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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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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
3 1262 08553 8410
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