation to my parents Joe and

Darlene Schroeder for their unwaivoring love and to my wife Stephanie

for her steadfast devotion.
















TABLE OF CONTENTS


ACKNOWLEDGEMENTS............

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

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

LIST OF ABBREVIATIONS.......

ABSTRACT..................


INTRODUCTION..................................................

REVIEW OF LITERATURE................... ...........................

Zinc...........................................................
Metallothioneins...............................
Hepatocyte-Stimulating Cytokines ..............................
Hepatocyte Model.......................................................

MATERIALS AND METHODS... ........ ................................

Animals .... ................................. ..................
Hepatocyte Isolation and Maintenance...........................
Experimental Designs...........................................
Materials and Analytical Techniques.............................

RESULTS.........................................................

Zinc Deficiency Study.......................................
Acute-Phase Zinc Metabolism Study...............................

DISCUSSION.................................. ......................

SUMMARY AND CONCLUSIONS........................................


APPENDIX..................

REFERENCES..............

BIOGRAPHICAL SKETCH.......


page

........................ iii

........................ v

........................ vi

........................ viii

........................ ix


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

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LIST OF TABLES


TABLE page

1 Zinc concentrations of rat liver and freshly isolated rat
hepatocytes ................................................ 30

2 Effect of medium zinc concentration on leakage of lactate
dehydrogenase activity from rat hepatocytes into culture
medium..................................................... 39

3 Zinc and glucocorticoid-dependence for interleukin 6
stimulation of metallothionein expression and cellular
zinc accumulation in rat hepatocytes........................ 47

4 Mineral composition of culture media and sera................ 68

5 Effects of cytotoxic compounds on cell survival and lipid
peroxidation of rat hepatocytes............................. 71













LIST OF FIGURES


FIGURE page

1 Effect of medium zinc concentration on zinc concentration
in rat hepatocytes..................................... 31

2 Effect of medium zinc concentration on zinc concentration
in rat hepatocytes.................................... 32

3 Effects of BSA and EDTA added to culture medium on zinc
concentration in rat hepatocytes cultured with differing
medium zinc concentrations................................ 34

4 Reduction of s-aminolevulinic acid dehydratase activity
in rat hepatocytes as a function of medium zinc
concentration ............................................. 35

5 Effect of medium zinc concentration on metallothionein
gene expression in rat hepatocytes......................... 37

6 Effect of medium zinc concentration on de novo synthesis
of hepatocyte proteins ........................ ............. 38

7 Zinc efflux from rat hepatocytes as a function of medium
zinc concentration.................. ............ ......... 41

8 Dependence of rat hepatocyte metallothionein mRNA and
metallothionein protein induction on cytokine
concentration ............................................. 42

9 Time course of metallothionein-1 and -2 mRNA and
metallothionein protein induction in rat hepatocytes by
interleukin 6.......................................... 44

10 Northern blot illustrating the effects of combinations of
zinc, dexamethasone, and interleukin 6 on metallothionein
mRNA concentrations in rat hepatocytes................... 46

11 Cytoprotection against iron (II)-nitrilotriacetic acid
and tert-butyl hydroperoxide-induced lipid peroxidation
in rat hepatocytes................ ....... ...... ....... 49









12 Cytoprotective effects of zinc, dexamethasone, and
interleukin 6 against iron (II)-nitrilotriacetic acid and
tert-butyl hydroperoxide-induced lipid peroxidation in
rat hepatocytes................................. ........... 50

13 Cytoprotection against carbon tetrachloride toxicity in
rat hepatocytes .................................. ..... 51

14 Interleukin 1-triggered up-regulation of metallothionein
gene expression and zinc metabolism in hepatocytes.......... 66

15 Northern blot illustrating the effects of combinations of
zinc, dexamethasone, and interleukin 6 on B-actin mRNA
concentrations in rat hepatocytes........................... 69

16 Northern blot illustrating the effects of combinations of
dexamethasone and interleukin 6 on metallothionein and
manganous superoxide dismutase mRNA concentrations in rat
hepatocytes ............................................... 70


FIGURE


page













LIST OF ABBREVIATIONS


ABBREVIATION MEANING

BSA bovine serum albumin
Cd cadmium
Ci Curie
Cu copper
d day
6-ALA-D s-aminolevulinic acid dehydratase
EDTA ethylenediamine tetraacetic acid
FBS fetal bovine serum
g gram
h hour
HSF hepatocyte stimulating factor
IL-la interleukin la
IL-6 interleukin 6
ip intraperitoneal
kg kilogram
LAF lymphocyte activating factor
LDH lactate dehydrogenase
mg milligram
min minute
ml milliliter
MT metallothionein
mol mole
ng nanogram
pl isoelectric point
pmol picomole
rhIL-la recombinant human IL-la
rhIL-6 recombinant human IL-6
sec seconds
SEM standard error of mean
aCi microcurie
9g microgram
<1 microliter
Amole micromole
Zn zinc


viii










Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

ROLES OF INTERLEUKIN 6, ZINC, AND METALLOTHIONEIN
IN CYTOPROTECTION

By

Joseph J. Schroeder

August 1990

Chairman: Dr. Robert J. Cousins
Major Department: Food Science and Human Nutrition

The objective of this research was to utilize hepatocytes as a

cellular model to study zinc deficiency, regulation of metallothionein

synthesis and zinc metabolism by acute-phase mediators, and

cytoprotection by inducers of metallothionein synthesis and zinc

accumulation. Rat hepatocytes were isolated by collagenase perfusion

and cultured in monolayers to address specific research objectives.

Hepatocytes were evaluated as a cellular model of zinc deficiency by

examining the effects of zinc-deficient culture medium containing 1 AM

zinc on a variety of zinc-dependent parameters. Hepatocytes cultured in

zinc-deficient medium maintained cell zinc similar to livers of

zinc-deficient animals even when high levels of the zinc-binding ligands

EDTA and BSA were present in the medium. Similarly, zinc-deficient

medium did not affect metallothionein, metallothionein mRNA, de novo

protein synthesis, or apparent membrane integrity but reduced activity

of the zinc-metalloenzyme a-aminolevulinic acid dehydratase (8-ALA-D).

In comparison, medium containing 16 or 48 AM zinc restored 6-ALA-D







activity and increased cell zinc and metallothionein expression. Since

they maintained cell zinc despite a partial loss of 6-ALA-D activity,

hepatocyte monolayers may be a good model to study soft tissue zinc

deficiency. Next, hepatocytes were utilized to evaluate the abilities

of cytokines to regulate metallothionein expression and zinc metabolism.

Interleukin la was examined because it increases hepatic metallothionein

expression when administered in vivo and interleukin 6 was studied

because it induces synthesis of other hepatic acute-phase proteins.

Interleukin 6 produced concentration- and time-dependent increases in

both metallothionein protein and mRNA while interleukin la had no

effect. Interleukin 6 also increased cellular zinc concentrations.

Each interleukin 6 effect required the glucocorticoid hormone

dexamethasone and was optimized by added zinc. Therefore, at the level

of the hepatocyte, interleukin 6 rather than interleukin la is a major

mediator of metallothionein expression and zinc metabolism. To

determine whether metallothionein induction and zinc accumulation could

provide cytoprotection, hepatocytes were pre-treated with combinations

of zinc, dexamethasone, and interleukin 6 to induce metallothionein and

then were exposed to cytotoxic compounds. Carbon tetrachloride-induced

cell death and lactate dehydrogenase leakage were reduced in hepatocytes

in which metallothionein and zinc accumulation were previously induced.

Thus, interleukin 6 provided cytoprotection via a mode consistent with

dependence upon increased cellular metallothionein and/or zinc.












INTRODUCTION

Metallothionein is a cysteine-rich, metal-binding protein that is

intimately involved in the metabolism of zinc. Not only does zinc bind

to and stabilize apometallothionein (Dunn et al., 1987), but dietary

zinc also transcriptionally regulates metallothionein expression

(Blalock et al., 1988). In addition, metallothionein synthesis is

induced by glucocorticoids and stress-related mediators with interleukin

1-like activity (Cousins, 1986). Metallothionein induction triggered by

interleukin 1 causes a transient depression of zinc in the plasma and

concomitant uptake of zinc by the liver, bone marrow, and thymus

(Cousins and Leinart, 1988; Huber and Cousins, 1988). The mechanisms by

which these changes are regulated at the cellular level are not known.

Based on metallothionein's purported ability to scavenge hydroxyl

radicals (Thornalley and Vasak, 1985) and zinc's stabilizing effect on

cell membranes (Bettger and O'Dell, 1981; Girotti et al., 1986),

cytoprotective functions for metallothionein and zinc have been

postulated (Thomas et al., 1987; Coppen et al., 1988; Abel and Ruiter,

1989). There is a need to better understand these functions.

In the present study, monolayer cultures of adult rat hepatocytes

were used as a cellular model to examine zinc deficiency, regulation of

metallothionein expression and zinc metabolism by the cytokines

interleukin 1 and interleukin 6, and cytoprotection by inducers of

metallothionein synthesis.












REVIEW OF LITERATURE

Zinc

Essentiality

Although Raulin first recognized the necessity of zinc for growth

of microorganisms in 1869 (Raulin, 1869), the essentiality of zinc in

animals was not demonstrated until 1934 by Todd and coworkers (1934).

They observed that rats fed zinc deficient but otherwise nutritionally

complete diets grew poorly and lost body hair. Later Tucker and Salmon

(1955) demonstrated zinc deficiency in pigs and O'Dell and colleagues

(1958) showed zinc deficiency in chickens. Zinc deficiency in humans

was first documented in the early 1960s by Prasad and coworkers in the

Middle East (Prasad et al., 1961; 1963).

Human zinc deficiency is most common in developing countries in

which cereal and leguminous foods make up the bulk of the staple diet

(Solomons and Cousins, 1984). The lower availability of zinc from these

foods as compared to meats has been attributed to the presence of

inhibitors of zinc absorption such as phytate (Reinhold, 1971; Reinhold

et al., 1973) and fiber (Pecoud et al., 1975). The possibility of zinc

deficiency in developed countries was emphasized by Hambidge and

coworkers (1972). They found that low zinc concentration in the hair of

school children living in Denver, Colorado, correlated with poor growth

and anorexia.







3

Symptoms of nutritional zinc deficiency include anorexia, growth

retardation, alopecia, dermatitis, reproductive dysfunction, skeletal

defects, mental lethargy, hypogeusia, and impaired immunocompetence

(Prasad et al., 1961; 1963; Mahajan et al., 1980; Fraker et al., 1982).

These symptoms have been reviewed in detail (Hambidge et al., 1986).

The biochemical bases for the symptoms of nutritional zinc

deficiency are not understood. The most notable biochemical change in

zinc deficient animals is decreased plasma zinc concentration. Some

deficiency symptoms are probably the result of altered enzyme activity

since zinc functions as a component of many zinc-metalloenzymes (Vallee

and Galdes, 1984). However, other functions of zinc are probably also

involved since the zinc concentrations of most soft tissues and the

activities of many zinc-metalloenzymes are not significantly altered,

even in severely zinc deficient animals (Kirchgessner et al., 1976). In

addition, many symptoms and physiological changes associated with

nutritional zinc deficiency are manifested more rapidly than changes in

zinc metalloenzyme activities (Bettger and O'Dell, 1981). These facts

stress the importance of further research regarding the cellular

metabolism of zinc.

Chemistry

Zinc is a first series transition metal. All transition elements

of the first series have an inner argon core surrounded by varying

numbers of valence electrons located in the 4s and 3d subshells. Zinc

is located at the end of the series with the electronic configuration

[Ar]4s23d1.







4

Since valence 3d electrons are of nearly equal energy and because

they are readily removed, most first series transition metals normally

exist in nature in more than one oxidation state (Phillips and Williams,

1966). However because of its d10 configuration, zinc prevails in the

+2 oxidation state. The Zn21 ion does not share many of the common

characteristics of most transition elements (Douglas et al., 1983). For

example, Zn2' is diamagnetic rather than paramagnetic because all of its

electrons are paired. Also Zn2" will not form colored compounds since

all of its subshells are filled and light can not shift an electron from

one energy level to another.

One characteristic that zinc shares with other transition elements

is the ability to bind ligands noncovalently to form coordination

complexes. This property is biologically important because ligands

dictate the absorbability of zinc, facilitate the transport and storage

of zinc, and impart to zinc the ability to perform vital biochemical and

physiological functions. The arrangement of ligands about a zinc

nucleus depends on the ligands involved. The most common arrangements

are the 4-coordinate tetrahedral and 6-coordinate octahedral geometries

(Nebergall et al., 1976).

The affinity of a ligand for a metal ion such as Zn2+ is most

easily understood when considered in terms of an acid-base interaction.

Due to its high charge density, Zn2 can act as a Lewis acid to withdraw

electrons from electron-rich functional groups of ligands.

Physiologically ubiquitous ligands which bind zinc tightly include

aspartate, glutamate, lysine, histidine, tyrosine, cysteine, arginine,

and N-terminal amino acids (Hughes, 1984). These amino acids contain







5

nitrogen, sulfur, and oxygen which are good Lewis bases (Basolo and

Pearson, 1967).

When several different ligands are present, competitive equilibria

result and a variety of zinc complexes are formed. The formation of

zinc complexes is also affected by a number of physiochemical factors

which are beyond the scope of this review. A combination of all of

these factors forms the basis for the biochemical and physiological

functions of zinc.

Functions

Zinc metalloenzvmes

As noted, zinc is a component of many metalloenzymes (Vallee and

Galdes, 1984). Carbonic anhydrase was the first zinc metalloenzyme to

be identified (Keilin and Mann, 1940). Today the list of known zinc

metalloenzymes contains members of all six enzyme classes and includes

RNA nucleotidyl transferases, alcohol dehydrogenase, alkaline

phosphatase, and a-aminolevulinic acid dehydratase (Vallee and Galdes,

1984). In these enzymes zinc provides structural integrity and/or

participates in catalysis. Zinc's involvement in a wide variety of

metabolic pathways via zinc metalloenzymes probably accounts for some of

the physiological symptoms associated with nutritional zinc deficiency.

Zinc fingers

Recently a novel DNA-binding motif was discovered by investigators

who were studying the amino acid sequence of Xenopus transcription

factor IIIA (Miller et al., 1985). The researchers recognized small

units repeated in tandem and subsequently proposed that each unit folds

around a Zn2+ ion to form individual structural domains. This motif,







6

referred to as a "zinc finger," is ubiquitous since other investigators

have found similar units in the amino acid sequences of other

transcription factors and viral proteins (Sunderman and Barber, 1988).

Several subclasses of zinc fingers have now been identified

(Sunderman and Barber, 1988). The classical arrangement of

transcription factor IIIA has a Zn2* ion coordinated to a doublet of

cysteine and a doublet of histidine in a tetrahedral arrangement. This

configuration has inner ligands separated by 12 or 13 amino acids. In

addition, at least two other subclasses have been found. The most

common features a sequence with the same spacing but contains two

doublets of cystiene residues. Less frequently a histidine doublet

separated by four or five amino acids from a cysteine doublet is

observed.

Zinc finger motifs may impart strength and specificity of

DNA-binding to regulatory proteins (Sunderman and Barber, 1988).

Specificity could be accomplished by varying the number of amino acids

in each domain, the number of amino acids between domains, and the

number of zinc fingers themselves. Strength of binding could be

modulated by varying the amino acid sequence of each domain.

Presently, the impact of nutritional zinc deficiency on zinc

fingers can only be hypothesized. Since zinc appears to play a

structural role in these transcription factors, improper folding may

occur in the absence of zinc. As a result, the sensitivity of gene

expression to hormones like glucocorticoids (Freedman et al., 1988),

would be reduced in zinc deficiency and cell dysfunction and concomitant

pathology could develop.








Cytoprotective role

A rapidly growing line of research suggests that in addition to its

role as a component of zinc metalloenzymes and zinc fingers, zinc also

exerts an essential cytoprotective role by stabilizing membrane

structure (Bettger and O'Dell, 1981). This role for zinc is supported

by the observation that lipid peroxidation is elevated in some tissues

of zinc deficient animals (Sullivan et al., 1980). Such a role could

explain the development of skin disorders as well as other symptoms

characteristic of zinc deficiency.

Bettger and O'Dell (1981) have suggested that this cytoprotective

function is the result of a direct effect of zinc on the cell membrane.

This hypothesis is based, in part, on studies which used erythrocytes

(Bettger et al., 1978; Bettger and Taylor, 1986; Chvapil et al., 1974).

Erythrocytes from zinc deficient rats exhibited increased osmotic

fragility compared to controls while erythrocytes from zinc adequate

rats exhibited decreased osmotic fragility when zinc was added. The

increased osmotic fragility of the erythrocytes from zinc deficient rats

was postulated to be a function of zinc concentration since the

membranes contained significantly less zinc than controls.

The hypothesis that zinc exerts a direct cytoprotective effect on

cell membranes was supported by Girotti and coworkers (1986). They

showed that the addition of Zn2 to erythrocyte ghosts concurrently with

xanthine, xanthine oxidase, and Fe3' significantly decreased H202 and 02'

dependent lipid peroxidation compared to controls which did not receive

added Zn2. Since xanthine oxidase activity was not affected, the







8

investigators concluded that Zn2" uptake by the membranes was

responsible for the cytoprotective effect.

Ludwig and Chvapil (1982) suggested that zinc may also stabilize

membranes indirectly. They hypothesized that zinc inhibits carbon

tetrachloride toxicity by stabilizing NADPH which, in turn, can inhibit

the microsomal drug oxidizing system which metabolizes carbon

tetrachloride to harmful CC13" radicals. Through propagation reactions

with other molecules, CC13' radicals can generate other radicals which

may cause membrane damage and cell death via peroxidation of

polyunsaturated fatty acids. Alternatively, an indirect cytoprotective

effect of zinc could potentially be mediated via the synthesis of the

zinc-binding protein metallothionein (Thornalley and Vasak, 1985).

Metallothioneins

The term metallothionein actually refers to a family of proteins

found ubiquitously in eukaryotic species. A unique property of

metallothioneins is that they bind stoichiometric quantities of heavy

metals such as cadmium, copper, and zinc. This property has led

investigators to hypothesize roles for metallothionein in regulating

heavy metal detoxification and essential metal homeostasis. Detailed

reviews of metallothioneins are available (Kagi and Nordberg, 1979;

Hamer, 1986; Dunn et al., 1987).

Physiochemistry

All metallothioneins are single-chain polypeptides of 60 to 61

amino acids with N-acetylmethionine and alanine at the amino and

carboxyl termini, respectively (Kagi and Nordberg, 1979). The primary

structure of metallothioneins is characterized by a high content of






9

cysteine residues ranging from 23 to 33 percent with no disulfide bonds,

histidines, or aromatic amino acids (Dunn et al., 1987). The

distribution of cysteinyl residues along the peptide chain is highly

conserved due to their involvement in binding metals.

Another distinctive characteristic of metallothioneins is their low

molecular weight (Dunn et al., 1987). Sequence data of mammalian

metallothioneins indicate a molecular weight for the native protein of

about 6000. Depending upon the metal composition, the actual molecular

weight can range from 6500 to 7000. X-ray crystallography has shown

that metallothioneins have an ellipsoid shape (Furey et al., 1986).

This asymmetry explains the apparent molecular weight of 10,000 usually

determined by gel filtration chromatography.

Metallothioneins contain two globular domains (Furey et al., 1986).

The domain within the carboxyl terminal end from amino acid 31 through

61 is referred to as "a" while the domain that spans the amino terminal

end from residue 1 through 30 is called "B". The "B" domain contains

nine cysteine residues and binds either three Zn2" or Cd2+ ions, or six

Cu1' ions (Nielson et al., 1985). The "a" domain contains eleven

cysteine residues and binds either four Zn2+ or Cd2+ ions, or five Cu'"

ions. The relative affinities of metallothioneins for these metals are

Cu''>Cd2+>Zn2, however Zn2+ is the most physiologically ubiquitous

(Hamer, 1986). Each Zn21 ion is bound to metallothioneins by four

cysteine thiolate ligands in a tetrahedral coordination complex (Hamer,

1986).

Metal binding reduces metallothionein degradation and increases

stability of the protein (Dunn et al., 1987). Thus the intracellular







10

concentration of metallothioneins is dependent upon zinc. When dietary

zinc is extremely low, intracellular levels of zinc are reduced and

metallothioneins in some tissues are degraded (Cousins, 1985).

The relative rates of turnover of metallothioneins with differing

metal compositions are not well understood. Zn-metallothioneins and

Cd-metallothioneins are resistant to degradation by selected cytosolic

proteases but susceptible to lysosomal breakdown (Feldman et al., 1978).

In contrast, Cu-metallothioneins are resistant to both cytosolic and

lysosomal degradation (Held and Hoekstra, 1979). A better understanding

of these mechanisms may provide insight into the functions of

metallothioneins.

Regulation by Metals

Metals that bind to metallothioneins also transcriptionally

activate metallothionein genes when the metals reach threshold levels

within cells. This has been demonstrated by administering metal salts

to intact animals by injection (Feldman et al., 1978) or feeding

(Blalock et al., 1988), or to cultured cells via the culture medium

(Failla and Cousins, 1978a). The relative abilities of metals to induce

metallothioneins are Cd2*>Zn2>Cul'.

Metals induce metallothionein expression by interacting with metal

regulatory elements in promoter regions located upstream from the

metallothionein structural genes (Hamer, 1986). The mechanism of this

interaction may involve metal binding to a nuclear regulatory protein

which in turn binds to the DNA sequence of the metal regulatory element

(Cousins et al., 1988).









Regulation by Hormones

Metallothionein synthesis is also up-regulated by hormones

associated with acute trauma including glucocorticoids, glucagon,

epinephrine, dibutyryl cAMP, endotoxins, and interleukin 1 (Cousins et

al., 1986). Interleukin 1 produces a tissue-specific redistribution of

zinc with a transient depression of zinc in the plasma and concomitant

uptake of zinc by the liver, bone marrow, and thymus (Cousins and

Leinart, 1988). Models constructed via the SAAM and CONSAM modeling

programs mathematically describe the relationship between these

hormonally-induced changes and metallothionein induction (Dunn and

Cousins, 1989). Thus, redistribution of body zinc stores by tissue-

specific hormonal regulation of metallothionein synthesis appears to be

an important part of the body's acute response to stress.

Several mediators of metallothionein synthesis probably act

directly at the level of the hepatocyte. A putative regulatory element

for glucocorticoid hormones in the promoter region of metallothionein

genes has been identified (Hamer, 1986). In addition, incubation of

hepatocytes with glucocorticoid hormones increases both metallothionein

concentration and zinc uptake (Failla and Cousins, 1978a; 1978b).

Glucagon and epinephrine also increase metallothionein concentration and

zinc uptake in cultured hepatocytes (Cousins and Coppen, 1987). The

effects of these polypeptide hormones may be mediated intracellularly

via cAMP since metallothionein genes contain potential cAMP regulatory

elements (Nebes et al., 1988). Also some studies suggest cAMP induces

metallothionein expression in cultured cells (Cousins and Coppen, 1987;







12

Nebes et al., 1988) however others indicate cAMP has no effect (Nebes et

al., 1990).

Endotoxin does not have a direct effect on metallothionein

induction in primary hepatocytes. Instead, the effect of endotoxin may

be mediated in some way via the cytokine interleukin 1. Interleukin 1

is a low molecular weight protein that is released from activated

macrophages, monocytes, and other cell-types in acute response to

infection as well as other trauma such as tissue injury and stress

(Dinarello, 1988). Once released interleukin 1 triggers the

up-regulation of a broad spectrum of systemic acute-phase responses

involved in host defense.

The mechanism by which interleukin 1 regulates metallothionein gene

expression and zinc metabolism in hepatocytes is not clear. A direct

action at the cellular level could involve multiple modes of signal

transduction (Dunn and Cousins, 1989; Nebes et al., 1988; Imbra and

Karen, 1987). In addition, interleukin 1 stimulates glucocorticoid

release via its corticotropin-releasing activity on pituitary cells

(Woloski et al., 1985). Therefore, interleukin 1 could affect

metallothionein synthesis, in part, via glucocorticoid hormones.

Other cytokines such as interleukin 6 (also referred to as

interferon B2 and hepatocyte stimulating factor), could mediate

interleukin 1 effects at the level of the hepatocyte. Like interleukin

1, interleukin 6 is a low molecular weight cytokine which is released

from a variety of cell-types including monocytes (Dinarello, 1988).

Interleukin 1 stimulates interleukin 6 synthesis in some cells (Van

Damme et al., 1987; Walther et al., 1988; Zhang et al., 1988).






13

Furthermore, interleukin 6 regulates the synthesis and secretion of a

variety of acute-phase hepatic proteins (Andus et al., 1988) and

synergizes with interleukin 3 to increase proliferation of hematopoietic

progenitor cells (Ikebuchi et al., 1987).

Functions

Homeostatic roles

A variety of homeostatic roles have been proposed for

metallothioneins. Metallothioneins may regulate zinc homeostasis by

functioning as a mucosal block to zinc absorption (Cousins, 1979)

similar to the original regulatory role proposed for ferritin in iron

absorption (Granick, 1946). Metallothioneins may also function in

ligand-exchange reactions as zinc donors for activation of

metalloenzymes and "zinc finger" motifs of DNA-binding transcription

factors (Otvos et al., 1989; Cousins and Hempe, 1990). Metallothioneins

could mediate zinc's effects on cellular processes analogous to the way

calmodulin mediates calcium's effects (Cheung, 1982).

Cytoprotective roles

Several cytoprotective roles for metallothioneins have been

proposed. First, because they are induced by and form complexes with

metals such as cadmium and copper, metallothioneins provide protection

from heavy metal toxicity (Hamer, 1986). The success of oral zinc

therapy in preventing copper toxicity in Wilson's disease patients

probably is due to sequestration of hepatic copper in metallothionein

induced by zinc (Lee et al., 1989). Studies utilizing cells transfected

with metallothionein genes indicate metallothioneins also provide

cytoprotection against alkylating agents (Kelley et al., 1988). In







14

addition, in vitro data suggest the zinc-thiolate clusters in

metallothioneins efficiently scavenge hydroxyl radicals (Thornalley and

Vasak, 1985). Therefore, metallothioneins may also function in the free

radical defense system as hydroxyl radical-scavengers.

Since tissue metallothionein concentrations are dependent upon

levels of dietary zinc (Blalock et al., 1988), the hydroxyl radical-

scavenger function can be placed within the context of zinc deficiency.

In animals with adequate zinc status, the basal level of metallothionein

expression may be sufficient to scavenge hydroxyl radicals and prevent

lipid peroxidation. However, in zinc deficient animals, reduced levels

of metallothioneins could account for increased lipid peroxidation

observed in some tissues (Sullivan et al., 1980).

The hydroxyl radical-scavenger role of metallothioneins can also be

placed within the context of events which occur during the body's

response to infection (Karin, 1985; Cousins, 1985). Following invasion

by bacteria or viruses, interleukin 1 is released from a variety of

cell-types (Dinarello, 1988). Interleukin 1 activates neutrophils and

macrophages leading to the release of large quantities of active oxygen

species including superoxide and hydroxyl radicals. These compounds

function to destroy the invading bacteria or virus. In the absence of

adequate cytoprotective mechanisms, these compounds could cause severe

damage to the host. Induction of superoxide dismutase by cytokines

provides one form of cytoprotection for cells (Wong and Goeddel, 1988).

Superoxide dismutase deactivates superoxide radicals. Induction of

metallothioneins triggered by interleukin 1 with concomitant movement of







15

zinc out of the plasma and into the liver and other specific tissues may

provide additional cytoprotection by deactivating hydroxyl radicals.

Recent support for the hydroxyl radical-scavenger hypothesis was

provided by a study that assessed the protective effect of zinc against

carbon tetrachloride hepatotoxicity in rats (Clarke and Lui, 1986).

Zinc (10 mg/kg) or saline was administered by intraperitoneal injection

24 and 2 hours before carbon tetrachloride administration (1 ml/kg, ip).

The rats were sacrificed four hours later and liver toxicity was

assessed by a variety of indices including serum aspartate

aminotransferase activity. Zinc afforded limited protection prior to

induction of metallothionein synthesis. Further, chromatographic study

of hepatic cytosols showed that metallothionein-bound zinc was

selectively depleted by carbon tetrachloride exposure.

Additional support for the radical-scavenger hypothesis was

provided by studies in which primary cultures of rat hepatocytes were

used to test the influence of zinc on free radical formation and lipid

peroxidation (Coppen et al., 1988). Peroxidation was induced in

cultures incubated with 1, 16, 24, 32, or 48 gM zinc by either

3-methylindole, tert-butyl hydroperoxide, or iron (II)-nitrilotriacetic

acid. Free radical formation was assessed by examination of electron

spin resonance spectra of appropriate spin trapped adducts and lipid

peroxidation was determined based on malondialdehyde production.

Results indicated that free radical formation and lipid peroxidation

were significantly reduced in cultures incubated with increasing amounts

of zinc where high levels of metallothionein were produced.







16

At least one inconsistency suggests the hydroxyl radical-scavenger

hypothesis is not valid. The bimolecular rate constant reported for

reaction of metallothionein with hydroxyl radicals (Thornalley and

Vasak, 1985) is one-to-two orders of magnitude greater than the

constant's diffusion-controlled, theoretical limit (Cantor and Schimmel,

1980). Therefore the bimolecular rate constant is probably incorrect.

Despite this inconsistency, the biological evidence is consistent with a

role of metallothionein in cytoprotection.

Hepatocyte-Stimulating Cytokines

The body responds to tissue injury, stress, and infection with a

series of local and systemic acute-phase reactions which arrest the

injury process, protect against further injury, and initiate repair

processes (Gauldie et al., 1989). Local responses include release of

arachidonate metabolites and vasoactive amines that alter vascular

permeability. Systemic responses include activation of phagocytic

cells, continued generation of arachidonate metabolites, and release of

many hormone-like polypeptides known as cytokines from various cell-

types. Cytokines stimulate proliferation of T and B lymphocytes,

promote development of cytotoxic T cells and antibody-producing plasma

cells, and activate macrophages and other inflammatory cells (Mizel,

1989). Cytokines also initiate changes in intermediary metabolism

including anorexia, fever, gluconeogenesis, glucose oxidation, decreased

fatty acid uptake by adipocytes, and increased hepatic synthesis of

fatty acids and acute-phase proteins (Klasing, 1988). Most hepatic

acute-phase proteins function as either antiproteinases, opsonins,

blood-clotting or wound-healing factors, or metal-binding ligands







17

(Gauldie et al., 1989). The principal hepatocyte-stimulating cytokines

responsible for inducing acute-phase protein synthesis are interleukin

1, tumor necrosis factor a, and interleukin 6.

Interleukin 1 and Tumor Necrosis Factor a

Interleukin 1 refers to a family of proteins released from

macrophages, monocytes, and other cell-types following stimulation by

phagocytosis or by immune complexes (Mizel, 1989). Two forms,

interleukin la and interleukin 1B, have been characterized in humans.

Each is the product of a different gene and is synthesized as a 33,000

molecular weight precursor which is processed to a 13,000-17,000

molecular weight mature form (Giri et al., 1985). The a and B forms of

interleukin 1 are most easily distinguished physiochemically by their

isoelectric points which are 5.0 and 7.0, respectively (Mizel, 1989).

Interleukin la and interleukin 1B bind the same receptor and have

the same potencies in bioassays but are differentially secreted

(Dinarello, 1988). Interleukin la may regulate autocrine events since

most of it remains in the cell or associated with the cell membrane. In

comparison, much of interleukin 1B is secreted resulting in circulating

levels as high as ten times that of interleukin la (Klasing, 1988).

Therefore interleukin 1B may regulate systemic events.

The physiochemical properties of tumor necrosis factor a (TNFa) are

similar to those of interleukin 1. In humans, TNFa is synthesized by

macrophages and monocytes as a 233 amino acid precursor and is processed

to a 17,000 molecular weight mature form with an isoelectric point of

5.3 (Le and Vilcek, 1987; Beutler and Cerami, 1986). Despite these

similarities, there is no significant homology between the amino acid







18

sequences of TNFa and either interleukin la or interleukin 18 and TNFa

binds to a different receptor (Klasing, 1988).

Initially, investigators believed that interleukin 1 and TNFa

regulated hepatic acute-phase protein synthesis directly since nearly

the entire acute-phase response could be mimicked in vivo by

administration of either cytokine (Dinarello and Mier, 1987). However,

recombinant forms of these cytokines elicit only partial responses in

cultured liver cells stimulating synthesis of a,-acid glycoprotein,

hepatoglobulin, and C3 complement in rat hepatoma cells (Baumann et al.,

1987) and suppressing synthesis of albumin in rat hepatocytes (Koj et

al., 1987). Hence, interleukin 1 and TNFa probably regulate other

acute-phase proteins indirectly by stimulating synthesis and release of

another cytokine such as interleukin 6.

Interleukin 6

Interleukin 6 synthesis is induced in macrophages, monocytes,

fibroblasts, and endothelial cells by interleukin 1, TNFa, and other

stimuli (Zhang et al., 1988). Depending upon the cellular source and

degree of glycosylation, the molecular weight of interleukin 6 often

ranges from 18,000 to 32,000 but molecular weights as high as 70,000

have been reported (Fuller and Grenett, 1989). The larger species may

represent precursor forms of interleukin 6. Like interleukin 1 and

TNFa, interleukin 6 exerts its biological effects'by interacting with a

specific saturable receptor.

In contrast to interleukin 1 and TNFa, interleukin 6 regulates a

broad spectrum of acute-phase proteins in both rat and human hepatocyte

cultures. For human hepatocytes, these proteins include serum amyloid







19

A, C-reactive protein, hepatoglobulin, a,-antichymotrypsin, fibrinogen,

a,-antitrypsin, and al-acid glycoprotein, as well as the negative acute-
phase proteins fibronectin, transferring, and albumin (Castell et al.,

1989). Therefore, interleukin 6 is the primary hepatocyte-stimulating

cytokine regulating acute-phase protein synthesis. Since interleukin 1

and TNFa also regulate a subset of acute-phase proteins and because they

can synergize or antagonize the effects of interleukin 6, these

cytokines probably function as accessory modulators which allow the

organism to achieve an effective homeostatic response to the specific

tissue injury, stress, or infection that has been encountered.

Hepatocyte Model

The experimental conditions of cell culture can be more closely

controlled than conditions of experiments using intact animals (Dagani,

1984). As a result, a variety of cell-types have been utilized to

study both zinc deficiency (Flynn and Yen, 1981; Flynn, 1984; Flynn,

1985; Bettger and Taylor, 1986; O'Dell et al., 1987; Falchuk et al.,

1975) and acute-phase protein synthesis (Baumann et al., 1987; Koj et

al., 1987; Castell et al, 1989).

Zinc Deficiency

Nonproliferating primary hepatocytes, cultured in zinc deficient

medium, provide a novel system to study zinc deficiency at the cellular

level. Kinetic studies of zinc uptake and exchange have shown that

cultured hepatocytes have an average zinc t, of 15 hours (Pattison and

Cousins, 1986). This suggests that turnover of zinc bound to some

cellular ligands is extremely rapid. Also, Guzelian and coworkers

(1982) reported that hepatocytes lose up to 70 percent of their zinc and






20

greater than 90 percent of the activity of the zinc metalloenzyme,

a-aminolevulinic acid dehydratase within 24 hours of culture in zinc

deficient medium. These changes parallel those observed in some animal

studies in which the hepatic zinc concentration and s-aminolevulinic

acid dehydratase activity were reduced during zinc deficiency (Faraji

and Swendseid, 1983). The changes differ from the results of other

studies which show that hepatic zinc levels are unaffected by zinc

deficiency (Taylor et al., 1988; Murthy et al., 1974; O'Dell et al.,

1976). Nevertheless, loss of zinc from specific intracellular pools

within hepatocytes could contribute to physiological manifestations of

zinc deficiency. The first objective of the present study was to use

hepatocytes as a cellular model to study zinc deficiency.

Acute-Phase Zinc Metabolism

Hepatocytes also provide a cellular model to study acute-phase

zinc metabolism. Glucocorticoids stimulate metallothionein synthesis

and zinc uptake in cultured hepatocytes (Failla and Cousins, 1978a;

1978b). In addition, hepatocytes are the major source of other acute-

phase proteins. Consequently, these cells have been used to examine the

effects of the cytokine mediators, interleukin 1, tumor necrosis factor

a, and interleukin 6 on acute-phase protein synthesis (Castell et al.,

1989). The second objective of this study was to utilize hepatocytes as

a cellular model to study the regulation of metallothionein synthesis

and zinc accumulation by acute-phase mediators. Also cytoprotection by

inducers of metallothionein synthesis and zinc accumulation was

examined.












MATERIALS AND METHODS
Animals

Male rats (Sprague-Dawley strain; University of Florida Breeding

Facility) weighing 150-250g were housed in stainless-steel, suspended

cages in a room with a 12 h light-dark cycle (0700 to 1900 h and 1900 to

0700 h, respectively). Rats were fed a standard commercial diet (Purina

rat chow, Ralston Purina, St. Louis, MO) and distilled water ad libitum.
Isolation and Maintenance of Rat Hepatocvtes

Rats were anesthetized with sodium pentabarbital (65 ag/kg; ip) and

hepatocytes were isolated between 0900 and 1100 h by collagenase

perfusion (Failla and Cousins, 1978b). Viability of isolated

hepatocytes was greater than 85% as judged by trypan blue exclusion.
The fresh hepatocytes were suspended (2.5 x 106 cells/3 ml) in

attachment medium which was 10% fetal bovine serum (FBS) and 90%

modified Waymouth's MB 752/1 medium (pH 7.4) that contained 15 mM HEPES

(N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid), 10 mM TES

(N-tris hydroxymethyll) methyl-2-aminoethane sulfonic acid), 30 mM

NaHCO3, 0.5 mM sodium pyruvate, streptomycin sulfate (0.1 mg/ml),

penicillin G (100 units/ml), gentamycin sulfate (50 ag/ml), alanine

(0.41 ;M), serine (0.53 aM), and insulin (1 ag/ml). Aliquots (3 ml)

were inoculated into 60-mm collagen-coated culture dishes and viable

parenchymal cells were allowed to attach selectively over a 3-h period

at 370C.






22

Following selective attachment, the medium was removed, the cells

were washed with a 3 ml aliquot of cell wash buffer (10 mM HEPES, 40 mM

NaCl, 7 mM KC1, 1 mg/ml glucose, pH 7.4), and the cells were cultured

with fresh, modified Waymouth's MB 752/1 medium supplemented with bovine

serum albumin (BSA, 2 mg/ml) and appropriate treatments. For

experimental culture periods lasting longer than 24 h, culture media

were renewed every 24 h.
Experimental Designs

Zinc Deficiency Study

For zinc deficiency experiments, zinc was extracted from FBS used

in the attachment medium so that the zinc concentration of the

attachment medium was approximately 1 gM (Table 4). Following

attachment, hepatocytes were cultured with BSA-supplemented media

containing 1 (the endogenous zinc concentration of BSA-supplemented

medium), 16, or 48 gM zinc. These zinc concentrations were chosen to

simulate zinc deficient, normal, and repleted plasma, respectively.

Supplemental levels of zinc were attained by adding zinc sulfate to the

media. After various culture periods, hepatocytes were harvested

(Pattison and Cousins, 1986) and appropriate measurements were made.

Acute-Phase Zinc Metabolism Study

For experiments utilizing hepatocytes as a model of acute-phase

zinc metabolism, cells were maintained after the attachment period in

BSA-supplemented medium for an additional 21 h before medium containing

hormones was added. The zinc concentration of the medium was increased

from 1 AM to either 16 or 48 gM by adding zinc sulfate. After various

culture periods, appropriate determinations were made.






23

Materials and Analytical Techniques

Extraction of Zinc from Sera and Media

Zinc was extracted from FBS used in the attachment media for zinc

deficiency studies via a batch procedure utilizing Chelex-100 resin

(Bio-Rad Laboratories, Richmond, CA). Chelex-100 was prepared as

described previously (Flynn, 1985) and applied to FBS in a ratio of 1:4

(w/v). After mixing briefly, the resin was removed by centrifugation

(5000 x g; 10 min), the pH of the FBS was adjusted to 7.4 with NaOH, and

the FBS was sterilized by filtration (0.45 um Millipore filter). The

zinc content of the FBS was then measured by atomic absorption

spectrophotometry (Table 4). A similar extraction procedure was

utilized to reduce the basal zinc concentration of BSA-supplemented

Waymouth's MB 752/1 medium (Table 4) used in one experiment (Figure 2).

Cytokine Preparations

Recombinant human interleukin la was provided by Hoffmann-La Roche

(Nutley, NJ). Specific activity was 2x107 lymphocyte-activating factor

(LAF) units (109 D10 units)/mg of protein. Recombinant human

interleukin 6 was provided by Genetics Institute (Cambridge, MA).

Specific activity was 106 hepatocyte-stimulating factor (HSF) units

(7x106 CESS units)/mg of protein.

Measurements

Cytoprotection

Cytoprotection measurements were made using hepatocytes previously

treated with zinc and/or hormones. Media were removed and hepatocytes

were cultured with BSA-supplemented medium containing iron (II)-

nitrilotriacetic acid, tert-butyl hydroperoxide, or carbon







24

tetrachloride. After appropriate culture periods, relevant measurements

were made including malondialdehyde, lactate dehydrogenase leakage, and

cell survival (ie. cell protein/dish).

Zinc

For each cellular zinc measurement, hepatocytes from 1-2 dishes

were washed twice with EDTA buffer (10 mM HEPES, 150 mM NaCl, 10 mM

EDTA, pH 7.4) prior to two washes with cell wash buffer. Then

hepatocytes were digested with 0.2% sodium dodecylsulfate in 0.2 N NaOH

and zinc was measured by atomic absorption spectrophotometry using a

set of zinc standards ranging in concentration from 0.1 to 1.0 ppm.

Media and sera zinc concentrations were measured directly by atomic

absorption spectrophotometry using similar zinc standards.

Zinc efflux

For zinc efflux measurements, hepatocyte zinc pools were pre-

labelled by including 65Zn2 (300 nCi/dish) in the attachment medium.

After attachment, hepatocytes were washed once with EDTA buffer and once

with cell wash buffer. Then BSA-supplemented medium containing

appropriate zinc treatments was added. Hepatocytes used for zinc efflux

measurements were harvested similarly to those used for total cellular

zinc measurements and 65Zn2 was measured by liquid scintillation

(Beckman LS 7500).

De Novo synthesis of hepatocyte proteins

Hepatocytes previously treated with various levels of zinc were

cultured with methionine-free medium containing the same levels of zinc

and 50 gCi of 35S-methionine per dish to examine de novo protein

synthesis. After 3 h, hepatocytes from 1 dish were harvested in 500 pl







25

of solubilization solution (9.3 M urea, 5 mM K2CO0) and sonicated. Then

25 gl of 4 mM phenylmethylsulfonyl fluoride, 10 j1 of Nonidet P-40, and

50 1l of 0.3 M dithiothreitol were added and particulate matter was

pelleted by centrifugation (500 x g).

Solubilized hepatocyte proteins were analyzed by two-dimensional

polyacrylamide gel elctrophoresis using a modification of the O'Farrell

procedure described by Roberts and coworkers (1984). Proteins were

separated in the first dimension according to their isoelectric points

in a pH gradient (pH 3.5-10) established using a mixture of ampholytes.

The tubular isoelectrically focused gel was incubated with equilibration

buffer (65 mM Tris, 1 % SDS, 1 % B-mercaptoethanol, pH 6.8) and affixed

atop a 10 % acrylamide slab for protein separation by SDS-polyacrylamide

gel electrophoresis. After electrophoresis, the gel was stained with

Coomassie Brilliant Blue dye and exposed to X-ray film to produce an

autoradiogram.

Metallothionein

Metallothionein was measured by the cadmium binding assay (Eaton

and Toal, 1982) using hepatocytes from 1-6 dishes. Hepatocytes were

harvested in 0.5-1 ml of 10 mM Tris-HC1 buffer (pH 7.4), homogenized

(Polytron with P10 generator, Brinkman), and centifuged (10,000 x g; 10

min; 4C). Then the supernatant was heated (1000C; 5 min) and

centrifuged (10,000 x g; 5 min). Next, 200 gl of cadmium solution (0.2

ng Cd and 0.5 gCi 19Cd per ml of 10 mM Tris-HCl buffer, pH 7.4) was

added to a 200 Ml aliquot of the supernatant and the mixture was

incubated (20 min; room temperature). Finally, 100 Ml of 2% hemoglobin

was added. Then the sample was heated (100C; 2 min) and centrifuged







26

(10,000 x g; 5 min). After this step was repeated, the concentration of

metallothionein was calculated using 19Cd measurements (Beckman Gamma

4000) of supernatants from the sample and appropriate controls.

Metallothionein mRNA

Total RNA was extracted from hepatocytes using the method of

Chomczynski and Sacchi (1987). Cells from 3-5 dishes were homogenized

in 1 ml of guanidinium isothiocyanate, protein was removed using a

phenol:chloroform:isoamyl alcohol mixture and RNA was precipitated with

ethanol. RNA was dissolved in sterile, distilled, deionized water and

the concentration of each sample was calculated using A26o. Dot blot

and Northern blot analyses were conducted as described (Blalock et al.,

1988), except 60-mer oligonucleotide probes specific for metallothionein

-1 and -2 genes and corresponding to bases 16-76 from the 5'terminus

(Anderson et al., 1987) were used for hybridization. The probes were

5'-end labeled with [Y -32P]ATP (DuPont/NEN) using T4 polynucleotide

kinase (Bethesda Research Laboratories) and purified by chromatography

(Sephadex G-50, Sigma) prior to hybridization. The specific activity of

each probe was routinely 3.0 gCi/pmol as measured by Cerenkov counting

(Beckman LS 7500). A 32P-labeled B-actin probe was used to verify

uniformity of hybridization.

For Northern blots, total RNA was electrophoresed in a 1.1% agarose

gel and transferred to a nitrocellulose filter (BA85; Schleicher and

Schuell). Dot blot analyses were used to quantitate metallothionein

mRNA. After hybridization, the 32P content of each dot was measured by

liquid scintillation counting (Beckman LS 7500). Molecules of

metallothionein mRNA per cell were calculated as previously described







27

(Blalock et al., 1988) using an RNA/DNA ratio of 4.0, 6.4 pg of DNA per

cell, and 100% efficiency of hybridization.

6-Aminolevulinic acid dehydratase

The activity of s-aminolevulinic acid dehydratase was measured by

the method of Gurba and colleagues (1972). Cells from 1-3 dishes were

harvested in 1 ml of phosphate buffer (0.04 M NaCl in 0.1 M sodium

phosphate buffer, pH 6.7), homogenized (Polytron with P10 generator),

and centrifuged (10,000 x g; 40C). Then 250 gl of supernatant was pre-

incubated with 25 g1 of phosphate buffer containing 8.75 #moles

B-mercaptoethanol for 1 h at 370C. After pre-incubation, 75 jl of

phosphate buffer containing 7.5 moles of 8-aminolevulinic acid was

added to initiate the reaction. The formation of porphobilinogen at

370C was terminated after 90 min by adding 350 ;l 0.1 M HgCl2 in 10%

trichloroacetic acid. Porphobilinogen formation was linear for at least

2 h. Following centrifugation (100 x g; 5 min; 4C), 500 1l of

supernatant was added to 500 ul of Ehrlich's reagent (0.13 M

dimethylaminobenzaldehyde and 4 M HC104 in glacial acetic acid). A555

was determined 30 min after color development was initiated and

porphobilinogen concentration was calculated by comparison to standards.

The specific activity of the enzyme is defined as nmoles

porphobilinogen/min/mg protein.

Malondialdehyde

Malondialdehyde concentrations were measured by the thiobarbituric

acid method as described by Buege and Aust (1978). Cells from 1-3

dishes were harvested in 2 ml of TCA-TBA-HC1 reagent (15% w/v

trichloroacetic acid, 0.375% w/v thiobarbituric acid, 0.25 N HC1), and







28

heated (1000C; 15 min). After cooling, the flocculent precipitate was

removed by centrifugation (1000 x g; 10 min). As53 was determined and

the malondialdehyde concentration of the sample was calculated using

535 = 1.56 x 105 M-cm1.
Protein

Protein concentrations were determined by the method of Lowry and

coworkers (1951). First, 1.0 ml of Lowry Reagent (0.58 mM NazCuEDTA,

0.18 M Na2CO3, 1% w/v sodium dodecylsulfate, and 0.1 M NaOH) was added

to a sample or standard which was previously diluted to 0.1 ml. After

the sample was mixed and incubated for 10 min, color development was

initiated by adding 0.1 ml of Phenol reagent (Sigma). Asoo was measured

30 min later and protein concentration was determined by comparison to a

set of BSA standards.

Lactate dehydrogenase

Lactate dehydrogenase activity leaked from hepatocytes into culture

medium was measured spectrophotometrically as the increase in NADH

concentration from the oxidation of lactate (Amador et al, 1963).

Briefly, 50 gl of culture medium was added to 1 ml of lactate

dehydrogenase reagent (50 mmol lactate, 7 mmol NAD, pH 8.9) and A340 was

monitored for several minutes. Lactate dehydrogenase activity was

calculated as a rate using E340 = 6.22 mM-Icm1'.

Statistical analyses

Data were subjected to analysis of variance and Duncan's multiple

range test (SAS Institute, 1985).













RESULTS

Zinc Deficiency Study

Effect of Extracellular Zinc and Zinc-binding Ligands on Cellular Zinc
Concentration

The zinc concentration in freshly isolated hepatocytes was similar

to that in whole liver (Table 1). Hepatocytes were cultured in medium

containing either 1, 16, or 48 AM zinc to assess the effects of

extracellular zinc on cellular zinc concentration (Figure 1).

Zinc-deficient (1 AM zinc) medium had no effect on the zinc

concentration in hepatocytes during the 3 h attachment period and for up

to 48 h of continuous culture. In contrast, zinc concentrations in

hepatocytes cultured after the attachment period in medium containing

either 16 or 48 AM zinc were significantly (P s 0.05) increased to 139

and 152% of the initial value, respectively, by 12 h. Zinc levels in

these hepatocytes remained elevated at 48 h of culture.

To attempt to deplete hepatocyte zinc, the zinc concentration in

the deficient medium was reduced to 0.3 AM by extracting zinc with a

chelating resin (Table 4). Similar to the results presented in Figure

1, zinc deficient medium (0.3 AM zinc) did not reduce the zinc

concentration of hepatocytes whereas medium containing 16 or 48 tM zinc

increased cellular zinc (Figure 2).

The zinc-binding ligands, BSA and EDTA, were added to culture

medium to attempt to reduce the availability of medium zinc and depress
























Table 1. Zinc concentrations of rat liver
and freshly isolated rat hepatocytes.

Tissue n Tissue zinc#


nmoles Zn/mg protein

liver 4 1.5 0.1

hepatocytes 19 1.7 0.1


#Values represent the mean SEM.































12


24


36


Culture Period, h


Figure 1. Effect
rat hepatocytes.
containing BSA (2
16, or (a) 48 gM.
cell zinc content
from at least two
in fresh cells.
value.


of medium zinc concentration on zinc concentration in
Hepatocytes were cultured in Waymouth's medium
mg/ml) and zinc at concentrations of either (.) 1, (o)
At various times, hepatocytes were harvested and the
was measured. Each point represents the mean SEM (ni7
experiments) expressed as a percent of the initial value
Significantly different (P s 0.05) from the initial


250

200


150

100


50

0


T*


** ----*--*
.-C .,e





II I I I I 0 I0I I I I I


48















250





0
> 200

r 150
oo

o 100 *'

S50


0 12 24
Culture Period, h





Figure 2. Effect of medium zinc concentration on zinc concentration in
rat hepatocytes. Hepatocytes were cultured in Waymouth's medium
containing BSA (2 mg/ml) and zinc at concentrations of either (.) 0.3,
(o) 16, or (,) 48 AM. At various times, hepatocytes were harvested and
the cell zinc content was measured. Each point represents the mean
SEM (n=4) expressed as a percent of the initial value in fresh cells.
*Significantly different (P < 0.05) from the initial value.







33

cellular zinc concentration (Figure 3). BSA (Figure 3A) was chosen

because it is the principal plasma zinc-binding protein and produces

saturable zinc-uptake kinetics in hepatocytes (Pattison and Cousins,

1986). EDTA (Figure 3B) was selected because it has been shown to limit

the availability of divalent cations to B and T lymphocytes (Zanzonico

et al., 1981). BSA and EDTA each acted in a concentration-dependent

fashion to reduce the increases in cellular zinc concentration produced

by medium containing 16 or 48 aM zinc. However, neither BSA nor EDTA,

at concentrations as high as 40 mg/ml and 98 gM, respectively, effected

the level of zinc in hepatocytes cultured in medium with 1 gM zinc. For

all subsequent experiments EDTA was not included in medium and BSA was

included at 2 mg/ml, the standard concentration recommended for

hepatocyte culture (Failla and Cousins, 1978b).

Effect of Extracellular Zinc Concentration on s-Aminolevulinic Acid
Dehvdratase Activity

Since zinc dissociates relatively easily from the thiol groups of

a-aminolevulinic acid dehydratase (s-ALA-D) (Tsukamoto et al., 1979),

the effects of medium zinc on the activity of this zinc metalloenzyme

were examined (Figure 4). In hepatocytes cultured in zinc deficient

medium, s-ALA-D activity was significantly (P i 0.05) reduced to 75% of

the initial value by 3 h where it was maintained for up to 24 h. In

comparison, in hepatocytes cultured after the 3 h attachment period with

medium containing either 16 or 48 gM zinc, s-ALA-D activity was restored

to about 95% of the initial value by 24 h. Beyond 24 h, s-ALA-D

activity decreased regardless of medium zinc concentration.












4.0
A 02 5 10 20 40 mg BSA/ml

3.0



2.0

0
I,-
0 1.0
E

N
0.0
0 B 0 4 16 32 48 6 M EDTA
E
C
J 3.0
N

2.0



1.0



0.0
1 16 48
Medium Zn, AM







Figure 3. Effects of BSA and EDTA added to culture medium on zinc
concentration in rat hepatocytes cultured with differing medium zinc
concentrations. Three h hepatocyte cultures were incubated in Waymouth's
medium containing zinc at concentrations of either 1, 16, or 48 gM. Media
also contained BSA (A) or EDTA (B) at the concentrations indicated. After
21 h hepatocytes were harvested and the cell zinc content was measured.
Each value represents the mean SEM (n=3).















150









I
0 12 24 6 48


0-

0 12 24 36 48
Culture Period, h






Figure 4. Reduction of s-aminolevulinic acid dehydratase activity in
rat hepatocytes as a function of medium zinc concentration. Hepatocytes
were cultured in Waymouth's medium containing BSA (2 mg/ml) and zinc at
concentrations of either (.) 1, (o) 16, or (A) 48 gM. At various times,
hepatocytes were harvested and 6-aminolevulinic acid dehydratase
activity was measured. Each point represents the mean SEM (na4)
expressed as a percent of the initial value in fresh cells.
Significantly different (P s 0.05) from the initial value.







36

Effect of Extracellular Zinc Concentration on Metallothionein Gene
Expression

Levels of metallothionein mRNA and metallothionein protein as

affected by medium zinc concentration are presented in Figure 5.

Similar to cell zinc concentration, neither metallothionein mRNA (Figure

5A) nor metallothionein protein (Figure 5B) were affected by zinc

deficient medium (1 gM zinc) during the 3 h attachment period and for up

to 48 h of culture. However, in hepatocytes cultured in medium

containing 16 or 48 itM zinc, both metallothionein mRNA and

metallothionein protein were proportionately increased within 24 h and

remained elevated at 48 h.

Effect of Extracellular Zinc Concentration on De Novo Synthesis of
Hepatocyte Proteins

To assess the effects of medium zinc on synthesis of other

proteins, hepatocyte polypeptides were labeled with 35S-methionine and

analyzed by two-dimensional polyacrylamide gel electrophoresis (Figure

6). Medium zinc concentration had no effect on Coomassie Blue stained

polypeptides or 35S-labeled polypeptides. Therefore, de novo synthesis

of hepatocyte proteins other than metallothionein was not affected by

varying levels of extracellular zinc.

Effect of Extracellular Zinc Concentration on Membrane Integrity

Membrane integrity was assessed by measuring LDH activity leaked

from hepatocytes into medium after culture with medium containing

various zinc concentrations (Table 2). Clearly medium zinc concentration

had no effect on the leakage of this cytosolic enzyme from hepatocytes

after either 24 or 48 h of culture.











60 M*
50 -
40 T
Q 30 0 ?
0 20 *o---
20
0

B T*
500
S400

0 300
C 200
^ 100
C .0- -


0 12 24 36 48
Culture Period, h




Figure 5. Effect of medium zinc concentration on metallothionein gene
expression in rat hepatocytes. Hepatocytes were cultured in Waymouth's
medium containing BSA (2 mg/ml) and zinc at concentrations of either (*)
1, (o) 16, or (,) 48 jM zinc. At various times, hepatocytes were
harvested and either metallothionein mRNA (A) or metallothionein protein
(B) was measured. Each point represents the mean SEM (n=3 for A; n=7
for B).











IEF Migration


acidic


basic


acidic basic

S. .'
1-^t


97 -


45 -


29- -


20 -


UA


97 -


45 -


29 -n .. -


20 -


Figure 6. Effect of medium zinc concentration on de novo synthesis of
hepatocyte proteins. Hepatocytes cultured for 18 h with BSA-supplemented
Waymouth's medium containing 1 (A), 16 (B), or 48 (C) ,M zinc were
cultured for an additional 3 h with methionine-free medium containing the
same levels of zinc and 35S-methionine. Then hepatocytes were harvested
and polypeptides were two dimensionally analyzed using isoelectric
focusing (first dimension) and SDS polyacrylamide gel electrophoresis
(second dimension). The Coomassie Blue stained polypeptides (1) and
autoradiograms of 35S-labeled polypeptides (2) are shown.


97 -


45 -


29 -


20 -


I-I
x



o
r-
s-
01

IV}
Qr
(^1


A2


-sI
'


,,


*W W-


j
j
fi;$


~3~F~
_~~___ ~





















Table 2. Effect of medium zinc concentration on leakage of lactate
dehydrogenase activity from rat hepatocytes into culture medium.

Culture Period Medium Zn LDH activity


U/mg protein'h

10.5 0.1

9.8 0.6

10.3 1.7


10.3 0.7

10.9 1.1

10.9 0.2


Hepatocyte cultures were incubated in Waymouth's medium containing
BSA (2 mg/ml) and zinc at the concentrations indicated. At the end
of culture periods, lactate dehydrogenase activity leaked into the
medium from hepatocytes was measured.


3-24


24-48






40

Effects of Extracellular Zinc Concentration on Zinc Efflux

Results of a study which used 65Zn2* as a tracer to examine the

effects of medium zinc concentration on zinc efflux from hepatocytes are

shown in Figure 7. Zinc efflux was reduced by medium containing 1 AM

zinc compared to efflux from cells cultured with medium containing

either 16 or 48 AM zinc.

Acute Phase Zinc Metabolism Study

Interleukin 6 Increases Metallothionein Gene Expression

To determine the effects of interleukin la and interleukin 6 on

metallothionein gene expression, hepatocytes were cultured for 24 h in

Waymouth's medium supplemented with various concentrations of the two

cytokines (Figure 8). The medium was also supplemented with 1 AM

dexamethasone since glucocorticoid hormones are often required for

cytokine effects (Baumann et al., 1984; Koj et al., 1984). Incubation

of hepatocytes with interleukin 6 led to concentration-dependent

increases in metallothionein mRNA (Figure 8A). A maximal increase in

metallothionein mRNA, approximately three times that of control, was

achieved with an interleukin 6 concentration of 10 HSF units/ml (10

ng/ml). In contrast, interleukin la at concentrations as high as 1000

LAF units/ml (20 ng/ml) had no effect on metallothionein mRNA. The

effects of these cytokines on metallothionein protein 24 h after

addition to the culture medium were also examined (Figure 8B). Similar

to its effect on the mRNA, interleukin 6 increased metallothionein

protein levels in a concentration-dependent manner. Again, a maximal

increase of approximately 3.5 times that of control cells was achieved

with interleukin 6 at 10 HSF units/ml (10 ng/ml). In contrast,

















C

2100 *
CL





5 25
o,
'-- 0


CO
0
0
pI i I I I a II I
0 4 8 12 16

Culture Period, h






Figure 7. Zinc efflux from rat hepatocytes as a function of medium zinc
concentration. Hepatocytes, labeled with 65Zn for three h during the
attachment period, were cultured in Waymouth's medium containing BSA (2
mg/ml) and zinc at concentrations of either (*) 1, (o) 16, or (-) 48 gM.
At various times, hepatocytes were harvested and 65Zn content was
measured. Each point represents the mean SEM (n=3).














U,
a,


E


800


600


400


200

1600


1200


800


400


IL-la 0 .01 .1 1 10 100 1000
IL-6 0 .001 .01 .1 1 10 100


units/mi




Figure 8. Dependence of rat hepatocyte metallothionein mRNA and
metallothionein protein induction on cytokine concentration. Twenty-
four h hepatocyte cultures were incubated with Waymouth's medium
containing BSA (2 mg/ml), I gM zinc, 1 gM dexamethasone, and either (o)
interleukin-la or (.) interleukin 6 at the concentrations indicated.
After 24 h the hepatocytes were harvested and either metallothionein
mRNA (A) or metallothionein protein (B) was measured. Each point
represents the mean SEM (n=4)


iA





.^/---*---^









'7
/
.... o.._o-0o-o-6 o








--" 0


O*== 0 -0--0 -0?1ooao


i i 1 1 I







43
increasing amounts of interleukin la had no effect on metallothionein

concentrations.

Hepatocytes were cultured for up to 48 h with either Waymouth's

medium alone, medium supplemented with dexamethasone, or medium

supplemented with dexamethasone and interleukin 6 at 10 HSF units/ml (10

ng/ml) to determine the temporal effects of interleukin 6 on

metallothionein expression (Figure 9). Cultures with interleukin 6

exhibited time-dependent increases in both metallothionein-1 (Figure 9A)

and metallothionein-2 (Figure 9B) mRNA. Interleukin 6 up regulated

expression such that mRNA levels for both metallothionein-1 and -2 were

increased over those in both control and dexamethasone-treated

hepatocytes within 3 h of culture. The maximal increases in each mRNA

were achieved after 12-18 h of culture. In addition, induction of

metallothionein-2 mRNA reached levels approximately three times that of

metallothionein-1 mRNA. Specificity of the individual oligonucleotide

probes is shown by adding the values at 24 h, 150 and 600 molecules per

cell, respectively. The sum agrees with the value of 750 molecules per

cell for the combined probe shown in the concentration-response

experiment (Figure 8A). Time-dependent increases in metallothionein

protein levels were also produced by interleukin 6 (Figure 9C).

Metallothionein was increased within 3 h of culture but did not reach

maximal levels until approximately 36 h.

Extracellular Zinc and Glucocorticoid Hormone Affect Interleukin 6-
Induced Metallothionein Expression and Cell Zinc Concentration

Zinc metabolism can be modulated via regulation of metallothionein

by glucocorticoids (Failla and Cousins, 1978a; 1978b; Etzel et al.,









300
A

200


100 /

U 00
J? 0 -------------------- -- -- --
S900 B

E 00


300 *

0 A-A-A-A-A A A

2500 *

S2000 I
0
1500

E 1000 *
500


0 12 24 36 48
Culture Period, h


Figure 9. Time course of metallothionein-1 and -2 mRNA and
metallothionein protein induction in rat hepatocytes by interleukin 6.
Twenty-four h hepatocyte cultures were incubated for up to 48 h with
either (.) Waymouth's medium, (o) medium containing 1 gM dexamethasone,
or (.) medium containing 1 gM dexamethasone and 10 HSF U interleukin
6/mi. All media contained BSA (2 mg/ml) and 1 AM zinc. At various
times, hepatocytes were harvested and either metallothionein-1 mRNA (A),
metallothionein-2 mRNA (B), or metallothionein protein (C) was measured.
Each point represents the mean SEM (n=4).







45

1979) and dietary zinc (Blalock et al., 1988). To examine the extent to

which extracellular zinc and glucocorticoid hormone affect interleukin

6-induced metallothionein expression, hepatocytes were harvested after

24 h of culture with various treatment combinations and RNA was

extracted for evaluation by Northern blot analysis (Figure 10). A

control experiment using a B-actin DNA sequence showed uniform abundance

of B-actin mRNA (Figure 15). Therefore, changes in the intensity of the

550 base band representing metallothionein mRNA correspond to

differences in treatments. Abundance of metallothionein mRNA was

increased by the addition of zinc to the culture medium. At both 1 AM

and 16,M zinc, the addition of interleukin 6 alone had little or no

effect, whereas dexamethasone (1 AM) increased the metallothionein mRNA

level dramatically. However, when interleukin 6 was added (10 HSF U/ml)

with dexamethasone, levels were increased above the corresponding

dexamethasone control cultures. Thus, metallothionein mRNA was most

abundant in hepatocytes cultured with a combination of added zinc,

dexamethasone, and interleukin 6.

The trends in metallothionein mRNA observed in the Northern blot

were confirmed when quantitated by Dot blot analysis (Table 3). Levels

increased 8.2 and 11.2 fold in response to interleukin 6 and

dexamethasone in hepatocytes cultured with 1 and 16 AM zinc,

respectively. Metallothionein protein levels and cellular zinc

concentrations are also shown. Clearly, the same trends are reflected

such that the highest levels of metallothionein and cell zinc were

observed in hepatocytes cultured with a combination of added zinc,

dexamethasone, and interleukin 6.













16uM Zn


Ctrl IL-6 Dex Dex
IL-+
IL-6


Ctrl IL-6 Dex Dex

IL-6


i4.



s.*.* .~


Figure 10. Northern blot illustrating the effects of combinations of
zinc, dexamethasone, and interleukin 6 on metallothionein mRNA
concentrations in rat hepatocytes. Twenty-four h hepatocyte cultures
were incubated with Waymouth's medium containing BSA (2 mg/ml) and the
treatment combinations indicated. Concentrations of dexamethasone and
interleukin 6 were 1 gM and 10 HSF U/ml, respectively. After 24 h the
hepatocytes were harvested and total RNA was evaluated by Northern blot
analysis.


1uM Zn


w
* -ft















Table 3. Zinc and glucocorticoid dependence for
stimulation of metallothionein expression and
accumulation.


interleukin 6
cellular zinc


Treatments MTmRNA MT Cell Zn


molecules/ ng/mg nmoles/mg
cell protein protein

1iM Zn 60a 66 1.8ab
+ IL-6 57a 69a 1.7a
+ Dex 275b 305a 2.2c
+ Dex + IL-6 495 1415b 2.6d

16AM Zn 115" 179a 2.0k
+ IL-6 115ab 202a 1.9ab
+ Dex 576c 933b 3.4e
+ Dex + IL-6 1289d 3964c 5.3f


Pooled SEM 54 196 0.1
n 3 3 4


Twenty-four h hepatocyte cultures were incubated in Waymouth's
medium containing BSA (2 mg/ml) and the treatment combinations
indicated. Concentrations of dexamethasone and interleukin 6
were 1 AM and 10 HSF U/ml, respectively. After 24 h the
hepatocytes were harvested and metallothionein mRNA (MTmRNA),
metallothionein (MT) protein, and cell zinc concentrations
were measured. Each value represents the mean SEM (n=3 or
4). Values with differing superscript letters are
significantly different (P s 0.05).







48

Interleukin 6, Glucocorticoid Hormone, and Zinc Affect Iron (II)-
Nitrilotriacetic Acid and Tert-Butyl Hydroperoxide-induced Lipid
Peroxidation

To determine whether inducers of metallothionein expression and

zinc accumulation provide cytoprotection, hepatocytes cultured for 24 h

with combinations of added zinc, dexamethasone, and interleukin 6 were

subsequently cultured with cytotoxic compounds. Interleukin 6,

dexamethasone, and zinc combinations significantly (P s 0.05) altered

iron (II)-nitrilotriacetic acid and tert-butyl hydroperoxide-induced

lipid peroxidation in hepatocytes (Figure 11). The main effect of each

mediator is shown in Figure 12. The addition of interleukin 6 or 48 AM

zinc significantly (P s 0.05) reduced iron (II)-induced lipid

peroxidation whereas dexamethasone increased peroxidation. In

comparison, only dexamethasone reduced tert-butyl hydroperoxide-induced

peroxidation.

Cytoprotection From Carbon Tetrachloride Toxicity is Consistent With
Dependence Upon Metallothionein Induction and Zinc Accumulation

Survival curves for hepatocytes cultured with combinations of

interleukin 6, dexamethasone, and zinc and subsequently exposed to

carbon tetrachloride are shown in Figure 13A. Hepatocytes pre-treated

with low extracellular zinc (1 AM) were the most susceptible to carbon

tetrachloride-induced damage with only 25% surviving the first 6 h of

exposure. The addition of interleukin 6 alone had no effect while the

addition of 48 AM zinc or dexamethasone provided partial protection,

significantly improving survival to 65 and 89%, respectively. In

contrast, full protection was provided by adding a combination of both

interleukin 6 and dexamethasone without additional zinc.











200


150

C
o 100
L
0




050
-o



S200
-or
0
I 150


100


50


0


Ctrl IL-6 Dex Dex Ctrl IL-6 Dex Dex
+ +
IL-6 IL-6
1 pM Zn 48 .M Zn
Treatment Combinations


Figure 11. Cytoprotection against iron (II)-nitrilotriacetic acid and
tert-butyl hydroperoxide-induced lipid peroxidation in rat hepatocytes.
Hepatocytes were cultured for 24 h in Waymouth's medium containing BSA (2
mg/ml) and zinc, dexamethasone, and interleukin 6 in the combinations
indicated. Concentrations of dexamethasone and interleukin 6 were 1 gM
and 10 HSF U/ml, respectively. After pre-treatments, hepatocytes were
cultured in BSA-supplemented Waymouth's medium containing iron (II)-
nitrilotriacetic acid (A) or tert-butyl hydroperoxide (B) for 1 h. Each
value represents the mean SEM (n=4). Values with differing letters are
significantly different (P s 0.05).











200
Al A2 A3

150

C T
"a) 100 -* .1. -
0





r-
-B 1 B2 B3

-200
50
Cn

Q) 0 -- ------,-- ----
11 82 83





100 *


0


1 48 0 1 0 10

Zn, aM Dex, /M IL-6, HSF U/ml
Pooled Treatments





Figure 12. Cytoprotective effects of zinc, dexamethasone, and interleukin
6 against iron (II)-nitrilotriacetic acid and tert-butyl hydroperoxide-
induced lipid peroxidation in rat hepatocytes. Data from Figure 11 were
pooled to determine the individual cytoprotective effects of zinc (1),
dexamethasone (2), and interleukin 6 (3) against iron (II)-
nitrilotriacetic acid (A) and tert-butyl hydroperoxide (B) induced lipid
peroxidation. Each value is the mean + SEM (n=16). Significantly
different (P s 0.05).









-c
._ 120 A

c 100 _
0
2 80

60 T--
40 -

20 -
0
0
B
16-_ T





0 0



0 6 12 18
Culture Period with CCI4, h



Figure 13. Cytoprotection against carbon tetrachloride toxicity in rat
hepatocytes. Twenty-four h hepatocytes were treated for 24 h with BSA-
supplemented (2 mg/ml) Waymouth's medium containing either (.) 1 AM zinc,
(A) 1 AM zinc and interleukin 6, (n) 48 AM zinc, (o) 1 AM zinc and
dexamethasone, or (*) 1 AM zinc, dexamethasone, and interleukin 6.
Concentrations of dexamethasone and interleukin 6 were 1 AM and 10 HSF
U/ml, respectively. After pre-treatments, hepatocytes were cultured for
up to 18 h in BSA-supplemented Waymouth's medium containing 5 mM CC14 and
140 mM DMSO or DMSO alone. Cell survival curves (A) were constructed by
expressing the amount of cell protein remaining on dishes exposed to CC14
as a percent of that on control dishes. Cell leakage curves (B) were
constructed using measurements of LDH activity leaked into the medium from
hepatocytes exposed to CCI4. Each point represents the mean + SEM (n=4).






52

The trends observed in survival were reflected by the leakage of

lactate dehydrogenase into the culture medium (Figure 13B). Hepatocytes

pre-treated with low extracellular zinc (1 MM) exhibited the greatest

leakage. The addition of interleukin 6 alone provided no protection

while the addition of either 48 aM zinc or of dexamethasone reduced

lactate dehydrogease leakage by 55 and 80%, respectively, over 18 h of

exposure to carbon tetrachloride. Again, the addition of a combination

of interleukin 6 and dexamethasone provided full protection, completely

eliminating carbon tetrachloride-induced leakage over 18 h of exposure.












DISCUSSION

The purpose of the zinc deficiency study was to evaluate hepatocyte

monolayer cultures as a cellular model of nutritional zinc deficiency.

The approach taken was to examine a variety of indices of zinc status

following culture of hepatocytes in medium containing zinc at levels of

1, 16, and 48 gM chosen to approximate the zinc concentrations in plasma

of zinc deficient, normal, and supplemented animals. Plasma zinc levels

equal to or exceeding 48 AM have been observed in zinc supplemented

humans (Oelshlegel and Brewer, 1977) and are common in rats injected

subcutaneously with zinc emulsions (Hill et al., 1984; Lee et al.,

1989). Plasma zinc levels as low as 1 gM have never been achieved under

practical dietary conditions. However, in severe nutritional zinc

deficiency, plasma zinc concentrations are reduced in rats from the

normal level of 16 to 24 AM to as low as 5 or 6 IM (Taylor et al.,

1988).

The results of the study indicate that hepatocytes cultured as

monolayers in zinc deficient medium maintain their zinc concentration

(Figures 1 and 2) even when extremely high levels of the zinc-binding

ligands EDTA and BSA are present in culture medium (Figure 3). This

finding conflicts with the results of Guzelian and coworkers (1982) who

conducted the only other study to examine the effects of zinc deficient

medium on hepatocyte monolayers. Using similar conditions to those in

the present study, the previous investigators observed a 60 to 70% loss







54

of cell-associated zinc within 24 h of culture. Considering the variety

and importance of zinc's biological functions, it is debatable whether

the hepatocyte could survive such a rapid and dramatic loss of zinc

unless a sizeable portion of the zinc constituted a reserve.

To explain the disparity in results between the two cell studies,

the possibility that the hepatocytes lost zinc during the isolation

procedure was investigated. However, this was not the case, since zinc

concentrations in whole livers were similar to those in freshly isolated

hepatocytes (Table 1). The possibility that medium containing a lower

level of zinc could deplete the zinc concentration of hepatocytes was

also investigated by treating culture medium with a chelating resin to

remove divalent cations (Table 4). After chelation, the calcium and

magnesium levels were restored and the medium was used for hepatocyte

culture. This zinc deficient medium containing 0.3 gM zinc also did not

lower the cellular zinc concentration (Figure 2). If one assumes the

level of zinc available to hepatocytes from the zinc deficient media

used in the present study was less than or equal to that of zinc from

zinc deficient plasma, hepatocyte monolayers reflect the ability of soft

tissues such as liver to maintain their cellular zinc concentration even

when severe signs of zinc deficiency are evident (Taylor et al., 1988).

The reduction in s-aminolevulinic acid dehydratase (s-ALA-D)

activity in hepatocytes cultured in zinc deficient medium suggests the

hepatocytes were mildly zinc deficient (Figure 4). The activity of this

zinc metalloenzyme was reduced by 25% within 3 h of culture in zinc

deficient medium, presumably reflecting a loss of zinc from essential

thiol groups of the enzyme. This observation parallels that of Guzelian







55

and colleagues (1982) who found a 95% loss of s-ALA-D activity within 24

h. a-ALA-D activity is probably a good relative index of zinc

deficiency up to the the first 24 h of culture since, in hepatocytes

cultured with medium containing zinc at levels of 16 and 48 AM, activity

was fully restored by this time (Figure 4). Beyond 24 h, however, use

of 5-ALA-D activity as an index is confounded since activity decreases

regardless of medium zinc concentration.

In normal liver, 6-ALA-D activity is one-to-two orders of magnitude

greater than the activity of aminolevulinate synthase, the rate-limiting

enzyme for heme biosynthesis (Meyer and Schmid, 1978). Therefore, the

reduction in 6-ALA-D activity for hepatocytes cultured in zinc deficient

medium may reflect a metabolic prioritization for zinc whereby zinc is

lost first from a location of lesser immediate metabolic importance.

The loss of zinc from 6-ALA-D and, perhaps, other low-priority cellular

locations was not sufficient to significantly effect the overall

cellular zinc concentration.

Different functions in other cultured mammalian cell-types may be

more sensitive to zinc deprivation than 6-ALA-D activity in hepatocytes.

For example, lymphocytes cultured in chelator-extracted medium

containing 0.8 AM zinc had reduced T-killer activity (Flynn and Yen,

1981). Also, chelator-extracted medium containing serum and 2.2 AM zinc

inhibited lymphocyte proliferation (Flynn, 1985). Cell proliferation

was not examined in the present study because hepatocytes in primary

culture do not replicate.

In studies in which intact animals were fed zinc deficient diets,

hepatic metallothionein expression decreased significantly (Taylor et






56

al., 1988; Blalock et al., 1988), whereas in the present study neither

metallothionein mRNA nor metallothionein protein levels in hepatocytes

were affected by zinc deficient medium. This observation underscores

that the zinc deficiency was mild. In comparison, cell zinc,

metallothionein mRNA, and metallothionein were each increased in

hepatocytes cultured with 16 or 48 aM zinc. These responses are similar

to those observed in intact animals fed supplemental zinc (Blalock et

al., 1988) or injected with zinc (Lee et al., 1989). The increases in

zinc accumulation caused by media containing 16 and 48 AM zinc are

linked to the increase in metallothionein expression in two ways. (i)

The promoter regions of metallothionein genes contain metal-regulatory

elements which are responsive to zinc (Hamer, 1986). Presumably,

transcription is increased by zinc via a trans-acting, nuclear protein

which binds zinc and interacts with the metal-regulatory element of the

DNA. (ii) Zinc binds to and stabilizes apometallothionein. As a

result, the protein's turnover is reduced (Dunn et al., 1987).

The medium containing 16 aM zinc was not expected to affect either

cell zinc concentration or metallothionein expression since it was

chosen to simulate the normal plasma zinc concentration. The zinc in

the medium may have been more available than zinc in normal plasma since

it was added in an ionic form as zinc sulfate. Nevertheless, sufficient

BSA (2 mg/ml) was present to bind all of the zinc. Medium containing

BSA produces saturable zinc uptake kinetics in hepatocytes which may

reflect hepatic zinc uptake in vivo (Pattison and Cousins, 1986).

Varying the level of extracellular zinc had no effect on de novo

synthesis of other hepatocyte proteins. This suggests that zinc-







57

responsive, metal-regulatory elements in the hepatocyte genome may be

unique to metallothionein genes. In contrast, glucocorticoid-regulatory

elements are ubiquitous since glucocorticoid hormones up-regulate the

synthesis of not only metallothionein (Hamer, 1986) but also a broad

spectrum of other hepatocyte proteins (Ivarie and O'Farrell, 1978).

Studies using intact animals and isolated cells have emphasized the

critical role of zinc in maintaining membrane integrity (Bettger and

O'Dell, 1981). In the zinc deficiency study, medium zinc concentration

had no apparent effect on membrane integrity as judged by lactate

dehydrogenase leakage. In comparison, previous work showed that

hepatocytes cultured in medium containing 1 gM zinc were more

susceptible to a variety of inducers of lipid peroxidation and free

radical formation than those cultured in medium containing higher levels

of zinc (Coppen et al., 1988). Extrapolating to zinc deficiency in

intact animals, these observations suggest that in the absence of other

complications hepatocyte membranes may function normally. However, in

an oxidative environment characteristic of stress or infection,

hepatocyte membranes and cell membranes of other soft tissues may be

susceptible to damage. This view is consistent with Taylor et al.

(1988). They found that the primary free radical defense system in the

liver of severely zinc deficient rats was not seriously compromised but

they cautioned that if free radical generation was increased, the

defense system might be inadequate for overall protection.

The mechanisms) by which soft tissues maintain cell-associated

zinc despite severe depressions in extracellular zinc concentrations is

not yet clear. Results of the present study indicate that maintenance







58

of liver zinc during zinc deficiency could be regulated, in part, by the

hepatocyte itself. The kinetic experiment using 65Zn2+ suggests that

cell zinc concentrations may be maintained in hepatocytes cultured in

zinc deficient medium by reducing zinc efflux (Figure 7). Presumably,

the purpose of reduced efflux is to balance reduced zinc uptake

(Pattison and Cousins, 1986). The application of computer modeling

techniques to zinc kinetic and pool size data derived from hepatocytes

could provide insights into the mechanisms) involved in this process.

Substantial interest has been generated regarding the mechanisms

that account for enhanced expression of acute-phase hepatic proteins in

response to tissue injury, stress, and infection. Interleukin 1, a

cytokine produced by activated macrophages and other cell-types,

triggers a broad spectrum of systemic acute-phase responses in vivo

including enhanced expression of hepatic acute-phase proteins and

increased synthesis of other cytokines such as interleukin 6 (Dinarello,

1988). Administration of recombinant human interleukin la to rats

induces the synthesis of hepatic metallothionein similar to other acute-

phase proteins (Cousins and Leinart, 1988; Huber and Cousins, 1988).

The increase in synthesis produces a tissue-specific redistribution of

zinc with a transient depression of zinc in the plasma and concomitant

uptake of zinc by the liver, bone marrow, and thymus. Similar changes

are triggered by dibutyryl cAMP, endotoxin, and other mediators with

interleukin 1-like activity (Cousins, 1985) and have been verified by

simulation and modeling techniques (Dunn and Cousins, 1989). Results of

the cytokine study examining acute-phase zinc metabolism in hepatocytes

show that interleukin 6 rather than interleukin 1 is a mediator of







59

metallothionein production and changes in zinc metabolism at the level

of the hepatocyte. Further, interleukin 6 induced changes provide

cytoprotection from carbon tetrachloride-induced hepatotoxicity via a

mode consistent with dependence upon increased cellular metallothionein.

Often the results of studies using cultures of various cell-types

to assess the effects of cytokines on acute-phase protein synthesis seem

to conflict. Differences in responsiveness to individual cytokines

between studies are due to factors related to specific cell-types or

lines or to differences in evaluation criteria such as measurement of a

specific protein versus its mRNA (Morrone et al., 1988). In the present

study, interleukin 1 had no effect on metallothionein mRNA in isolated

hepatocytes (Figure 8) whereas Karen et al (1985) have shown that

interleukin 1 increases metallothionein mRNA in Hep G2 hepatoma cells.

The present results are consistent with those of others (Andus et al.,

1988; Castell et al., 1988) who found that interleukin 6 affects the

synthesis of a broad spectrum of acute-phase proteins in hepatocytes

while interleukin 1 regulates only a few.

Interleukin 6 gave a maximal increase in metallothionein protein at

10 HSF units/ml (10 ng/ml). This concentration agrees well with the

value of 30 HSF units/ml reported for maximal induction of other acute-

phase proteins (Andus et al., 1988). The increase in metallothionein

expression is probably dependent upon changes initiated at the level of

transcription since metallothionein mRNA was maximally induced by the

same level of interleukin 6.

Glucocorticoids may play an important role in regulating the acute-

phase response. Both interleukin 1 and interleukin 6 stimulate the






60

release of corticotropin from cultured pituitary cells, suggesting that

these cytokines increase glucocorticoid levels in vivo (Woloski et al.,

1985). Glucocorticoids stimulate metallothionein synthesis in

hepatocytes both in vivo (Etzel et al., 1979) and in vitro (Failla and

Cousins, 1978a; 1978b). This ability is due to glucocorticoid

responsive elements in the promoter region of the metallothionein genes

(Hamer, 1986).

In the present study, glucocorticoid (dexamethasone) was required

for interleukin 6 to up-regulate metallothionein synthesis.

Glucocorticoid-dependency has also been recognized for interleukin 6

regulation of other acute-phase proteins (Baumann et al, 1984; Koj et

al, 1984). Since interleukin 6 alone stimulates manganous superoxide

dismutase gene expression in primary hepatocytes (with W. Dougall and H.

Nick; Figure 16), glucocorticoid hormones are not required for synthesis

of functional interleukin 6 receptors. A possible explanation for the

glucocorticoid-dependency for interleukin 6 stimulation of expression of

metallothionein and some other acute-phase proteins is that

glucocorticoids bind to and alter the conformation of the promoter

regions of the genes via their receptor proteins. Then interleukin 6 or

a second messenger can act via their respective nuclear regulatory

proteins. This glucocorticoid-dependency for interleukin 6 regulation

in primary hepatocytes may reflect a need for a basal level of

glucocorticoid which normally bathes the liver in vivo to facilitate

expression of some liver functions. Alternatively, a possible effect of

increased levels of circulating glucocorticoids on the liver during

inflammation may be to shift the target tissues of interleukin 6 from







61

cell populations such as monocytes to other cells such as hepatocytes

(Amrani et al., 1986; Bauer et al., 1989).

The ability of increased levels of extracellular zinc to facilitate

interleukin 6-induced metallothionein production and cellular zinc

accumulation (Table 3) can be attributed to two mechanisms. (i) Zinc

binds to and stabilizes interleukin 6-induced apometallothionein so that

the protein's turnover is reduced (Dunn et al., 1987). (ii) The

promoter regions of the metallothionein genes contain metal regulatory

elements that are responsive to zinc (Hamer, 1986).

If one assumes that each molecule of metallothionein binds seven

atoms of zinc, the increases in cellular zinc in hepatocytes cultured

with either added zinc or dexamethasone are accounted for by the

increases in cellular metallothionein (Table 3). In comparison, the

increases in cellular zinc in hepatocytes cultured with dexamethasone

and interleukin 6 together are less than would be expected for the

corresponding increases in metallothionein. Therefore, the addition of

dexamethasone and interleukin 6 together may trigger a change in the

intracellular distribution of zinc such that the portion of zinc not

associated with metallothionein is reduced.

The reason for induction of hepatic metallothionein synthesis and

zinc accumulation by acute-phase mediators is not yet clear. Chvapil

and colleagues (1976; 1977) believe the purpose of decreasing plasma

zinc is to increase the phagocytic activity of macrophages and the

functional activities of other circulating cells. Another potential

benefit of moving zinc out of the plasma and into organs such as the

liver is to enhance zinc's availability to these tissues. Based on







62

zinc's role in stabilizing membranes (Bettger and O'Dell, 1981; Girotti

et al., 1986) and metallothionein's purported role as a radical

scavenger (Thornalley and Vasak, 1985), it has been proposed that zinc

and/or metallothionein may play important intracellular roles as

antioxidants by protecting hepatocytes and other cells during infection,

stress, or tissue injury when host-generated cytotoxic oxygen species

are produced in large quantities (Thomas et al., 1987; Coppen et al.,

1988; Abel and Ruiter, 1989). In the present study, several cytotoxic

compounds were screened for their abilities to increase lipid

peroxidation or reduce cell survival (Table 5). Subsequently, these

cytotoxic compounds were applied to hepatocyte cultures pre-treated with

inducers of metallothionein synthesis and zinc accumulation to determine

whether the inducers could provide cytoprotection.

The protective effects of interleukin 6 and zinc against iron (II)-

induced lipid peroxidation and of dexamethasone against tert-butyl

hydroperoxide-induced lipid peroxidation are probably not related to

cellular metallothionein and zinc because they do not correlate well

with metallothionein induction and zinc accumulation (Table 3). Zinc

probably protects hepatocytes from iron (II)-induced lipid peroxidation

by inhibiting iron uptake (Coppen et al., 1988) while interleukin 6 may

protect the cells by inducing manganous superoxide dismutase synthesis

(Figure 16). The inhibition of tert-butyl hydroperoxide-induced lipid

peroxidation by dexamethasone could be due to a plethora of

glucocorticoid effects in hepatocytes.

Only cytoprotection against carbon tetrachloride hepatotoxicity

showed a strong correlation with levels of cellular metallothionein and







63

zinc. This result fits the theoretical framework of a functional role

of zinc and/or metallothionein in membrane stabilization.

Metallothionein can be envisioned as providing stabilization either

directly as a radical scavenger or by binding CC13' radicals or

indirectly as a zinc donor to membrane sites or to specialized

components such as the cytochrome P45s system. Zinc has been shown to

inhibit this system by stabilizing NADPH (Chvapil et al., 1976; Ludwig

et al., 1980; Jeffrey, 1983).












SUMMARY AND CONCLUSIONS

Hepatocytes cultured in zinc deficient medium maintained their zinc

concentration similar to livers of zinc deficient animals despite an

extracellular environment sufficiently zinc deficient to cause partial

loss of zinc-dependent 4-aminolevulinic acid dehydratase activity.

Therefore, hepatocyte monolayers may be a good cellular model to study

the effects of zinc deficiency on the metabolism of livers and other

soft tissues. Apparent membrane integrity, metallothionein expression,

and de novo protein synthesis were unaffected by zinc deficient medium

suggesting that in the absence of other complications such as tissue

injury, stress, or infection, the defense system of the liver is

adequate for overall protection.

Studies which utilized hepatocytes to examine acute-phase

regulation revealed that, at the cellular level, interleukin 6, rather

than interleukin 1, is a major physiological determinant of

metallothionein expression and zinc metabolism. These effects of

interleukin 6 require glucocorticoids and are optimized by increased

levels of extracellular zinc. Since interleukin 1 does not elicit

changes in metallothionein expression at the cellular level, measures of

metallothionein mRNA, metallothionein protein, and cell zinc

concentrations in hepatocytes may provide sensitive bioassays to

functionally discriminate between interleukin 1 and interleukin 6

activity.







65

Interleukin 6 also induced changes which provided cytoprotection

from carbon tetrachloride hepatotoxicity in a manner consistent with

dependence upon increased cellular metallothionein and/or zinc. Since

metallothionein is regulated similarly to other acute-phase proteins and

may function in cytoprotection, it should be classified as an acute-

phase protein. If metallothionein and/or zinc have important

cytoprotective roles, a dietary zinc deficiency compounded by chronic

infection, stress, or tissue injury could have an adverse effect on

liver metabolism. Also, in conditions of chronic elevation of

interleukin 6, the dietary zinc supply may be an important determinant

in the physiological manifestations induced by this cytokine.

The findings of these studies, taken together with the results of

other investigations, may provide a mechanism and reason to explain

interleukin 1-triggered up-regulation of metallothionein and zinc

accumulation in hepatocytes during the acute-phase response (Figure 14).

Under normal conditions and in zinc deficiency, hepatic membrane

stability is adequate. However, during tissue injury, stress, and

infection when levels of host-generated cytotoxic oxygen species are

high, hepatic membranes are at risk for damage, particularly in zinc

deficient animals. During the acute-phase response, interleukin 1 is

released from macrophages (Dinarello, 1988) and, in turn, stimulates the

release of corticotropin which causes adrenal steroidogenesis (Woloski

et al., 1985). Glucocorticoids can act on hepatocytes to increase

metallothionein production as well as to feedback-inhibit the release of

interleukin 1 from macrophages (Woloski et al., 1985). Interleukin 1

also stimulates the synthesis of interleukin 6 by some cell-types (Van






















Tissue Injury

Stress Endotoxin


SMacrophag


Interleukin- 1
e .. .. Monocytes, etc -Interleukin- 6-.


Pituitary-- ACTH-AdrenIl- Glucocorticoids-


Hepatocyte
Nucleus
P MT Gene

UTmRNA
IM / Translation

Mietallothionein
Zn Pool

Cytoprotection


<______________


Figure 14. Interleukin 1-triggered up-regulation of metallothionein
gene expression and zinc metabolism in hepatocytes.


\


Dietary Zn --






67

Damme et al., 1987; Walther et al., 1988; Zhang et al., 1988). In the

presence of glucocorticoids, interleukin 6 increases metallothionein

production and zinc uptake by hepatocytes. These interleukin 6 effects

are optimized by increased levels of extracellular zinc. The functional

role of increased hepatic metallothionein production and zinc

accumulation may be to provide cytoprotection.












APPENDIX


Table 4. Mineral composition of culture media and sera.

Medium or serum Zinc Magnesium Calcium


(SM) -------(mM)-------
Fetal bovine serum 45.0

Chelated fetal bovine serum# 5.0 --- ---

Waymouth's medium 0.7 2.9 0.8

Chelated Waymouth's medium# 0.3 0.3 0.2

Repleted Waymouth's medium* 0.3 2.3 0.8


#Serum or medium was treated with Chelex-100 resin to
remove divalent cations.

Medium was treated with Chelex-100 resin and then
magnesium and calcium were restored to their initial
levels by adding MgCl2 and CaCl2, respectively.






















luM Zn

Ctrl IL-6 Dex Dex
L-6


16uM Zn


Ctrl L-6 Dex


,*1r


~.1




4l~
.. A'


Figure 15. Northern blot illustrating the effects of combinations of
zinc, dexamethasone, and interleukin 6 on B-actin mRNA concentrations in
rat hepatocytes. Twenty-four h hepatocyte cultures were incubated with
Waymouth's medium containing BSA (2 mg/ml) and the treatment
combinations indicated. Concentrations of dexamethasone and interleukin
6 were 1 gM and 10 HSF U/ml, respectively. After 24 h the hepatocytes
were harvested and total RNA was evaluated by Northern blot analysis.


Dex
-6
L-6


AI


* ,


i, .

::;~
i~ .~
.r, tl~ui~l.


















Control IL-6 Dex Dex
+
IL-6
.. .... -.1 ;,.-: .

,i -












S14,500 b





---550 b





Figure 16. Northern blot illustrating the effects of combinations of
dexamethasone and interleukin 6 on metallothionein and manganous
superoxide dismutase mRNA concentrations in rat hepatocytes. Twenty-
four h hepatocyte cultures were incubated with Waymouth's medium
containing BSA (2 mg/ml), 1 AM zinc, and the treatment combinations
indicated. Concentrations of dexamethasone and interleukin 6 were 1 AM
and 10 HSF U/ml, respectively. After 24 h the hepatocytes were
harvested and total RNA was evaluated by Northern blot analysis. The
14,500 and 550 nucleotide bands represent manganous superoxide dismutase
and metallothionein mRNA's, respectively.














r-
0



*-)
- a C C >
> 0 00 >

o o


0

+)i a 7)
S0 -0
S- > r- > r- r-




X0 4 10 06 0 0
- r-
0 C- 0 -
> L.) .-

(0 0 0 ,- 0 -
5- (0 (0 a (0 a) aa
x C3 E U EU U
0

a)
0.
-o

t- 0
0. S----.























0 4
--- 1 .C C





- 0=















SCL












4- Ea aS
4- o 4o m > .
LA- S,. -_
0*0 --





4 o 0 0

0 0













--) a
-. .S- DI V)
a)




- U L i-









C > O
Li a 5.i Li

0 t- C
a- 0 C 4 ..0



SS- a) 0 0 M
- ) *.- .4. U












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BIOGRAPHICAL SKETCH

Joseph James Schroeder III, the eldest of Joe and Darlene

Schroeder's four children, was born in Columbia, Missouri, on March 9,

1960. He received his early education at West Boulevard and Russell

Boulevard Elementary Schools and West Junior High School. Joseph

graduated with honors from Rock Bridge High School in 1978 and was

awarded a Curator's Scholarship to attend the University of Missouri

where he earned the B.S. degree in biochemistry in 1983. In 1985 Joseph

married Miss Stephanie Lynn Zeek of Lake Ozark, Missouri, and shortly

thereafter completed the M.S. degree in nutrition under Dr. Dennis T.

Gordon. In the same year he was awarded a U.S.D.A. predoctoral

fellowship to pursue the Ph.D. degree in nutrition under Dr. Robert J.

Cousins at the University of Florida. He was conferred the degree in

August 1990. Among other distinctions he achieved during his education,

as a doctoral student Joseph received the American Institute of

Nutrition 1989 Graduate Student Research Award for an outstanding paper

of original research.






I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of Doctor
of Philosophy.

Robert J. Cousins
Boston Family Professor of Human Nutrition

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of Doctor
of Philosophy.

J sse F. Gregory III) J
Professor Food Science and Human Nutrition

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of Doctor
of Philosophy.

avi-dE. Richardson
Associate Professor of Chemistry

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of Doctor
of Philosophy.
"* i

Gary E. Rodrick
Associate'Rrofessor of Food Science and
Human Nutrition

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of Doctor
of Philosophy. -

-"-Rachel M. Shireman
Professor of Food Science and
Human Nutrition






This dissertation was submitted to the Graduate Faculty of the
College of Agriculture and to the Graduate School and was accepted as
partial fulfillment of the requirements for the degree of Doctor of
Philosophy. )


August 1990


Dean, ge of Agricu
Dean, Co 'ege of AgricultuOp


Dean, Graduate School





























































UNIVERSITY OF FLORIDA


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