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1293 00779 4575

 

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3MZL2333 or: 3 z» 33.

:_ Umvu. 5:33;

 

 

This is to certify that the

dissertation entitled
THE ASSESMENT OF EPIDERMAL GROWTH FACTOR BINDING
AND PROTEIN KINASE ACTIVITY AS BIOMARKERS OF
2 , 3 , 7 , 8-TETRACHLORODIBENZO-P-DIOXIN TOXICITY
IN RAINBOW TROUT

presented by

JOHN LESLIE NEWSTED

has been accepted towards fulfillment
of the requirements for

Doc toral degree in Environmental Toxicology/
Fisheries and Wildlife

033%? 7333333
OZ

 

 

Major profM

Date ?’/’ ?/

MS U L: an Affirmative Action/ Equal Opportunity Institution 0-12771

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES Mum on or before due duo.

DATE DUE DATE DUE DATE DUE

 

   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU It An Afflrm‘lvo ActionIEqual Opportunity Indhutlon

cWMt

 

 

A _‘.. a,_._____‘ 4..-

THE

THE ASSESSMENT OF EPIDERMAL GROWTH FACTOR BINDING
AND PROTEIN KINASE ACTIVITY AS BIOMARKERS OF
2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN TOXICITY

IN RAINBOW TROUT

BY

JOHN LESLIE NEWSTED

A DISSERTATION '

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Fisheries and Wildlife
and
Institute for Environmental Toxicology

1991

 

 

Haloge
SYDthetic
dibenzodio
toxicity,
thair rela
HABS are
Species '13
in the Gr:
that are 3
“product
stages 0
environme
isosterQC
of animaj
°°mmon m
IECeptOr.
in Gel/913
mediated
of the bi]
marker 0f

“(X
W4

557—'

ABSTRACT
THE ASSESSMENT OF EPIDERMAL GROWTH FACTOR BINDING
AND PROTEIN KINASE ACTIVITY AS BIOMARKERS OF
2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN TOXICITY
IN RAINBOW TROUT
BY

JOHN LESLIE NEWSTED

Halogenated aromatic hydrocarbons (HAHs) are a group of
synthetic contaminants that includes polychlorinated-
dibenzodioxins (PCDDs) and biphenyls (PCBs) . Due to their
toxicity, tendency to bioaccumulate in aquatic food chains,
their relative persistence and.widespread distribution, these
HAHs are of particular concern to the health of aquatic
species in the Great Lakes. Several studies have shown that
in the Great lakes, salmon accumulate HAHs to concentrations
that are sometimes sufficient to cause adverse effects in the
reproduction, development and survival of the early life
stages of lake trout. .Although. HAHs exist as complex
environmental mixtures, many HAHs are approximate
isostereomers and produce similar toxic symptoms in a variety
of animals. Research has shown that HAHs work through a
common mode of action that is mediated through the Ah
receptor. Some RAH-elicited responses are similar to changes
in development and are related to alterations in hormone-
mediated systems. In RAH-treated mammals, "down regulation"
of the binding of epidermal growth factor (EGF) is a' sensitive

marker of exposure in tissues and cells.

ii

 

In this
the binding
rainbow tro:
stimulated t
have an EC?
and saturab
that seen 1
activity th
receptor anc
tYrosine kix
kinase inhi‘

Reduct
dependent In:
The reduet
Protein ki;
alteration
sWific to;
with a rain
alterations
co"elated

In this study we have examined the relationship between
the binding of EGF and TCDD-induced lesions in the liver of
rainbow trout. Characterization of EGF binding and the EGF-
stimulated tyrosine kinase activity showed that rainbow trout
have an EGF receptor-like protein that binds EGF in a specific
and saturable manner and has a molecular weight similar to
that seen in mammals. Upon binding, EGF elicited a kinase
activity that autophosphorylated tyrosine residues on the
receptor and exogenous polypeptide substrates. ESP-stimulated
tyrosine kinase activity was significantly reduced by tyrosine
kinase inhibitors.

Reduction in the binding of EGF was time and dose
dependent manner TCDD-exposed rainbow trout and RTE-149 cells.
The reduction in binding was accompanied by an increase in
protein kinase C and tyrosine kinase activities. The
alteration in ESP-binding and protein kinase activities were
specific for Ms that could induce cytochrome P-450. Studies
with a rainbow trout hepatoma cell line, RTE-149, showed that
alterations in EGF binding and protein kinase activity were
correlated to increases in cell proliferation and DNA

synthesis.

iii

Dedicated to my wife, Pam.

 

I woul
Kevern, Mor
Giesy for
doctoral s
professor,
a scientist
and opport'
also like
Providing 1
did.

Thank
would alsc
more thing
life times
mY eVerlas
thank all
pr°jects a

the years‘

ACKNOWLEDGEMENTS

I would like to thank my graduate committe, Drs. Niles
Kevern, Monte Hayes, Robert Roth, Steven Bursian and John
Giesy for their time and guidance over the course of my
doctoral studies. I would also like to thank my major
professor, John Giesy, for his guidance in my development as
a scientist and professional and for allowing me the freedom
and opportunity to pursue my scientific rainbows. I would
also like to thank Bob Hoke for his friendship and for
providing the inspiration for me to finish my degree before he
did.

Thanks also go to my family for their faith and love. I
would also like to thank my wife, Pam. She has put up with
more things than.most.people could ever*put up‘with in several
life times while I have completed my doctorate. To her I give
my everlasting thanks and love. Additionaly, I would like to
thank all the people in the laboratory for their help in my
' projects and for the friendship that they have given me over

the years.

LIST OF TAP.
LIST 01" FIG

GRHRAL INT
Proble
Haloqe
TCDD i
TCDD T
TCDD
TCDD
TCDD E
Histo;
Bioche
Holecu
Objec
Refere

 

Chara

t

T
Introd
Materi

TABLE OF CONTENTS

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

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

GENERAL INTRODUCTION . . . . . . . . . . . . . . . . . 1
Problem Definition . . . . . . . . . . . . . . . . 1
Halogenated Aromatic Hydrocarbons . . . . . . . . 4
TCDD in Great Lakes Fish . . . . . . . . . . . . . 6
TCDD Toxicity . . . . . . . . . . . . . . . . . . 7
TCDD Effects in Fish . . . . . . . . . . . . . . . .8
TCDD Effects on Fish Early Life Stages . . . . . . 9
TCDD Effects on Juvenile Fish . . . . . . . . . . 10
Histopathologic Effects in Fish . . . . . . . . . 11
Biochemical Effects of TCDD Toxicity . . . . . . . 13
Molecular Mechanisms of TCDD Toxicity . . . . . . 17
Objectives and Significance . . . . . . . . . . . 23
References . . . . . . . . . . . . . . . . . . . . 26

CHAPTER 1
Characterization of Epidermal Growth Factor Binding

to the Hepatic Plasma Membranes of Rainbow
Trout(Qng9;nyn§ng§ mykiss) . . . . . . . . . 43
Introduction . . . . . . . . . . . . . . . . . . 44
Materials and Methods . . . . . . . . . . . . . . 46
Materials . . . . . . . . . . . . . . . . . . 46
Fish . . . . . . . . . . . . . . . . . . . . 46
Plasma Membrane Isolation . . . . . . . . . . 46
Binding Assays . . . . . . . . . . . . . . 47
Binding and Statistical lAnalysis . . . . . . 48
Results . . . . . . . . . . . . . . . . . . . . . 48
Plasma Membrane . . . . . . . . . . . . . . 48
Optimization of Binding . . . . . . . . . . 49
Saturation Analyses . . . . . . . . . . . . . 60
Discussion . . . . . . . . . . . . . . . . . . . . 69
References . . . . . . . . . . . . . . . . . ._. . 73

CHAPTER 2

Epidermal Growth Factor' Receptor-Protein, Kinase
Interactions in the Liver'Membranes of Rainbow
Trout (Oncorhynchus mykiss) . . . . . . . . . 77

Introduction . . . . . . . . . . . . . . . . . . . 78

Materials and Methods . . . . . . . . . . . . . . 80
Materials . . . . . . . . . . . . . . . . . . 80

vi

 

OHM

 

Resul

(an: m mmdmgggr'fimofim
r1

Discus
Refers

CEAPTER 3

Effec‘

Intro:
Mater;

Results . . . . .

Fish . . . . . . . . . . . . .
Plasma Membrane Isolation . . .
EGF Binding Assay . . . . .

Standard Phosphorylation Assays
Peptide Phosphorylation Assays
Phosphoamino Acid Analysis . .
Western Analyses . . . . . . .
Statistical Analysis . . . . .

Binding of 125I-EGF to trout membranes
Time Course of Membrane Phosphorylation
Effect of Mg and Mn on Phosphorylation .
Effects of ATP and EGF Concentrations on
Phosphorylation . . . . . . . . . . . .

Effect of Nucleotides on Membrane

Phosphorylation . . . . .
Hormonal Specificity of Phosphorylation . . .
Substrate Specificity in Membrane

Phosphorylation . . . . . . . . . . . .
Effect of Kinase Inhibitors on
Phosphorylation . . . . . . . . . . . .

Discussion . . . . . . . . . . . . . . . . . . . .
References 0 O O O O O O O O O O O O O O O O O 0

CHAPTER 3

Effect of 2 , 3 , 7 , 8-Tetrachlorod ibenzo-p-dioxin

Introduction . . . . . . . . . . .
Materials and Methods . . . .

Results . . . . . . .

Discussion . . . . . . . . . .
References . . . . . . . . . .

CHAPTER 4

(TCDD) on the Epidermal Growth Factor Receptor
in Hepatic Plasma Membranes of Rainbow Trout

Fish . . . . . . . . . . .
Treatment of Fish . . . . .
Plasma Membrane Isolation .
EGF Binding Assay . . . . .
Tyrosine Kinase Assay . . .
Protein Kinase C Assay, . .
Immunoprecipitation of EGF R
Phosphoamino Acid Analysis
MFO Activity Assay . . .

Statistical Analysis . .

cep s

Effects on EGF Binding .

TCDD Effects on Kinase Act v

tY

oopoooooomooooooo

ooooooooflooooooo
O

ooooooooflooooooo

cop-coco

The Effects of TCDD on Epidermal Growth Factor

Binding and Protein Kinase Activity in the
Rainbow TRout Cell Line RTE-149 . . . . . . .

vii

81
81
82
82
84
84
85
86
86
86
89
94

97

104
106

108

108
110
118

122
123
128
128
128
129
129
129
130
131
132
133
133
133
133
143
154
159

167

 

 

 

Introduction . . . .
Methods . . . . . . .
Chemicals . . .
Cell Culture .
Cell Membrane Isolation
Cell Proliferation . .
Measurement of DNA Synthes
EGF Binding Studies . .
Tyrosine Kinase Assay .
Protein Kinase C Assay
Immunoprecipitation . .
Statistical Analysis .

o o o o o 0 E00 0 o o o o
o o o o o o o o o o o o o
o o o o o o o o o o o o o
o o o o o o o o o o o o o
o o o o o o o o o o o o o
o o o o o o o o o o o o 0

09000000000000.

Results . . . . .
Modulation of DNA.Synthesis and Cell rowth.by
TCDD O O C O O O O O O 0

Effect of TCDD on EGF binding . . . . .

Effect of TCDD on Protein Kinase Activity
Discussion . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . .
Summary . . . . . . . . . . . . . . . . . . .

viii

168
171
171
171
172
172
173
173
174
175
176
177
177

177
182
189
195
201
207’

Tabl:

LIST OF TABLES

Table Page

1. Kinetic analysis of EGF binding to rainbow
trout hepatic plasma membranes. . . . . . . . 54

2. Effects of competing peptides and hormones on
binding of [125I1-EGF to rainbow trout hepatic
plasma membranes 0 O O O O O O O O O O O O O 59

3. Summary of the dissociation constants (KD) and
maximal binding capacity (Bmax) of [125IJ-EGF
to RBT membranes as determined by Scatchard
analysis . . . . . . . . . . . . . . . . . . 61

4. Competition among nucleosides in the
phosphorylation of RT-HPM with labeled
triphosphate nucleosides. . . . . . . . . . . 105

5. Efficacy of Hormones to stimulate
phosphorylationof RT-HPM . . . . . . . . . . 107

6. Phosphorylation of exogenous proteins by
RT-HPM in the presence and absence of EGF . . 109

7. Effect of protein kinase inhibitors on the EGF
stimulated phosphorylation of RT-HPM . . . . 111

8. Comparison of EGF binding affinities and
binding capacities in plasma membranes of
control and TCDD-treated rainbow trout . . . 139

9. Effects on in yixg administration of various
toxicants on [1251]-EGF binding to RT-HPM . . 144

ix

 

10.

ll.

12.

13.

14.

LIST OF FIGURES

Figure
1. Mechanism of action of halogenated aromatic
hydrocarbons . . . . . . . . . . . . . . . . . . .
2. Molecular model for the role of the EGF receptor in
TCDD tOXiCity O. O O I O O O O O O O O O O O O O O
3. Association of 1251-EGF to RBT liver plasma
membranes 0 O O I O O I O O O O I O O I O I O O O
4. Dissociation of bound 1251-EGF from RBT hepatic
plasma membranes . . . . . . . . . . . . . . . . .
5. Effects of cations on the binding of EGF. . . . .
6. Saturation curve of 125I-EGF binding to RBT hepatic
plasma membranes . . . . . . . . . . . . . . . . .
7. Kinetic analysis for site-to-site interaction of
EGF binding to RBT hepatic membranes . . . . . . .
8. Representative displacement curves of 125I-EGF
from RBT hepatic membranes by EGF, an EGF-fragment
and TGF'G o o o o o o o o o o o o o o o o o o o o
9. Time course of lzsI-EGF binding to RT-HPM . . . .
10. The incorporation of [32PJ-phosphate into RT-HPM
from labeled ATP in the presence of EGF . . . . .
11. Time course for membrane dephosphorylation in the
presence or absence of EGF . . . . . . . . . . . .
12. Effects of“ Mga' and. Mna' concentrations on -EGF
stimulated phosphorylation of RT-HPM . . . . . . .
13. Effect of ATP on the phosphorylation of rainbow
trout membranes . . . . . . . . . . . . . . . . .
14. Effect of EGF concentration on the phosphorylation

of rainbow trout hepatic plasma membranes . . . .

Page

19

22

51

53

58

63

66

68

88

91

93

96

99

101

Figure

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

West
stint

Dose-
125I-

Scat:
and 1

Compa
activ
in TC

Chang
(PKC)
EGF-r
0f t:
TCDD/;

32-?
immunl
study

Dose-

kinas.
the h.

Effec‘
Proli

Effec.
111C011

C°nCe1
to RT]

TCDD-1

Scatcl

LIST or FIGURES (cont.)

Figure

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

Western and autoradiographic analyses of EGF-
Stimu1ated RT-HPM o o o o o o o o o o o o o o o o

Dose-response relationships for the binding of
1251-EGF and TCDD exposure in RT-HPM . . . . . . .

Scatchard analysis of IZSI-EGF binding to control
and TCDD-treated RT-HPM . . . . . . . . . . . . .

Comparisons (as percentages) of EGF binding, EROD
activity, condition factor and liver somatic index
in TCDD-treated and control rainbow trout . . . .

Changes in the binding of EGF, protein kinase C
(PKC) and tyrosine kinase (PTK) activities, and
EGF-receptor (EGF-R) phosphorylation as a function
of time after’ a single i.p. injection (10 ug
TCDD/kg) . . . . . . . . . . . . . . . . . . . . .

32-P incorporation into phosphoamino acids of
immunoprecipitated EGF-receptor in a time course
stUdy O O O O O O O O O O O O O O O O O O O O O O

Dose-response relationships between TCDD, protein
kinase activity and EGF-receptor phosphorylation in
the hepatic plasma membranes of rainbow trout . .

Effect of TCDD on the stimulation in cell
proliferation in RTH-149 cells . . . . . . . . . .

Effect of TCDD on the stimulation of’[1H]thymidine
incorporated in RTE-149 08118 c o o o o o o o o o

Concentration-dependent inhibition of EGF binding
to RTH-14 9 by TCDD O O O O O O O O I O I I C O _ O O

Time-dependent inhibition of binding of EGF to
TCDD-treated Rm- 14 9 cal 1 s O O O O O O O O O O O O

Scatchard analysis of EGF binding data. . . . . .

xi

Page

103

135

138

141

146

150

152

179

181

184

186

188

Figure

270 Effe'
tyro:
(EGF«
cell:

28. Effec
tyros
phos;

 

 

LIST OF FIGURES (cont.)

Figure

27.

28.

Effect of TCDD on protein kinase C (PKC) and
tyrosine kinase (PTK) activities and EGF receptor
(EGF-R) phosphorylation in confluent RTH-149

cells . . . . . . . . . . . . . . . . . . . . . .

Effect of TCDD dose on protein kinase C (PKC) and
tyrosine kinase (PTK) , and EGF receptor (EGF-R)
phosphorylation in confluent RTH-149 cells . . . .

xii

Page

191

193

lm

t1
Gr.
PIE

McL.

GENERAL INTRODUCTION

WW
During the late 19408 and throughout the 1950s the lake

trout (Minus W) populations in the Laurentian
Great Lakes were decimated by overfishing, sea lamprey
predation and degradation of spawning habitat (Wells and
McLain 1973). Despite massive restocking programs, attempts
to restore self-sustaining lake trout populations in the Great
Lakes have been only successful in Lake Superior and Lake
Huron (Swanson and Swedberg, 1980). In areas of where the
reproduction of lake trout is not self-sustaining, lake trout
reach sexual maturity, produce viable gametes and successfully
spawn. However, only a negligible number of their young
survive (Jude et a1. 1981; Nester and Poe, 1984; Marsden et
a1. 1988). The lack of self-supporting lake trout populations
is of great concern to many of the Great Lake states in that
sport and commercial fisheries are an important part of their
economies (Brown, 1982). Regulatory agencies are also
concerned by the possibility that the failure of these fish
populations to be self-sustaining may be indicative of future
problems in human health for populations that live near

coastal areas and who consume Great Lakes fish (Fein et al.

2
1984). This concern is based on the observation that field
and laboratory studies have suggested a possible role of
halogenated aromatic hydrocarbons (HAHs) in the increase in
salmoninae early life stage mortality (Berlin et al. 1981: Mac
1988).

The problem of increased mortality of Great Lakes
salmoninae became evident during the early 19803 when several
hatcheries in the Great Lakes region observed an unexplained
increase in rearing mortality in chinook.salmon (gaggznyngngs
tsnggytsgna) fry. An examination of this problem determined
that the cause of the mortality was not due to bacterial,
fungal or‘viral infection (Flagg, 1982). Chemical analysis of
the fry showed that moribund fry had greater concentrations of
several DDTs and PCBs than did live fry. In subsequent
studies, it was also demonstrated that sampling location was
the most significant influence on observed fry mortality. Mac
et a1. (1985) found that the concentration of total PCBs and
organochlorine pesticides was positively correlated with early
life stage mortality of lake trout eggs collected from the
Great Lakes. In that study, the eggs collected from Lake
Michigan contained the greatest concentrations of PCBs and
organochlorine pesticides, while eggs collected from Lakes
Huron and Superior contained lesser concentrations. Rates of
mortality of fertilized eggs from Lakes Superior, Huron and
Michigan was 4, 22, and 30% respectively, while mortality of

the fry equaled 41, 52, and 96%, respectively. When fry that

 

 

develope
exhibite
lethargy
syndrome
observed
they die
collected
the labor
other stuc
sample 10
Parameters
establishe
(1986) dem
°°rrelatio

and early ;

°n1Y about

 

3

developed from eggs collected from Lake Michigan died they
exhibited loss of equilibrium, erratic swimming behavior and
lethargy which is characteristic of "swim-up" or "drop-out"
syndrome (Walker et al. 1991) . Abnormal behaviors were
observed in fry that died from Lakes Superior and Huron when
they died. In a similar study, coho salmon eggs were
collected from Lakes Michigan, Ontario and Erie and reared in
the laboratory (Morrison et al. 1985a; 1985b). As in the
other studies, mortality observed.in the frwaas correlated to
sample location but no relationships between intrinsic egg
parameters and inorganic and organic contaminants could be
established. Using principle component analysis, Giesy et al.
(1986) demonstrated that there was a statistically significant
correlation between the concentration of toxaphene and PCBs
and early life stage mortalities of chinook salmon. However,
only about 30% of the total variance was explained by the
principle components indicating that there are other variables
involved in the observed mortalities. Recently, Mac et a1.
(1988) directly correlated the concentration of a single toxic
PCB congener 3,3',4,4'-tetrachlorobiphenyl in Lake Michigan
lake trout with egg mortality but did not examine this
relationship in sac-fry.

To date, laboratory and field evidence indicates that the
presence of persistent environmental contaminants are
implicated for the mortality that has been documented in the

contaminated regions of the Great Lakes. These contaminants

 

 

Ca

En-
Sol

SYr

4

include polychlorinated dibenzo-p-dioxins (PCDDs),
polychlorinated. dibenzofurans (PCDFs) and. polychlorinated
biphenyls (PCBs) which may act in a combined fashion to cause
the poor survival of early life stages of some species of
salmoninae and as a consequence reduce the reproductive
success of these species in the Great Lakes (Walker et al.
1991).

HQLQSSDQEQQ AIQEQIIQ BXQIQQQIDQES

PCDDs, PCDFs and PCBs belong to a family of
polyhalogenated aromatic hydrocarbons. These compounds are
poorly metabolized, very persistent and lipophilic. Thus,
they bioaccumulate in the food chain (Tanabe et al. 1989).
There are 75, 135, and 209 possible congeners of the PCDDs,
PCDFs and PCBs respectively (Poland and Knutson 1982). Of
these compounds, only 21 congeners are considered extremely
toxic and these compounds constitute the planar halogenated
hydrocarbons (PHHs) (Safe et al. 1985; EPA 1989). 2,3,7,8-
tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic of these
compounds and is considered the prototype for the toxicity of
“the other PHH congeners (Poland and Knutson, 1982).

TCDD is a chlorinated, planar tricyclic halogenated
carbon-oxygen compound that is extremely toxic to most animal
species (Harris et al. 1973) . Inputs of TCDD into the
environment come from three principal sources. The first
source includes chemical manufacturing where TCDD appears as

synthesis intermediates and by-products. 3 Specific examples of

 

61

D2

5
these sources include the production of 2,4,5-T and Silvex
herbicides where PCDDs occurred in the ppb to ppm.range (Rappe
et al. 1978: Esposito et al. 1980). Trace concentrations of
TCDD have also been found in samples of bleached pulp, in
bleached kraft pulp effluents and in some products
manufactured from the bleached pulp (Anon 1987). The second
source is the combustion of chlorinated hydrocarbons. PCDDs
are formed.by alkaline hydrolysis of chlorophenolate salts at
temperatures that exceed 180%: (Blair 1983; Ballschmiter et
al. 1988). Burning of coal, chlorophenols and/or solid waste
can result in the formation of PCDDs (Long and Hanson 1983).
Evidence for this source is that PCDDs have been found in
flyash and flue gas emissions of municipal and industrial
incinerators (Lustenhouwer et al. 1980). The third source of
PCDDs incorporates the<disposal of the wastes of the first and
second source and includes chemical waste dumps, sludge from
incinerators, sediments and contaminated soils. Transport
from these sites can occur by aerial transport, ground water
leaching, surface water runoff or’through accidental release.
Examples of the later type of release include the Seveso
incident (Reggiani 1980) and the accidental spraying of waste
oil contaminated with 2,3,7,8-TCDD on dirt roads and horse
arenas at various locations in Missouri including Times Beach
(Carter et al. 1975). It has been hypotheSized that
atmospheric deposition of particulate-bound contaminants

produced by combustion is the major source of PCDDs in the

 

 

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6

produced by combustion is the major source of PCDDs in the
Great Lakes system (Czuczwa and Hites 1986).
12012 in Great lakes Fish

Concentrations of many PCDDs, including 2,3,7,8-TCDD,
have been found in Great Lakes salmoninae. Petty et al.
(1983) analyzed six species of Great Lakes salmon and found
that an averaged 33.4 ng/kg TCDD per whole body weight for all
the fish sampled. In Lake Ontario, lake trout averaged 30.8
ng TCDD/kg and 22 ng TCDD/kg wet tissue weight in livers and
fillets, respectively (Tosine et al. 1983) while commercial
fish taken during 1980 contained 2.0 to 38.5 ng TCDD/kg in 21
of 62 samples with detectable concentrations (Ryan et al.
1984). These findings were collaborated by the results of
O'Keefe et al. (1983) where Lake Ontario lake trout had TCDD
concentrations ranging from 51 to 107 ng/kg body weight. To
establish a statistical baseline for the comparison between
each of the Great Lakes, De Vault et al.(1989) analyzed lake
trout taken from each of the Great Lakes and Lake St. Clair
for PCDDs and PCDFs. They found total PCDD concentrations
averaging 7.2 ng/kg b.w. in Lake Superior, 64.5 ng/kg b.w.in
Lake Ontario, 1.0 ng/kg in Lake Superior and 48.9 ng/kg in
Lake Ontario. Due to their extreme hydrophobicity and very
low environmental concentrations, there is currently no
information on the water concentration of PCDDs in the Great
Lakes (Czczwa and Hites 1984). Whether the observed Great

Lakes fish TCDD concentrations are sufficient to induce the

7
observed toxic symptoms in salmoninae of these lakes is still
not known at this time.
T321212 12819.13!

The toxicity of PHH is determined by their structure
(Safe, 1990). Results of acute and chronic toxicity studies
have reported that the tetra- to hexachloro 2,3,7,8-
substituted PHH analogs are more active than those congeners
which contain less than four lateral chlorines substituents
(Poland and Knutson, 1982) . Moreover, as the degree of
chlorine substitution at the nonlateral positions increase in
PHHS with lateral chlorines there is a decrease in the
observed toxicity. Structure activity studies have shown that
the ideal PHH ring structure is planar and covers an area of
3 X 10 A. Of the PHHs, 2,3,7,8-TCDD is the most toxic
isostereomer and has been used as a model PHH to examine the
molecular mechanism of toxicity for this class of compounds.

The observed toxicity of TCDD is dependent on a number of
factors including the dose, the age, strain, species and sex
of the animals tested (Safe 1986). LDSOs from several animal
species range from 600 to 2,000 ng/kg b.w.in the guinea pig,
22,000 to 45,000 ng/kg b.w. in the rat, 25,000 to 50,000 ng/kg
b.w.in the chicken, 70,000 ng/kg b.w. in the monkey, 115,000
ng/kg b.wx in the rabbit, 114,000 to 284,000 ng/kg b.w. in the
mouse, 21,000,000 ng/kg b.w. in the bullfrog and 1,157,000 to
3,000,000 ug/kg b.w.in the hamster (Kociba and Schwetz 1982;

Tucker et al. 1982) . TCDD toxicity differences are also

 

8
observed within the same species which females being more
sensitive than males of the same reproductive maturity
(Schwetz et al. 1973).

Two signs observed in.most animals acutely poisoned with
TCDD are loss of body weight (wasting syndrome) and lymphoid
depletion. Body weight loss can be as much as 50% of the
critical body weight but does not appear to be strictly
related to a reduction in food intake (Gasiewicz et al. 1980;
McConnell 1980) . The mechanism of TCDD-induced lymphoid
tissue loss is believed to be the result of an alteration of
thymus epithelium which results in the alteration in the
maturation and the loss of T-lymphocyte precursors (Vos and
Moore 1974; Greenlee et al. 1985) . Other TCDD effects include
liver necrosis, enlargement of the liver due to hyperplasia
and hypertrophy of parenchymal cells and fatty infiltration
(McConnell 1980). Commonly observed TCDD-induced effects in
mammals is hyperplasia and hyperkeratosis of the epidermis
resulting in an acnegenic response and the production of
comedones and cysts (Kimbrough 1983). In man, chloracne is
most commonly observed in individuals exposed to TCDD (May
1973).

EDD 21139.28 in £13.11

Fresh water fish are uniquely sensitive to the effects of
TCDD. Unlike mammals, fish do not show the wide range in
sensitivity to TCDD. Of six species of fresh water fish from

five families, only a five-fold difference in TCDD LDSOs was

9

observed (Kleeman et al. 1988). The LD50s for these species
were not affected by the exposure route in that both intra-
peritoneal injection and waterborne exposure yielded similar
L050 values (Spitsbergen et al. 1988b). The LD50 range for
these fish was similar to that observed in the most sensitive
mammal, thelguinea.pig (Schwetz et al. 1973). However, early
developmental stages of fish are more sensitive to TCDD than
the adult fish. In rainbow trout(QngQ:nyngng§ 323155), TCDD
exposure of swim-up fry resulted in a concentration of 900
ng/kg and 45% mortality (Mehrle et al. 1988). In newly
hatched rainbow trout sac fry, the LD50 was 300 ng/kg (Walker
1991) while juvenile trout had an LD50 of 16 ug/kg (Kleeman et
al. 1988). Thus, newly hatched sac-fry are 3 and 30 times
more sensitive to the lethal effect of TCDD than trout swim-up
fry and juveniles, respectively.
T9120 Effects sen Fish Earl): Life Stages

Early life stages fish have been shown to be very
sensitive to the effects of waterborne TCDD (Helder, 1980;
Helder, 1981). Rainbow trout eggs, sac fry, and juveniles
'exposed directly to TCDD in water ( 0.1 ng/l) for 96 h
exhibited reduced survival. Exposure of sac fry to 10 ng/l
for 96 h caused 100% mortality. For older fry, 100 ng/l was
required to cause 100% mortality. In exposed eggs, these
concentrations caused underdevelopment, hemorrhages, edema and
malfunctions of the jaws (Helder, 1981). Northern pike eggs
exposed 0.1, 1.0, and 10 ng/l for 24 h exhibited a 23%

 

 

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Thes
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nortl
was c
in eg

(Mac

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(199:
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Occur
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10

retardation in the development rate relative to control eggs.
These concentrations also caused delayed mortality of fry. .A
exposure to 10 ng TCDD/l resulted in 100% mortality. Eggs of
northern pike exhibited a tail-first hatching phenomena that
was dose-dependent. This finding was similar to that observed
in eggs sampled that are hatched from Lake Michigan lake trout
(Mac 1988).

To relate the concentration of TCDD in eggs to the
toxicity manifested during early develOpment, Walker et al.
(1991) exposed lake trout eggs to TCDD. Hatchability was
reduced at egg concentrations greater than or equal to 226 ng
TCDD/Kg wet weight. The greatest TCDD-related mortality
occurred at the sac-fry stage. The No Observable Adverse
Effect Level for cumulative mortality was 34 ng/kg b.w. while
the Lowest Observable Adverse Effect Level was 55 ng/Kg b.w.
Concentrations of TCDD in eggs that caused 50% mortality above
control at swim-up (LD50) was 65 ng/Kg b.w. While the
greatest TCDD-induced mortality occurred during the sac-fry
stage, a greater percentage of the larvae (8-17%) died half-
hatched when concentrations equaled or exceeded 226 ng TCDD/kg
b.w.
mo Effects 911 mu: Fish

Juvenile fish are also very sensitive to the toxic
effects of TCDD. Guppies exposed to TCDD in water for 24 h
and then observed for 69 d exhibited an increase in fin

deterioration (necrosis and fungus infection). Toxic

 

n.

a!

11

threshold for the fin deterioration was estimated to be
between 0.08 and 0.8 ng TCDD per gram wet weight (Miller et
al., 1979). Fathead minnows exposed to 0.0, 0.1, 1.0, 10 and
100 ng TCDD/l for 1, 2, 3, or’4 d exhibited mortality that was
time of exposure and dose-dependent (Adams et al., 1986). In
juvenile rainbow trout, the Maximumr Acceptable Toxicant
Concentration was between 1 and 38 pg TCDD/L while the No
Observable Adverse Effect Level was less than 38 pg TCDD/L in
rainbow trout (Mehrle et al. 1988). TCDD induced significant
alterations in feeding behavior and general activity levels at
38 pg TCDD/L. While rainbow trout body TCDD concentrations
were not measured in Mehrle's study, Kleeman et a1 ( 1986)
determined for rainbow trout that no overt signs of toxicity
were observed until body burdens reached a threshold of 150-
250 ng TCDD/kg. As was observed in mammalian studies,
lethality in fish was delayed 1 to 3 weeks post exposure.
W £213.95 in man

The effects of TCDD exposure in laboratory studies using
fish are very similar to the effects observed in laboratory
studies with mammals. In both mammals (McConnell 1980: Poland
and.Knutson 1982) and fish (Kleeman et a1 1986; Spitsbergen et
al. 1988 a,b), the primary sites of TCDD-induced lesions are
epithelial and lymphomeyloid tissues. TCDD exposure results
in altered epithelial differentiation and proliferation
(metaplastic/hyperplastic) with different organ systems
affected depending on the species of test animal. The

12

generalized edematous response, thymic involution and lymphoid
depletion, leukopenia, thrombocytopenia, hyperplastic lesions
of bile ducts and gastric mucosa, and fibrous pericarditis
observed in fish have also been previously reported in either
laboratory mammals (McConnell 1980) or birds (Allen 1964;
Gilbertson 1982) . Slow metabolism and elimination of TCDD in
rainbow trout compared to that of mammals may explain the
appearance of hyperplastic lesions in rainbow trout bile ducts
and gastric mucosa after single TCDD exposures (Kleeman et al.
1986) .

Fin margin necrosis appears to be a common indicator of
TCDD exposure in fish. TCDD is known to enhance terminal
differentiation in cultured human epidermal cells (Osborne and
Greenlee 1985) . Terminal differentiation in fish skin exposed
to TCDD could decrease regenerative capacity and result in
lesser resistance to shear stress of swimming. Separation of
epidermal cells as a result of immune responses is not
considered a plausible explanation for fin necrosis since
epidermal inflammation is typically absent (Spitsbergen et al.
1988 a).

Exposure of lake trout early life stages to TCDD results
in several well-defined lesions that correspond to lesions
observed in eggs collected from the Great Lakes (Spitsbergen
et al. 1991). In eggs exposed to 400 ng TCDD/L, embryos
develop normally until one week prior to hatch. At this time

retrobulbar, meningeal and subcutaneous edema become evident.

13

Following hatching, sac-fry develop severe subcutaneous edema
with cessation of blood circulation in the yolk sac and body.
Necrosis of the retina, brain and spinal cord were also
present in the embryos. Thus, the cardiovascular system
appears to be the initial tissue affected by TCDD in
developing trout. Other lesions in the brain, retina and
liver develop as a result of circulatory derangements
expressed as anemia and hypoxia (Spitsbergen et al. 1991).

As in mammals, the type and severity of lesions observed
in fish do not account for the observed mortality. The
"wasting syndrome'flalso does not play a role in fish mortality
since some species exhibit no weight loss after TCDD exposure
(Spitsbergen et al. 1988 b) and most necropsied fish exhibit
adequate body reserves of fat. While the mechanism of TCDD-
induced lesions in both mammals and fish remains unknown, it
appears to be an evolutionarily conserved phenomenon because
of the similarity of observed effects in' a number of
phylogenetically disparate groups.

Biochemical EIIEQSS QB TCDD 193191;!

TCDD and other PHHs cause diverse biochemical responses
in animals that include the induction of both phase I and
phase II drug-metabolizing enzymes (Poland et al. 1979:
Goldstein and Safe 1989). TCDD induces both cytochrome P-
4501A1 and cytochrome P-4501A2 hemoproteins and their
associated microsomal monooxygenases which include

ethoxyresorufin O-deethylase (EROD) and aminopyrine

14

demethylase (APDM). TCDD also induces glutathione S-
transferase (Baars et al. 1980) , glucuronyl transferase (Owens
1977) and several other enzymes including ornithine
decarboxylase (Nebert et al. 1980) , epidermal transglutaminase
(Puhvel et al. 1984) and DTldiaphorase (Beatty and Neal 1976).

Many of the effects of TCDD are similar to those
associated with modulation of hormone-mediated responses. The
"wasting syndrome" observed in TCDD toxicity is similar to
that seen in animals that have altered hormone metabolism.
TCDD-mediated reduction of thyroid hormones in rats and
tadpoles has been correlated with toxic endpoints such as
mortality' and reduction. in. growth. and a retardation of
development (McKinney 1985). Moreover, thyroid hormones may
have a role in modulating the toxicity of TCDD in that
radiothyroidectomy protected rats against TCDD-mediated T-cell
immunotoxicity, mortality and body weight loss (Rozman et al.
1985). TCDD-induced lesions are also similar to several
glucocorticoid-induced effects such as lymphoid involution,
edema,lipid mobilization (McConnell 1980: Safe 1986),
'elevation of steroid metabolizing enzyme activities (Poland
and Knutson 1982) and teratogenesis in mice (Birnbaum et al.
1985) . Other TCDD-hormone associated effects include the
reduction of progesterone and estrogen (Romkes et al. 1987:
Kleeman et al. 1990) and Vitamin A (Hakansson et al. 1986).
In animals, hormone levels are regulated through

intercellular signalling systems while their biological

15
activity is mediated through cellular receptors. TCDD and
other PHHs have been shown to alter the membrane integrity of
cells through lipid peroxidation (Alsharif et al., 1990).

TCDD can affect membrane function in target organs of
exposed mammals. In rats, a single subacute TCDD dose can
reduce the hepatic biliary excretion of ouabain which was
correlated to an inhibition of Cazl-Mgz' and Na1-K‘ ATPases of
the biliary plasma membrane (Eaton and Klaussen 1979: Peterson
et al. 1979 a, b). SDS-PAGE electrophoresis analysis of’TCDD-
treated animal membranes revealed significant alterations in
the band profile of plasma membrane proteins concentrations
(Brewster et al., 1982). Receptor binding assays of TCDD-
treated membranes ' revealed a reduction in the binding of
insulin, glucagon, concanavalin A and epidermal growth factor
to their receptors (Matsumura et al. 1984).

Alterations of membranes have also been implicated in
TCDD-induced hyperlipidemia (Swift et al. 1981). The
hyperlipidemia is characterized by the accumulation of
cholesterol esters and triglyceride-carrying lipoproteins in
the blood. It is associated with the reduction of low density
lipoprotein receptors in adipocytes (Bombick et al. 1984) and
the reduction in lipoprotein lipase (LPL) activity (Brewster
et al. 1984). Both of these systems are active in the control
of cholesterol, free fatty acid and concentrations of

triglyceride in the blood (Chapman 1980).

16

Of the intrinsic plasma membrane proteins studied to
date, alteration of EGF binding to plasma membrane receptors
has been shown to be the most sensitive to TCDD exposure
(Madhukar et al. 1984) . In the transformed keratinocyte cell
line (SOC-12F) , TCDD was found to reduce the binding of EGF in
a dose-dependent manner (ED50-1 uM) (Hudson et al. 1985;
Osborne and Greenlee 1985). The reduction in the binding of
EGF was the result of a loss in high affinity binding sites
(Hudson et al. 1985) . Polycyclic aromatic hydrocarbons (PAHs)
and phorbol esters have also been shown to reduce the binding
of EGF in several cell lines (Ivanovic and Weinstein 1982;
Karenlampi et al. 1983) . The reduction in EGF binding was
compound-specific and transient (< 24 hours). Only
benz(a)anthracene, 3-methylcholanthrene, and benzo(a)pyrene
reduced binding while p.p'-DDT, phenobarbital and testosterone
had no significant effect on binding (Ivanovic and Weinstein
1982) . However, PAHs and phorbol esters only caused a short
term reduction in binding (<24 hours) while TCDD significantly
reduced binding (40% of control) for up to 10 days after the
removal of TCDD from the culture media (Hudson et al. , 1985) .
The dose-dependent and compound specific reduction in the
binding of EGF seen in various cell types has also been
observed in rats, mice, guinea pigs and hamsters exposed to
TCDD (Madhukar et al. 1984) . The formation of cleft palate in
newborn mice has also been attributed to alteration of EGF

receptor homeostasis in the fusion of the shelves of the

l7

palate (Abbott and Birnbaum 1990). .Alteration.of‘EGF2receptor
homeostasis has also been linked to hyperkeratinization of the
epidermis of TCDD-treated animals (Osborne and Greenlee 1985) .
W Mania: 2f 19.0.12 123.152.1112

One common effect seen in all TCDD-responsive animals is
the induction of microsomal cytochrome P-450 and its
associated mixed-function oxidase activities. Early studies
of the induction of hepatic aryl hydrocarbon hydroxylase
(h_AHH) activity by homologous series of PHHs revealed a
relationship between PHH structure and their ability to induce
AHH activity (Poland and Knutson 1982). Further
investigations showed that in genetically inbred aryl
hydrocarbon (Ah) responsive (C57BL/6) and nonresponsive
(DEA/2) mice and their backcrosses there was at least one
order of magnitude difference in their sensitivity to TCDD-
induced AHH acitivity (Poland and Glover 1980). In
comparison, 3-methylcholanthrene was inactive as an inducer in
the nonresponsive mice. Genetic studies with the inbred
strains and their backcrosses demonstrated that.AHH induction
is inherited as a simple autosomal dominant trait (Poland et-
al. 1974). These studies were the basis for the theory that
the induction of AHH activity was receptor- mediated and that
the reduced responsiveness of DEA/2 mice was due to a defect
in the Ah receptor. Subsequent studies have confirmed that
the differential responsiveness of the inbred mice strains was

due to differences in affinity of TCDD for a cytosolic

18

Figure 1. Mechanism of action of halogenated

aromatic hydrocarbons (M. Denison).

 

 

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20

receptor, Ah or TCDD receptor (Poland and Knutson 1982; Okey
et al. 1989). Subsequent research revealed that the ability
of PHHs to bind to this receptor correlated well with its
ability to induce AHH activity and to induce toxicity such as
thymic involution, wasting syndrome land epidermal
hyperkeratinization (Poland and Knutson 1982; Safe, 1990) . As
a result of these studies, a common mode of action has been
suggested for PHH compounds that is based on a model that
incorporates alteration of receptor-mediated gene expression
(Figure 1) (Whitlock 1987). Initial binding of TCDD to the
cytosolic Ah receptor is followed by atransformation where a
heat shock protein dissociates from the TCDD:AhR complex and
. occupied receptor complexes accumulate in the nucleus.
Binding of the nuclear complexes to specific DNA sequences or
dioxin responsive elements (DREs) located in the 5'-upstream
region from the CYP1A1 gene lead to the enhancement of CYP1A1
gene expression and the subsequent accumulation of cytochrome
P-450 specific mRNA (Whitlock 1990).

Other TCDD-induced lesions have been hypothesized to be
the result of altered proliferation and differentiation
(Bombick 1986). In this model (Figure 2), alteration of EGF-
binding homeostasis is the key event in the disruption of
normal cell growth and differentiation. As is seen in
cytochrome P-450 induction, the interaction of the TCDD:Ah-
receptor complex with DREs is suspected. to be the initial

event of the toxic response. However, instead of cytochrome

21

Figure 2. Molecular model for the role of the EGF-

receptor in TCDD toxicity. (Bombick, 1986).

 

22

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23

P-450 induction being the next event, the activation of
specific oncogenes is the catalyst for the observed toxic
lesions. Oncogene products are protein kinases and the
elevation in kinase activity has been shown to be important in
the activation and inactivation of cellular proteins and
enzymes that control cell growth and differentiation (Hunter
1984; Coggin 1986). . Studies supporting this model have
demonstrated that TCDD induces ppéo'" and p21'“, both of which
are related to disruption of growth processes. Furthermore,
Abbott and Birnbaum (1990) have shown that TCDD exposure
altered the regulation of growth factors and their receptors
in cleft palate formation in mice.
W and Significance

' Fish are very sensitive to the toxic effects of TCDD.
Many of the TCDD-induced lesions observed in fish are also
observed in mammals that suggests that the mode of toxic is
similar between these species (Spitsbergen et al. 1988 a,b).
One common TCDD-induced effect is the induction of hepatic
cytochrome P-450s that are functionally similar to those
observed in mammals (Stegeman 'and Kloepper-Sams 1987).
Moreover, the Ah receptor has been identified in rainbow trout
(Heilmann et al. 1988) and in the rainbow trout cell line RTH-
149 (Lorenzen and Okey 1990). Based on this information, it
appears that the mechanism of TCDD induction in fish is

mechanistically similar to that in mammals. However, there is

24
little information on molecular mechanisms of TCDD- induced
toxicity in fish.

The objectives of this dissertation are to examine the
applicability of a mammalian model of TCDD-induced toxicity to
rainbow trout that is based on the alteration of EGF-receptor
homeostasis (Figure 2). This model will serve as the
characterization and assessment of TCDD-induced alterations in
the binding of EGF and the stimulation of protein kinase
activity as biomarkers of TCDD toxicity in rainbow trout.

We will test the null hypothesis that there is no effect
on TCDD on the binding of EGF or on the activity of protein
kinases in hepatic plasma membranes of rainbow trout. We will
use two-tailed tests since we do not know what effect TCDD
will have on these parameters.

TCDD was chosen as the prototype PHH because of its
extreme toxicity and the large amount of toxicological
information available on TCDD effects to rainbow trout.
Specific objects include:

1. To quantify and characterize the binding of EGF to the
hepatic plasma membranes of rainbow trout.

2. To examine the relationship between the binding of EGF
and the activation of protein kinase activity in the
hepatic membranes of trout.

3. To examine the relationship between TCDD dose and time on
the binding of EGF and protein kinase activity in the

liver of rainbow trout.

25

4. To correlate the relationship between other TCDD-induced
responses and the alteration of EGF binding in rainbow
trout.

5. Use a rainbow trout hepatoma cell line , RTE-149, as an
in 2131.9 model to examine the effect of TCDD on the
binding of EGF and to other processes of cell
proliferation and differentiation.

The studies were grouped into two sections. The first
set of studies characterized the binding, physiology and
biochemistry of the EGF receptor in hepatic tissues of rainbow
trout. The second set of studies was designed to examine the
effect of TCDD on the EGF receptor in the hepatic plasma

membrane of rainbow trout and in the cell line RTH-149.

26
LIST 0? REFERENCES

Alsharif,N.Z., Grandjean,C.J., Murray,W.J. and. Stohs,S.J.
(1990). 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-
induced decreases in the fluidity of rat liver membranes.
zengbigtigg 20: 979-988.

Abbott,B.D., and Birnbaum,L.S. (1990). TCDD-induced altered
expression of growth factors may have a role in producing
cleft palate and enhancing the incidence of clefts after
coadministration of retinoic acid and TCDD. mm
Appl; RnazmagglL 106:418-432.

Allen, J.R. (1964). The role of "toxic fat" in the production
of hydropericardium and ascites in chickens. Am‘l‘ yet;
Egg; 25:1210-1219.

Anon. (1987) . Assessment of potential health risks from dermal
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transferase activity of rat liver cytosol. W
EhgzmggglL 27: 2487-2494.

Ballschmiter,l<. , Braunmiller,I. , Niemczyk,R. and Swerev,M.

(1988) . Reaction pathways for the formation of polychlor-

27

dibenzo-p-dioxins (PCDDs) and dibenzofurans in combustion
processes. II. Chlorobenzenes and chlorophenols as
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dioxins-dibenzofurans in flame chemistry. CW 17:
995-1001.

Beatty, P.W. and Neal, R.A. (1976) . Induction of DT-diaphorase
by 2 , 3 , 7 , 8-tetrachlorodibenzo-p-dioxin. Mam, W

Rm Cm 68: 197-204.
Berlin, W.H., Hesselberg,R.J. and Mac,M.J. (1981). Growth and

mortality of fry of Lake Michigan lake trout during
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Hydreeerbenesass:Tameka:inntheakeereduetiem:andhfiurxixel

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40
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CHAPTER 1

CHARACTERIZATION OF EPIDERMAL GROWTH FACTOR BINDING

TO THE HEPATIC PLASMA MEMBRANES OF RAINBOW TROUT

(W W)

INTRODUCTION

Epidermal growth factor (EGF) is a 6,045 dalton, single
chain polypeptide which was first discovered in the mouse
submaxillary gland (Cohen, 1962). EGF is classified into a
group of cytomodulatory factors that are hormone-like in their
behavior and biological potency. EGF, when administered to
mammals, produces several significant biological responses in
epithelial and epidermal cells (Carpenter and Cohen, 1979).
Lo 1119, EGF induces proliferation and keratinization of
epidermal cells, stimulation of corneal and intestinal cell
regeneration, hepatic hypertrophy and hyperplasia, and the
inhibition of gastric and thyroid hormone secretion (Ahren,
1987: Soderquist and Carpenter, 1983). lojgitro, EGF induces
ion transport in cells, the turnover of phosphotidylinositol
and ATP, the induction of ornithine decarboxylase, and the
activation of RNA, DNA and protein synthesis (Cohen, 1983).
EGF exerts its biological effects through a cell surface
transmembrane receptor (Adamson and Rees, 1981).

The mouse EGF receptor is a single chain glycoprotein
(Mr= 170,000) that has no subunit structure, but can be
separated into three functional domains. A heavily

glycosylated external domain contains the EGF recognition site

44

45

and is involved in ligand binding; 'The second.domain consists
of a transmembrane segment that functions in anchoring the
receptor to the cell and in signal transmission. 'The
cytoplasmic domain contains an intrinsic tyrosine kinase.
This kinase can phosphorylate specific tyrosine residues on
the receptor itself and other intracellular proteins and is
believed to be the mechanism by which this receptor mediates
its mitogenic effects (Das, 1983; Downward et al., 1984).

The purpose of the present study was to characterize the
EGF receptor in the liver of adult rainbow trout. Binding of
EGF to tissues in fish has been demonstrated in several fish
species (Naftel 1989). In a phylogenetic survey of 1RSI-EGF
binding, EGF specific binding was demonstrated in three bony
fishes (lithium hetereelitte. §teh9temue ehflseee. and
W M) and one cartilagenous fish (Woo
oooio) (Naftel, 1989). In the fishes which bound EGF, the
greatest amount of binding was seen in the liver.
Comparative binding experiments between the dogfish.(uo§tolo§
oonig) and mouse liver preparations showed that the EGF
association and saturation binding curves were nearly
identical. Here we report the binding characteristics of the
EGF-specific high affinity, limited capacity binding sites on

the plasma membranes of rainbow trout liver.

46
MATERIALS AND METHODS
Hateriale
Receptor grade EGF, dexamethasone, glucagon, insulin,
cortisol, FSH, LH, and prolactin were purchased from Sigma
Chemical Co. (St. Louis, MO). Transforming growth factor-a
(TGF-a) and EGF-fragment were purchased from Calbiochem (San

125

Diego, CA). I-EGF (100-150 uC/ug) was obtained from ICN

Radiochemicals (Irvine, CA).
Eleh

Rainbow trout (Oncozhyoohos mykioo: RBT) were obtained
from Baldwin Fish Farming Enterprise, Big Rapids, MI. Fish
were held at 14 : 1° in 1,000 l tanks supplied with a
continuous flow of oxygenated, dechlorinated tap water. The
tanks were cleaned twice a week and the fish were fed (Purina
trout chow) to satiation three times weekly. The photoperiod
was kept constant at 12:12 hr, light:dark.
Elam um Is_olat12h

Hepatic plasma membrane fractions were prepared by the
following procedure. Fish were killed by a sharp blow to the
’head and their livers were immediately removed and placed in
ice-cold 0.25 M sucrose-SOmM Tris-HCl (pH 7.2) buffer. The
liver was dissected free of the gall bladder and connective
tissue, blotted dry and weighed. The livers were then finely
minced with scissors and gently homogenized in a loose fitting
Dounce homogenizer (2 passes) in 0.25 M sucrose-SOmM Tris-HCl,

pH 7.2, (1.5 w/v) containing the following protease

47
inhibitors: aprotinin (100 kallikrein units/m1) , leupeptin (10
ug/ml) and PMSF (2mM) . The homogenate was passed through
double layered gauze and the membranes were separated by
differential centrifugation as described by Lutz (1973) . The
purified membranes were resuspended to about 4-6 mg protein
per ml in 0.25 M sucrose-SOmM Tris-HCl (pH 7.2) which
contained protease inhibitors and aliquots were stored under
liquid nitrogen. Protein analysis was by the method of Lowry
et a1 (1951) with bovine serum albumin (BSA) as the standard.

mm

The binding of 125

I-EGF to rainbow trout hepatic
membranes was carried out by a modification of the procedure
of Carpenter (1985). Briefly, aliquots of membrane protein
(50 pg) were incubated in a Krebs-Ringer bicarbonate buffer
developed for salmon (Wolf, 1963), pH 7.4 with 0.1% BSA.
125I-EGF was added to each tube in the presence (nonspecific
bound) or absence (total bound) of a 1,000-fold excess of
unlabeled EGF. The assays were typically conducted at 24° for
180 min unless otherwise stated. The binding assays were
terminated by dilution with ice-cold buffer and the bound

125I-EGF was separated from the free 125

I-EGF by rapid
filtration through Whatman GF/F glass fiber filters. Each
filter was washed three times with 3 ml of ice-cold buffer and
counted for radioactivity in a Packard autogamma counter (70%
efficiency). Specific binding was calculated as the

difference between total and nonspecific binding and expressed

48

as fmol 125I-EGF bound/mg protein.

Binding and Statietieal Analxele

The binding curves were fitted and factors extracted by
use of the LIGAND/KINETIC computer program package
(McPhearson, 1985) . The association (K1) and dissociation (K_
1) constants were calculated by the KINETIC program and are

1 1 and min-1, respectively (Rodbard,

expressed as M- *min-
1984). The equilibrimm dissociation constant (KD) and the
apparent maximum binding capacity (Bmax) were calculated with

the LIGAND program (Munson and Rodbard, 1980). The KD and

125

B were expressed as nM and fmol of I-EGF bound per mg

max

protein, respectively. Individual saturation binding curves
were plotted according to the methods of Scatchard (1949) and
Klotz (1982).

Data were expressed as the mean 1 standard error of the
mean (SEM). Treatment differences were examined with either
Students-T test or by Duncan's New Multiple Range test (Steel
and Torrie, 1980). Differences were considered significant at
p5 0.05 for all statistical procedures.

RESULTS
Blaema Henhrane

The hepatic plasma membrane isolation procedure used
Yielded the majority of the EGF binding activity in the
fraction between the 10% and 32% layers of the sucrose

gradient. This fraction corresponds to the hepatic plasma

membrane fraction isolated and characterized by Lutz( 1973)

 

49

from rainbow trout. Due to the efficiency of our procedure
and the high yield of plasma membrane relative to liver
weight, we used this method in all later studies. The
duration of membrane storage did have a significant effect on
the binding characteristics in that after one month we
observed a 5% loss of specific binding activity; Thus, in all
of our assays we only used plasma membranes that had been
stored for less than one month.
Qatlnlzetism 9f fleeing

Preliminary studies were performed to identify optimal
conditions for EGF binding assays with rainbow trout (RBT)
membranes. Time course studies of the binding of EGF with
receptors exhibited temperature dependence for binding
kinetics. The rate of binding at 24° was more rapid than that
observed at lesser"temperatures (Fig. 3). A steady state for
binding was reached after approximately 200 min at both 14°
and 24P'while at.49 steady state was not observed until after
350 min. Analysis of the association data by KINETIC showed
that a two site model gave a statistically better fit than

that of a single site model. At 24°, the apparent association
rate constant (K11) for the high affinity site was 7.2 x 108M-

1*min-1 while the apparent rate constant for the low affinity

7

site (K12) was 1.01 x 10 M-1*min-1. The association rate

Constants at 4° and 14° were also calculated but only the 4°
rate constant was found to differ statistically from the value

Observed at 24° (Table 1) .

50

Figure 3. Association of ¥25I-EGF to RBT liver plasma

membranes. liver plasma membranes (50 pg) which
were incubated in triplicate for designated times
at 4°(o), 14°(A), and 24°(l) in 0.2 ml Krebs-Ringer
bicarbonate buffer (pH 7.2) with 0.1% BSA.
Incubations were started with the addition of

1° M (100,000 cpm) 1251-3617. Non-specific

8.3x10-
binding was determined in duplicate in the presence

of a 1,000-fold excess unlabeled EGF.

51

 

 

200

14f.-
4
24° .
—e

40

 

 

 

 

 

1 O

40’
I

l
O
(I)

160-
120-

(tIIaioxd Btu/393 10m)
DNICINIH 011103.13

350

250

I 200
TIME (min)

 

400

300

150

100

50

F

52

125

Figure 4. Dissociation of bound I-EGF from RBT hepatic

plasma membranes. After a standard binding assay,
125I-EGF bound to the membranes was displaced by
the addition of 4 ml of buffer which contained a
1000-fold excess unlabeled EGF. The amount of
bound activity was determined at various time
intervals by filtration and each activity

measurement was corrected for nonspecific binding.

Assays were conducted at 4°(o), 14°(A), and 243(l).

 

53

 

 

180 -

 

 

 

24°

1

T—fi

 

 

. I
o I h
.I
7“— r f I
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In N O) (D n
1— 1—

mezo-Id SUI/£03 tow)

DNICINIH DLrIIOEcIS

160

1 20
TIME (min)

T2

54

Table 1. Kinetic analysis of EGF binding to rainbow trout

hepatic plasma membranes.

 

 

 

Temperature Association Dissociation KD
(°¢) (M-l*min-1) (min-1) (M)
4 site 1 3.63x108 1.541410"3 4.22410"11
Site 2 3.61x106 2.33::10"1 6.4x10-8
14 Site 1 9.38x108 2.03::10"3 2.3x10'11
site 2 2.28x107 2.07::10"1 9.1::10'9
24 Site 1 7.24x108 2.29::10’2 3.2x10"11
Site 2 1.02x107 1.64x10-1 1.6x10'9

 

55

To examine the reversibility of RSI-EGF binding to

rainbow trout hepatic plasma membranes, dissociation

0

experiments were conducted at 4°, 14 , and 24° (Fig. 4). The

125I-EGF from the membranes was rapid with a

dissociation of
half-life of about 22 min. KINETIC analysis of dissociation
data statistically fit a two site model. At 24°, the
dissociation rate constant for the high affinity site (K_11)

was 2.29 x 10_2 min.1 while the low affinity site ( K_12) was

1.46 x 10-1 min-1. Using the dissociation and association
rate constants, estimates of the equilibrium dissociation
constants (KO) were calculated using equation 1 (Table 1)

KO = K_1/K1 (1)

125I-EGF with greater quantities of hepatic

Incubation of
plasma membrane protein showed that the level of total and
nonspecific binding were directly related to protein
concentration. This relationship was linear for both total
and nonspecific binding over a concentration range of 10 to
150 pg/200 pl assay volume. At greater concentrations of
protein, the relationship between nonspecific binding and
protein became nonlinear with nonspecific binding being
proportionally greater than that observed for total' binding.
This resulted in lowered protein-normalized specific binding
values.

Using a wide range buffer system which contained citrate,
Phosphate, borate and diethylbarbiturate counterions (Hock and

HDIlenberg, 1980) , the maximum specific binding was observed

 

56
at pH 7.2. The level of specific binding remained relatively
constant between pH 6.5 to 7.5 with only a 5% loss from the
optimum binding which occurred in this pH range. Above pH 7.5
or below pH 6.5 there was a dramatic, irreversible loss of
specific binding.

The presence of cations can affect the binding of EGF
with its receptor (Hock and Hollenberg, 1980) . In rainbow
trout, both monovalent (Na+,K+) and divalent (Ca2+, Mng,
Mn2+) cations resulted in a 1.5 to 2.0-fold increase in
specific binding over control values (Fig. 5). Divalent
cations were found to be more effective than the monovalent
cations since optimal binding was observed at a lower ionic
strength (0.05) in the presence of divalent cations than in
the presence of monovalent cations (0.2) . The stimulatory
effects of the individual cations at maximal effective
concentration were not additive and as a result, combinations
of various cations did not increase the observed specific
binding maxima above that seen with individual cations.

To confirm the specificity of the EGF ligand for the
'membrane receptor, a number of hormones and peptides were used
in displacement assays (Table 2). Of the hormones tested,
only unlabeled EGF, TGF-a and an EGF-fragment effectively

displaced the 125

I-EGF from the receptor. These results are
Similar to those observed in studies of mammals in that the
EGF receptor exhibits little cross-reactivity with other

bioactive peptides such as insulin, glucagon or FSH.

57

Figure 5. Effects of cations on the binding of EGF. (A)
Monovalent cations and (B) divalent cations.
Protein was incubated in 20 mM HEPES buffer (pH
7.2), containing 0.1% BSA and the indicated
concentrations of either NaCl, KCl, MgClz, CaClz,

or MnClZ. Each solution was incubated with 8x10-

10 125

M I-EGF in the presence or absence of 1000-

fold excess unlabeled EGF.

 

58

 

 

 

 

 

 

 

 

 

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59

Table 2. Effects of competing peptides and hormones on

binding of 12

membranes .

5

I-EGF to rainbow trout hepatic plasma

 

 

 

before

Competitors Concentration % Displacement
(pg/m1)
None 0 0
*4!
EGF 1 70
**
TGF-a 1 64
4*
EGF-Fragment 1 67
Insulin 50 5
Prolactin 50 5
Glucagon 50 3.5
LH 50 2.5
FSH 50 5
Cortisol 50 8
Dexamethasone 50 5
a.

Competitors were preincubated with membranes for 15 min

125I-EGF was added to start the reaction.

Significantly different from the control at p5 0.05.

II

60

Temperature is an important variable in the control of
EGF binding kinetics in mammalian species (O'Keefe et al.,
1974). This is of particular concern for fish which are
poikilothermic. To examine the effect of temperature on EGF
binding to rainbow trout membranes, association, dissociation
and saturation studies were conducted at 4°, 14°, 24° and 37°.
At 37°, for the first 10 min, the binding of EGF proceeded at
a rate that was not significantly different from that observed
at 24°. However, after this time , there was a dramatic loss
of specific binding such that over 70% of the maximal observed
binding at 10 min was lost in the following 10 min. The loss
of specific binding at 37° was irreversible in that when
these membranes. were preincubated at 24°, no specific binding
was observed. There was a small effect of temperature on EGF
binding kinetics and binding capacity (Tables 1 and 3) .
However, these effects were not of equal magnitude as that
which have been observed with mammalian receptors (Hook and
Hollenberg, 1980).

SateratienAnalme

The binding of 125

I-EGF to RBT hepatic membranes was
saturable (Fig. 6A) with nonspecific binding in the saturation
assays averaging 55% of the total binding. The half-maximal
binding in these assays was 1.5x10-9M 125I-EGF. Klotz plots
(data not shown) confirmed the saturation of the receptors

(Klotz, 1982). Scatchard plots were curvilinear (Fig.6B)

which indicates the presence of a heterogeneous population of

61

Table 3. Summary of the Dissociation constants (K and

125

D)

maximal binding capacity (Bmax) of I-EGF to RBT

membranes as determined by Scatchard analysis.

 

 

Temperature Kb Bmax _
(°C) (M) (fmol/mg protein)
. -11
4 Slte 1 2.81x10 1.74
site 2 2.624410"9 181.57
14 Site 1 4.78x10-11 11.40
Site 2 5.222410"9 282.60
24 Site 1 6.01x10-11 15.67
8

Site 2 3.13x1o' 259.07

 

Figure 6. Saturation curve of

62

125I-EGF binding to RBT

hepatic plasma membrane. Liver membranes (50 pg)
were incubated with increasing concentrations of
125I-EGF in 0.2 ml Krebs Ringer-bicarbonate buffer
(pH 7.2) with 0.1% BSA. After 120 min at 24°,
specific binding (fmol EGF bound/mg of protein) was
determined as outlined in "Material and Methods".
(Insert) Representative Scatchard plot of 125I-EGF

bound to hepatic plasma membranes of RBT.

ASS x 5 22252328 ewe
on on 8 on a... .8 cm 3 .o

 

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64

receptors. The equilibrium dissociation constants (K0) and
binding capacities (BM) at 24° were calculated to be 6.01 x
10-11 M and 15.7 fmol EGF/mg protein respectively for the high
affinity site and 3.13 x 10"8 M and 259.1 fmol EGF/mg protein
respectively for the low affinity site. To confirm that the
curvilinear nature of the Scatchard analysis was due to
multiple receptor sites and not due to site-to-site
interaction, the method of DeMeyts et al. (1976) was used.
This procedure is based on the kinetics of 125I-EGF
dissociation from its receptor in the presence of an
" infinite" dilution or dilution with the addition of an excess
of unlabeled EGF. Site-to-site interactions are indicated
when the dissociation rate of 125I-EGF statistically differs
between the two treatments. Results of this analysis indicate
the curvilinear Scatchard analysis was due to a heterogeneous
Population of receptors (Fig. 7). This conclusion agrees with
analyses conducted with LIGAND, which showed that the data
were best fit to a two receptor site model. Furthermore, Hill
Plots of the EGF binding data resulted in slope values that
were not significantly different from unity which indicates a
lack of site-to-site interaction (Cuatrecasas and Hollenberg,
1976).

Displacement of labeled EGF from fish receptors with EGF,
an EGF-fragment and TGF-a was conducted to confirm receptor
affinities (Fig. 8). Analysis of the displacement data by
LIGISND showed that a two site model best fit the data.

65

Figure 7. Kinetic analysis for site-to-site interaction of
EGF binding to RBT hepatic membranes. Membrane

125

protein (500 ug/ml) was incubated with I-EGF

(3.144410'lo

M) in the absence or presence of
unlabeled EGF (9.0x10-7M) for 180 min at 24° in a
total volume of 2.5 ml. .At the end of the
incubation, specific binding was determined with a
40 pl aliquot from each tube by filtration. This
value was considered as 100% at time 0. After the
incubation, 40 pl aliquots were diluted in 4 ml
buffer in the presence or absence of (1.6x10-7M)
unlabeled EGF. At indicated intervals, three tubes
of each set were filtered and counted. The
radioactivity on the filters, expressed as a
percentage of the radioactivity at 0 min, . are

plotted as a function of time after dilution (o)

and dilution plus unlabeled EGF (I).

66

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oop

 

(01111111151 "117111111 .10 11:10 %)
0111011151 OIJIDEdS .103

67

125

Figure 8. Representative displacement curves of I-EGF

from RBT hepatic membranes by EGF‘ (e) , an EGF-
fragment (4) and TGF-a (I). Nonspecific binding
was determined in the presence of a 1000-fold
excess of unlabeled EGF. Data are plotted as the
relative decrease of saturable binding in the
presence of different concentrations of unlabeled

displacers.

68

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69

Displacement Of 125I-EGF by unlabeled EGF had an IC

50
of1.48x10-']‘1 M for the high affinity site and an IC50 of
1.03x10-9 M for the low affinity site. The EGF-fragment,

which contains the binding portion of the EGF ligand,

125

displaced the I-EGF with about equal affinity as that of

the unlabeled EGF. The high affinity site had an IC50 of
11

M while the low affinity site had an IC50 of
1.59x10"9 M. Displacement with TGF-a was similar to that

2.01x10'

observed with unlabeled EGF. IC for the high affinity site

50
was 1.12x10-11 M while the ICso for the low affinity site was
9.11x10-9 M. The results of the displacement analyses

indicate that both EGF and TGF-a compete for the same receptor
sites with about equal affinity. Furthermore, the
displacement assays confirm our previous finding of the

existence of two EGF receptor classes in RBT liver membranes.

DISCUSSION

This is the first published report on the
characterization of EGF binding sites in RBT. Of particular
significance is that the binding of mouse EGF to trout
receptors is very similar to that observed in mammals. In
RBT, EGF-receptors consist of two classes of receptors that
have different, but fixed binding affinities (King and
Cuatrecasas, 1982). The apparent dissociation constant for
the high and low affinity sites of EGF receptors in RBT fall

within the ranges reported for mammals (Gregoriou and Rees,

70

1984). For human liver, the apparent dissociation constant

(19,) for the low affinity site was 1.4 x 10"9

10

M while that for
the high affinity site was 1.08 x 10- M (Lev-Ran et al.,
1984). The observation of two receptor classes differs from
that of Naftel (1989) for dogfish liver. Reasons for this
«discrepancy are unknown. Based on the results of the kinetic
(experiments (DeMytes et al., 1976), the observed
curvilinearity of the Scatchard plot was due to different
receptor classes and not an artifact of our binding protocol.

The EGF kinetics of association and dissociation with
receptors in RBT are similar to those observed in mammalian
‘tissues. The rate constants for association (K1) are 1.56 x

8 1 1 8 1 1

10 M- *min- and 6.0 x 10 M- *min- for human placenta and

mouse liver, respectively. The rate constants for dissociation

2 -1 2 -1 for human

(K_1) are 3.7 x 10- min and 3.7 x 10- min
placenta and mouse liver, respectively (Hock and Hollenberg,
1980: O'Keefe et al., 1974). Unlike mammals, temperature
caused only a small shift in RBT binding kinetics over the
temperature range we studied.

EGF bound maximally to RBT liver membranes at pH 7.0 and
at slightly basic conditions (pH 7.5). No other information
is currently available for comparison in fish but, human
placenta receptors bind with a pH optimum of pH 7.6. Like
fish, the EGF binding with receptors remained stable over the

same pH range, pH 6.0 to pH 7.5 (Hook and Hollenberg, 1980).

71

The ionic requirements of EGF-binding are similar to
those reported for human placental tissue. Optimum binding
was observed at an ionic strength (I) for monovalent cations
of 0.1 for human placenta while optimal binding in RBT was
observed at an ionic strength of 0.2. For divalent cations,
optimal binding of EGF occurs at an ionic strength of 0.015
for placental membranes while in RBT it was 0.05. While the
cations are not required for EGF-binding in RBT and human
placental tissues, they do increase the binding maximum above
that of the control values.

The binding of EGF to RBT receptors is very specific in
that only unlabeled EGF, an EGF-fragment and TGF-a displaced
the 125I-EGF. TGF-a is a single chain polypeptide that is
structurally and functionally related to EGF and may be
important in fetal development (Marquardt et al., 1984: Pike
et al., 1982). In displacement studies with rainbow trout
hepatic membranes, TGF-a and unlabeled EGF displaced 125I-EGF
from the receptors with about equal efficacy in that they had
comparable ICSOs. These results are consistent with
'observations of the behavior of EGF in mammalian tissues.

Before a binding site can be considered a
physiologically relevant receptor, various criteria must be
met. These criteria include: 1) that the ligand bind to the
receptor with high affinity: 2) that the binding be saturable
and reversible: 3) that there be a high degree of chemical

specificity for the receptor in regards to binding to the

72
receptor by other ligands, and 4) the binding of the ligand
results in a physiological effect (Laduron, 1984). The first
three criteria have been met in this study. To study the
fourth criteria we are now examining the effect of EGF’binding
on the activation of a tyrosine kinase that is associated with
the EGF receptor in RBT hepatic membranes. However, the EGF
binding properties of the RBT hepatic membranes in this study

lzsI-EGF

were consistent with the premise that the site of
binding represents a form of EGF receptor that is homologous

to that found in mammalian EGF-receptors.

73

REFERENCES

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receptors. Molo Cello Biooneno 34, 129-152.

.Ahren, B. (1987). Epidermal growth factor (EGF) inhibits
stimulated thyroid hormone secretion in the mouse.
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Carpenter, G. (1985). Binding assays for epidermal growth
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Carpenter, G. and Cohen, S. (1979). Epidermal growth factor.
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Cohen, S. (1983). The epidermal growth factor (EGF). Canoe;

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Das, M. (1983). Epidermal growth factor receptor and

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74
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DeMeyts, P., Bianco, A.R., and Roth, J. (1976). Site-site
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Downward, J., Parker, P., and Waterfield, M.D. (1984). Auto-
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Gregoriou, M. and Rees, A.R. (1984). Properties of a

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Klotz, I.M. (1982). Numbers of receptor sites from Scatchard
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Lev-Ran, A., Huang, D., Josefsberg, z., Barseghian, G.,

Kemenyy M., Meguid, M., and.Beatty, D. (1984). Binding'of

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[awry

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Marq

McPh

Runs

Raft

O'Ke

Pike

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Lutz, F. (1973). Isolation and some characteristics of liver
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(1984) . Rat transforming growth factor type 1: Structure

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McPherson, G.A. (1985). Analysis of radioligand binding

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binding systems. Anglo Bioonono 107,220-239.

Naftel, J. (1989). Personal communication.

O'Keefe, E.J., Hollenberg, M.D., and Cuatrecasas, P.

(1974). Epidermal growth factor: Characteristics of

specific binding in membranes from liver, placenta and

other target tissues. Arehl.fiieehen1 fiioonyoo 164, 518-

526.
Pike, L.J., Marquardt, H., Todaro, G.J., Gallis, B.,

76
Casnellie, J.E., Bornstein, P., and Krebs, E.G. (1982).
Transforming growth factor and epidermal growth factor
stimulate the phosphorylation of a synthetic tyrosine-
containing peptide in a similar manner. J; Biol; gnono
257,14628-14631.

Rodbard, D. (1984). Lessons from the computerization of a
radioimmunoassay: An introduction to basic principles of
modelling. In " Computers in Endocrinology", (D. Rodbard
and G. Forti, Eds.) Raven Press, New York.

Scatchard, G. (1949). The attraction of proteins for small
molecules and ions. Ann; Nile Aooo‘ Solo 51, 660-672.

Soderquist, A.M., and Carpenter, G. (1983). Developments in
the mechanism of epidermal growth factor activation:
Activation of protein kinase by epidermal growth factor.
Ego; £229; 42, 2615-2620.

Steel, R.G. and Torrie, J.H. (1980). "Principles and
Procedures of Statistics". 2nd Ed. McGraw-Hill Book.Co.,
New York.

Wolf, K. (1963). Physiological salines for fresh-water

teleosts. The frog; Eléh gnlto 25, 135-140.

CHAPTER 2

EPIDERMAL GROWTH FACTOR RECEPTOR-PROTEIN KINASE

INTERACTIONS IN THE LIVER MEMBRANES OF RAINBOW

TROUT (W W)

p01
in

Coh
spe
cel
eff
oua
ara
Ree
gas

198

SUr
ran
rec

dff

a 1;
Sch

INTRODUCTION

Epidermal growth factor (EGF) is a 6,045 Dalton
polypeptide that has mitogenic and growth stimulatory activity
in cells of epidermal and epithelial origin (Carpenter and
Cohen, 1979: Gospodarowicz, 1981). Binding of EGF to a
specific receptor located on the plasma membrane of responsive
cells will cause a number of biological responses. In gitro
effects include increased amino acid transport, activation of
ouabain-sensitive Na+/K+ ATPase activity , stimulation of
arachidoniate metabolism and 'DNA synthesis (Gregoriou and
Rees, 1984). In yiyo responses include the inhibition of
gastric secretion, altered endocrine function (Ritvos et al. ,
1988) , and the stimulation of growth hormone synthesis
(Schonbrunn et al., 1980). These responses are mediated by
surface receptors whose affinities (K0) for EGF are in the
range of 10° M'1 to 5x109 M'1 for a major class of low affinity

'1 for a minor class of high

receptors and 1010 to 1011 M
affinity receptors (Gregoriou and Rees, 1984).

The EGF receptor is a transmembrane glycoprotein that has
a ligand-dependent tyrosine kinase activity (Carpenter, 1987:
Schlessinger, 1988) . The mature receptor is synthesized from

a precursor (134 KDa) which is N-glycosylated to 170 KDa.

78

Wh
th‘
d0‘
re
bi
ac
tr
wh
be
an
10
ac
bi
si
SY

19

0f
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3&1
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rec
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re;

79

While it has no subunit structure, it can be divided into
three major domains based on structure and function. The
domains are as follows: ( 1) The N-terminus extracellular
region which is heavily glycosylated constitutes the ligand
binding domain: (2) a unique hydrophobic transmembrane region
acts in anchoring the receptor and is also involved in signal
transduction: and (3) the C-terminus or intracellular region
which contains a tyrosine kinase. In mammals, this kinase has
been shown to phosphorylate cytosolic proteins as well as
autophosphorylating four tyrosine residues located at 1068,
1086 1148 and 1173 (Margolis et al., 1989). It is the
activation of the tyrosine kinase that is essential for the
biological function of the EGF receptor and for its role in
signal transduction, receptor trafficking, stimulation of DNA
synthesis and cellular transformation (Moolenaar et al.,
1988).

We have previoulsy reported that hepatic plasma membranes
of rainbow trout bind mouse 125I-EGF (Newsted and Giesy, 1991) .
The binding of 125I-EGF to the membranes was peptide-specific,
saturable, reversible and of high affinity. Scatchard and
kinetic analysis of the binding data indicated - multiple
receptor sites of differing affinity. Apparent dissociation
constant for the high affinity (KD- 2.3x10'11 M) and low
affinity (K,,=9.1x10'9 M) sites were in agreement with values
reported for mammalian species. Maximal binding capacities

were 13 fmol EGF bound/mg protein and 270 fmol EGF bound/mg

protein
respecti‘
EGF cova
showed a
in sampl<
In
phosphor
in the k
Paramete
incubati
and Dept
have als
the Phos
Studies,
binding
demonstr
an intr.
biOchemi

mammal-18

80

protein for the high and low affinity receptor sites,
respectively. Furthermore, SDS-PAGE electrophoresis of 125I-
EGF covalently linked to the receptor in trout membranes
showed a band that corresponded to 150 KDa that was displaced
in samples incubated with 1,000 fold excess of unlabeled EGF.

In this study we characterized an EGF-stimulated
phosphorylation reaction and its relationship to EGF binding
in the hepatic plasma membranes of rainbow trout (RT-HPM).
Parameters that we have evaluated include the effect of
incubation buffer and time, ATP concentration, other hormones
and peptides and EGF on the phosphorylation of RT-HPM. we
have also examined the protein tyrosine kinase associated with
the phosphorylation induced by the binding of EGF. These
studies, in conjunction with studies that characterized the
binding of EGF in RT-HPM (Newsted and Giesy, 1991) have
demonstrated that rainbow trout have an EGF receptor that has
an intrinsic tyrosine kinase activity that is similar in
biochemical activity' to ‘that. which is observed in :many

mammalian species and cell lines.

MATERIAL AND METHODS
Hateriale
Receptor grade EGF, insulin, prolactin, glucagon, FSH,
LH, dexamethasone, angiotensin II, [ValSJ-angiotensin II,
phosvitin, casein, and histone H1 type III were purchased from

Sigma (St. Louis, MO.) . Transforming growth factor-0 (TGF-a)

was purcha
200 uC/ug)
(Boston,

dihydroxy«
tryphosti1
aminobena
protein k.
protein

Laborator-

receptor

81

was purchased from Calbiochem (San Diego, CA). ”51

-EGF (150-
200 pC/pg) and 32P[1]ATP (3000 Ci/mmol) were purchased from NEN
(Boston, MA). Protein kinase inhibitors methyl 2,5-
dihydroxycinnamate (MDHC), lavendustin A, genistein,‘
tryphostin, RCMA-lysozyme, 2 hydroxy-5-(2,5-dihyroxybenzyl)
aminobenzoic acid (HDAA) and PKC inhibitor (19-36) as well as
protein kinase substrates RR-src and acetylated- myelin.basic
protein (Ac-MBP) were purchased from Gibco-BRL (Gibco
Laboratories, Grand Island, NY). Phosphotyrosine and EGF-

receptor antibodies were purchased from Oncogene.

Eieh

Rainbow trout (Qnoornynonns nykioo) were obtained from
Stony Creek Trout Farm, Grant MI. Fish were held at 12 i 1 C
in 1,000 liter tanks supplied with a continuous flow of
oxygenated, dechlorinated tap water. The tanks were cleaned
twice a week and fish were fed (Silvercup trout Food) to
satiation twice weekly. The photoperiod was 16:8 hr
(light:dark).

Elaena Membrane Ieelatien
RT-HPM were prepared by the method of Newsted and Giesy

(1991). This method was based on the isolation of crude
membranes by differential centrifugation followed by a final
purification step by centrifugation through a sucrose

gradient. The purified RT-HPM were resuspended to about 4-6

 

mg protein
containing
ug/ml) and

the RT-HPM

 

analysis we

serum 8113111

3133 m
Bind-
Procedure
“actions
Hepes (T
No“Speci
°°ntain,
assaYs
buffer
by rapi
Each f_
and tb
authBE
Galen?

bindi

%

fiha

82
mg protein in 50 mM Tris-HCl,(pH 7.2) 0.25 M sucrose buffer
containing aprotinin (100 kallikrein units/ml), leupeptin (10
pg/ml) and phenylmethylsulonyl fluoride (2 mM). Aliquants of
the RT-HPM were then stored under liquid nitrogen. Protein
analysis was by the method of Lowry et al (1951) with bovine

serum albumin (BSA) as the standard.

HereinainsAesax

Binding assays were conducted by a modification of the
procedure of Carpenter (1985). Briefly, triplicate binding
reactions were carried out in 100pl volumes containing 20 mM

12°I-EGF.

Hepes (pH 7.4), 0.1% BSA, 50 pg RT-HPM, and
Nonspecific binding was measured in triplicate tubes which
contained a loo-molar excess of unlabeled EGF. The binding
assays were terminated by dilution with ice-cold binding
buffer and the bound 125I-EGF was separated from the free EGF
by rapid filtration through Whatman GF/F glass fiber filters.
Each filter was washed three times with 3 ml ice-cold buffer
and the filters were counted for radioactivity in a Packard
autogamma counter (70% efficiency). Specific binding was
calculated as the difference between total and nonspecific

125

binding and expressed as fmol I-EGF bound/mg protein.

standard Rheeoherrlatien Aeeaxe
Assays of RT-HPM phosphorylation were conducted in a

final volume of 60 pl in microfuge tubes. Reaction mixtures

contai
MgSO,,
(2.0x]
the p]
reacti
C for
#1 al
were i
(10 m]
phOSpI
min)

SCint:
cockt;

quant;

was 31
(Laemj
Was n
with
Stain.
with .
exDos.
inCQr]
the

s°int

83

containing 20 mM Hepes buffer (pH 7.2), '5 mM MnSO‘, 20 mM
11950,, 100 pM Na3v0,, 0.05% Nonidet-P40 and 10 pM “VP-ATP
(2.0x105 cpm) were placed on ice and incubated for 10 min in
the presence or absence of 0.5 pM EGF. The phosphorylation
reaction was initiated by the addition of ATP and incubated 4
C for 15 min. The reaction was terminated by pippetting a 50
pl aliquot onto Whatman P81 phosphocellulose filters which
were immediately immersed in a beaker of 75 mM phosphoric acid
(10 ml/filter). Filters were washed 3 times (10 min each) in
phosphoric acid at room temperature, extracted with acetone (5
min) and air dried. The dried filters were placed in
scintillation vials and 7 ml of Safety Solv scintillation
cocktail was added to the' vials. Radioactivity was
quantitated by liquid scintillation counting.

In a separate set of phosphorylation assays, the reaction
was stopped by the addition of 30 pl of 4x SDS sample buffer
(Laemmli, 1970) and heated at 100 C for 5 min. Each mixture
was run on 7.5% SDS-polyacrylamide gels. The gels were fixed
with acetic acid:water:methanol (10:40:50 by volume) and
stained with Coomassie Blue R-250. After extensive washing
with 7% methanol/7% acetic acid (v/v) , the gels were dried and
exposed to X-ray film for autoradiography. To quantitate 32P
incorporated into specific proteins, bands were excised from
the dried gel and radioactivity counted by liquid

scintillation counting.

was an

Tyros.
filter papt
KPH (25 #9?
buffer in

substrate .

   
  
  

reaction w
ATP, 1000-

min at 0°C

 

 

84

Pentiae anesthemtlatien Assays
Tyrosine kinase activity was quantitated by use of a
filter paper assay (Pike, 1987) . Triplicate aliquants of RT-
HPM (25 pg) were incubated in standard phosphorylation assay
buffer in the absence and presence of a phosphorylation
substrate. The final volume of the reaction was 60 pl. The
reaction was initiated by the addition of ATP (50 pM [1-32P]
ATP, 1000-2000 cpm/pmol) and the tubes were incubated for 15
min at 0°C. Reactions were terminated by addition of 150 pl
5% trichloroacetic acid-1.65 Mm KHZPO, and 10 pl of 10 mg
BSA/ml. Samples were centrifuged at 10,000xg for 15 min at 4
C. Two 70 pl aliquants of supernatant were spotted onto 2.3
cm filter disks (Whatman P81). The filters were washed 3
times for 10 min each at room temperature in 75 Mm phosphoric
acid, once with acetone and air dried. Filters were then
counted by liquid scintillation counting. Protein kinase
activity was calculated as the difference in 32P incorporated
in the presence and absence of substrates and expressed as

pmol 32P incorporated per min per mg protein.

EheenheanineAeidAnalxeie

Phosphoamino acid analysis was based on a modified
procedure of Ushiro and Cohen (1986) . szP-labeled membranes
from the standard phosphorylation assays were hydrolyzed in
100 pl 6 N HCl at 100 C for 2 h in sealed tubes. The

hydrosylates were dried under vacuum and dissolved in 25 pl of

water CC
phospho:
chromat<
system c
acid/wa1
ninhydr:
autorad:
amino a
spots j

scintil}

Lem
RT-
prepare,
Laemml 1
wells
electro]
electro]
Perform
SDS‘PAG1
(100 v 1
contain:
Tris/ O . g
powdere<
01.79,..nigI

5 min ar

85
water containing phosphoamino acid standards (phosphotyrosine,
phosphoserine and phosphothreonine). Samples were
chromatographed on cellulose thin layer plates with a solvent
system consisting of n-butyl alcohol/isopropyl alcohol/formic
acid/water (3:1:1:1, v/v) . The standards were located by
ninhydrin while radioactive material was located by
autoradiography using X-ray film. 3zP-incorporated into the
amino acids was quantified by scrapping the radiolabeled
spots into scintillation vials and counting by liquid

scintillaton .

Eastern Analyeie at Rheenherxlated Membranes

RT-HPM were phosphorylated in a standard assay and
prepared for electrophoresis by heating at 100 C for 5 min in
Laemmli SDS sample buffer. RT-HPM (50 pg) was applied to
wells of the 4.5% stacking gel and subjected to
electrophoresis on a 7.5% resolving gel in a Mini Protean
electrophoresis unit (Biorad, Melville, NY). Immunoblots were
performed according to the method of Towbin et al. (1979) .
SDS-PAGE separated proteins were electophoretically transfered
(100 V for 1 h) to nitrocellulose. The nitrocellulose sheets
containing the transfered proteins were rinsed in 0.02 M
Tris/0.5 M NaCl (TBS), pH 7.4 for 5 min, blocked in 5%
powdered non-fat milk in TBS containing 0.5% Tween 20
overnight at 4 C. The sheets were rinsed 3 times with TBS for

5 min and then incubated with the primary antibody for 1 h at

86
room temperature with gentle shaking. The sheets were then
rinsed three times with TBS and incubated with horseradish
peroxidase-linked IgG (Immunoselect system, Gibco, Grand
Island, NY). Visualization and quantification were performed

by autoradiography followed by densitometry.

statistieal Analysis

Results are expressed as means i S.D. for each measured
parameter. Statistical analyses were performed using
Student's T test, analysis of varience and planned paired
comparisons (Steel and Torrie, 1980). The level of

significance to detect differences between treatments was

p < 0.05.
RESULTS
binding at Efrzner te treat membranes
125

The rate of binding I-EGF to RT-HPM was rapid and
temperature dependent (Fig. 9). At 24 C, steady state binding
was reached at approximately 200 min with a very rapid
'increase in EGF binding occurring in the first 25 min. At 4
C, the binding of EGF to RT-HPM proceeded at a much slower
rate with steady state being attained by 350 min. Half
maximal binding at 0 C occurred by 1 h while it was attained
by 15 min at 24 C. At 0C the rate of EGF binding for the
first 30 min was 1.03 fmol EGF bound/min/mg protein while the

rate was 3.13 fmol EGF bound/min/mg protein at 24 C.

87

Figure 9. Time course of us

I-EGF binding to RT-HPM. Binding

reactions were initiated by the addition ofmI-EGF

(8.3 x10"°M, 100,000 cpm) to standard reaction

mixtures. At the indicated times, triplicate tubes

for total and nonspecific binding were quantitated.

 

 

 

 

 

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89
Time tenrse 9.: Membrane RheenhenLlatien

The incubation of RT-HPM with [1329]“? resulted in the
incorporation of radioactivity into phosphoric acid insoluble
material at 0 C and 24 C (Fig. 10). The addition of EGF to
the reaction at both temperatures stimulated a 4- to 5-fold
increase in the phosphorylation of endogenous membrane-
associated.proteinsu In the presence of'EGF, the initial rate
of phosphorylation was increased from 24.2 pmole [32P1phosphate
per min per mg protein at 0 C to 36.1 pmole fnPJphosphate per
min per mg protein at 24 C (Fig. 10A and 10B). While the
difference in the phosphorylation between the two temperatures
was small, it was stastistically significant (p < 0.05). At
both temperatures, the‘addition.of EGF'to the samples resulted
in a stimulation of phosphorylation that reached a maxmimum
level by 120 min.

The rate of dephosphorylation of 32P0‘ from RT-HPM was
rapid (Fig. 11). After 40 min at 0 C, approximately 15% of
the radioactivity incorporated into membranes in the basal
phosphorylation reactions was displaced by the unlabeled ATP.
Approximately 25% of the radioactivity was displaced in the
EGF-treated membranes incubated at 0 C with unlabeled ATP.
While the extent of dephosphorylation was affected by the
presence of EGF, the rate of dephosphorylation between the two
treatments was kinetically and statisitcally similar. In both
EGF-stimulated and basal phosphorylation reactions there was

a significant loss in radiolabeled phosphate from RT-HPM due

90

Figure 10. The incorporation of (”PJ-phosphate into RT-
HPM from labeled ATP in the presence of EGF.
A. Incubations were carried out at 0 C: B.

Incubations were carried out at 24 C.

mate into E.
asence of ..
it at 0 C:i

24 C.

 

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I'd 160
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02
52. 4° ‘
o L L a l L. L L L L J L L
O 20 4O 50 80 100 120
TIME (111.111.)
r 1 I f r 1 r F
200 - 3 +EGF ‘

d

 

 

[SB-P -INCO 01111111)
] [pmollfi
.1;
O

 

 

 

 

 

80 .1
w d
0 . L
0 20 4O 60 80 100 120
TIME (min)

Figure

11.

92

Time course for membrane dephosphorylation in
the presence or absence of EGF.
Dephosphorylation was monitored by adding 1.5
mM ATP. At the indicated times, triplicate
tubes with and without ATP were removed and

. 32
the membrane assoc1ated P was measured.

93

om

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NOLLVHOdHOONI 381

94
to phosphatase activity. Incubations conducted at low
temperatures, 0 to 4 C did not reduce the amount of
phosphatase activity. To reduce phosphatase activity in our
phosphorylation reaction, we included 100 pH of orthovanadate
that is a potent inhibitor of phosphatase activity (Pike,

1987).

Effeet af 1193’ e119. mi on W

EGF-stimulated phosphorylation reactions in RT-HPM
require the presence of Mg2+ or Mn?” (Fig. 12) . In the absence
of added metal ions, only trace phosphorylation activity
wasobserved in EGF-stimulated RT-HPM. The optimum Mgzi
concentration for EGF-induced phosphorylation reactions was
approximately 60 mM while for Mnait was 30 mM. The
stimulatory effect of Mn°+ and "92+” in EGF-mediated-
phosphorylations are seen at all concentrations tested. In
reactions conducted with an’, the values of basal
phosphorylation remained small relative to EGF-stimulated
values over the concentration range. This was not seen with
”92+ where the values of basal phosphorylations increased with
increasing M92. concnetrations relative to EGF-stimulated
values. In the presence of 10 mM EDTA, EGF stimulated and
basal phosphorylations were completely inhibited. The combined
effect of “92+ and Mn2* on EGF-stimulated phosphorylation was

nonadditive with nonEGF-stimulated reactions values remaining

constant over the cation concentration range tested. No

Figure 12.

95

Effects ofMg2+ and Mn2+ concentrations on EGF
stimulated-phosphorylation of RT-HPM.
Standard assays were carried out at 0 C for 30
min except that the assay‘ buffer' did not

contain any divalent ions.

96

 

 

m

b D

m

”Bole
zoFéommooz

a
2

 

m w

1

fauna

l
100

MAGNESIUM CONCENTRATION
[mM]

120

80

20

 

 

 

  

 

        
   

20 30 50 60
MANGANESE CONCENTRATION
[mM]

10

 

 

w . m . m . m . m . m
2 2 2 cl al
70353
ameéodmooaufinma

97
effects on EGF-induced phosphorylations were noted with the

addition of Ca” (0.1 to 20 mM), Na+ or K’ (10 to 200 mM).

Effects of ATP and £91: eoneentratiens en Rhesbhemlatien

Both nonEGF- and EGF-stimulated phosphorylation reactions
were increased at ATP concentrations of 10 to 120 pM (Fig.
13). Subtraction of the nonEGF- from EGF-stimulated RT-HPM
reactions showed that EGF-induced a Constant 1.25-fold
increase in phosphorylation over the ATP concentrations used
in this study. Lineweaver-Burk plots of the phosphorylation
data exhibited a Km of 1.25 x10°M with a Vmax of 318 x10°M
per min in EGF stimulated membranes whereas the nonEGF-treated
membranes had a Km of 1.19 x10°M and a Vmax of 273 xloflM per
min. This suggests that EGF did not significantly alter the
Km of the phosphorylation reaction for ATP but increased the
Vmax of the reaction.

The phosphorylation of hepatic plasma membranes of
rainbow trout was dependent on the concentration of EGF (Fig.
14). Maximal stimulation of the phosphorylation of RT-HPM
occurred at about 22 nM EGF while half maximal stimulation
occurred at approximately 4 nM EGF. At 100 nM EGF, there was
some inhibition of the phosphorylation of RT-HPM.

The properties of rainbow trout HPM were studied in the
Presence or absense of EGF by three methods: SDS-acrylamide
Electrophoresis, autoradiography and. western blot analysis

(Fig; 15). In lanes E and F, three major phosphorylated

Figure 13.

98

Effect of ATP concentration.on phosphorylation
of rainbow trout membranes. ATP was added at
the indicated final concentrations with
labeled ATP (1 pCi/ tube) and the assays were

conducted at 0 C for 30 min.

99

OO—
_

~23
22.252528 a:

on 00 o¢

 

 

hum+

_ . q q _

 

O
‘-

O
N

on

[d—zs]

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CIEIVHOdHOONI-

100

Figure 14. Effect of EGF concentration on the
phosphorylation of rainbow trout hepatic

plasma membranes.

 

 

 

 

 

o L .2
N
[SGIONd
OEIVHOdHO0N1—[d—zs]

l
O
1-

 

60 80 100
EGF CONCENTRATION
[nM]

40

20

Figure

15.

102

Western and autoradiographic analyses of EGF-
stimulated RT-HPM. Membranes were subjected
to the standard phosphorylation procedures in
the presence and absence of EGF for 30 min at
0 C. Lanes: A. A-431 membranes probed with
AB-4 antibody; B. rainbow trout membranes
probed. with. AB-4 antibody: C and D,
phosphotyrosine antibody western of rainbow
trout membranes phosphorylated with (C) and
without (D) EGF; E and F, autoradiography of
membrane components phosphorylated in the

presence (E) and absence (F) of EGF.

103

f EGF-

jected

ms in

min at

{d with

a has: 3.279....—
. s . .

on.o§<

Ibranes

nd DI

     
    

:ainboi’

    

 

' + . 1

so:
after... 5v<

C) and
in the

why at

 

104

bands can be observed that have molecular weights of
approximately 150,000 116,000 and 66,000 daltons. No other
phosphorylated bands were observed in either lane of the
autoradiograph. Westerns of RT-HPM using a phosphotyrosine
monoclonal antibody showed a major band at 180,000 daltons
with a minor set of doublets at approximately 130,000 and
116,000 daltons. Western analyses conducted on duplicate gels
with an EGF-receptor monoclonal antibody (AB-4) exhibited
protein bands at 150,000, 116,000, 105,000 and 66,000 daltons.
The 180,000 band seen in lanes C and D corresponds to the EGF
receptor band observed in lane A for A-431 membranes.

Phosphoamino acid analysis was conducted on the 180,000
protein band to examine further the increase in EGF receptor
phosphoamino acid concentration. Results showed that in EGF-
stimulated samples there was a greater than 2-fold increase in
phosphotyrosine. The level , of phosphothreonine and
phosphoserine in this band remained relatively constant with

only about a 10% increase in their labeled phosphate content.

Effeet 9i Hmeleetides en Membrane Rhesmherxlatien

All protein kinase-mediated phosphorylation reactions are
dependent upon the presence of triphosphate nucleotides (Cohen
et al., 1980). In our assays, the presence of either cyclic
AMP or cyclic GMP did not affect the phosphorylation of RT-HPM
(Table 4). Also, in the presence or absence of EGF, the

addition of cyclic nucleotides, (10-3 to 10'5 M) did not

105

Table 4. Competition among nucleosides in the phosphorylation

of RT-HPM with labeled triphosphate nucleosides.

 

 

 

Labeled Unlabeled 32P Incorporated
Nucleoside Nucleoside -EGF +EGF
[gamma-”P]ATP 853 1329
ATP 693 689

GTP 857 1003

CAMP 874 1260

CGMP 853 1210

[alpha-RP1ATP 1033 1045
ATP 1319 1300

GTP 1165 1176

CAMP 1097 1108

CGMP 1053 1060

Reaction mixtures were incubated with either or

[1-32P1ATP (30 pH, 1.15 x10° cpm) in the presence or

absence of unlabeled nucleosides.
were added at a 20-fold excess

initiating the reactions.

(600 pM)

Unlabeled nucleosides

prior to

106
stimulate the phosphorylation activity in RT-HPM
preparations. GTP, which in many biochemical reactions can
substitute for ATP, also did not alter the phosphorylation of
basal or EGF- stimulated rainbow trout membranes.

In. mammals, EGF-induced phosphorylation of the EGF
receptor and of membrane proteins is dependent upon the
presence of [1-RPJATP. To examine the specificity of RT-HPM
phosphorylation labeled and unlabeled triphosphates were used
in a standard assay. Phosphorylation of membrane components
by [V-RPJATP is significantly reduced by a 20-fold excess of
unlabeled ATP (Table 4) while GTP reduced the level of
phosphorylation to a lesser extent. [a-szPJATP was
anineffective donor of phosphate groups to RT-HPM either in

the presence or absence of EGF.

Hormonal fineoifioitx in Treat Membranes

A wide variety of peptide hormones were used to examine
the hormonal specificity of the activation of RT-HPM
phosphorylation (Table 5). Basal phosphorylation activity of
'rainbow trout membranes was not affected by insulin,
prolactin, glucagon, FSH, LH and dexamethasone. EGF, and EGF-
fragment (EGFMB) and a-TGF were effective in stimulating the
phosphorylation of rainbow trout membranes. 'These compounds
have previously been shown to have biological activity in the
activation of phosphorylation of A-431 membranes (Carpenter et

al., 1979) as well as having the ability to bind to the EGF

107

Table 5. Efficacy of hormones to stimulate phosphorylation

 

 

 

of RT-HPM.°
Hormone :nP-Incorporated
(0PM)
None 354 (30)
EGF 672 (110)'
Insulin 345 (15)
Prolactin 346 (98)
Glucagon 313 (51)
FSH 321 (25)
LR 326 (41)
Dexamethasone 329 (46)
TGF-a 394 (35)'
EGF-fragment i ' 452 (41)'
Reactions were carried out at 0°C for 30 min. EGF, EGF-

fragment and.TGF-a were added at a final concentration.of
40 ng/tube. All other hormones were added at 100
ng/tube. Results are reported as averages of triplicate
determinations.

° Numbers in parentheses are standard deviations

* Significantly different from control at p < 0.05.

108

receptor in RT-HPM (Newsted and Giesy 1991).

sobetrate Soeoifieitx in Membrane Rhosnhorxlation

The activation an EGF-stimulated tyrosine kinase was
examined using several specific and nonspecific kinase
substrates (Table 6). RT-HPM readily phosphorylated [ValSJ-
angiotensin II, angiotensin II, RR-Src, phosvitin, and to a
lesser extent casein and histone. Angiotensin I and the
protein kinase C substrate, Ac-MBP, were not significantly
phosphorylated in the presence of EGF. In mammalian EGF-
receptor systems, [ValSJ-angiotensin II is a better
phosphorylation substrate than angiotensin II, however,
rainbow trout membrane EGF receptors show no such preference
in that the overall extent of phosphorylation of
eithersubstrate was similar. Using equimolar concentrations
of substrates, the rates of EGF-stimulated phosphorylation of
substrate were examined in RT-HPM. Of the substrates tested,
RR-Src exhibited the greatest rate of phosphorylation (0.11
pmol°2PO,. incorporated/min/mg) while Histone H1 Type III had
the least (0.004 pmol 32P0‘/min/mg protein). The rates of
phosphorylation were also similar for [ValSJ-angiotensin II

and angiotensin II, 0.58 and 0.61 pmole/min/mg, respectively.

Effeetofisinaselnhibitorsonmosbhomlation
To further characterize the phosphorylation activity of

RT-HPM, we examined the effect of various kinase inhibitors on

109

Table 6. Phosphorylation of exogenous proteins by RT-HPM in

the presence and absence of EGF'.

 

Substrate Phosphorylation
(% of Control)

 

[Val°]-Angiotensin II 227 (35)'
Angiotensin II 239 (41).
Angiotensin I 95 (25)
Histone H1 Type III 105 (11)
Phosvitin 144 (23)'
Casein 122 (13)'
RR-SRC 347 (52)'
PKC Substrate (19-35) ' 109 (15)

 

° Phosphorylation assays were conducted at 0 C for 30 min.

The exogenous protein substrates were added at a final
concentration of 100 pM except for phosvitin, histone and
casein which were added at a final concentration of‘l mg/ml.
The results presented are averages of triplicate
determinations (S.D) of percent differences from control
‘values of tubes that did not contain EGF.

* Significance determined at P < 0.05.

110
the ability of RT-HPM to phosphorylate RR-Src in the presence
of EGF (Table 7). Of the kinase inhibitors studied, methyl
2,5-dihydroxycinnamate (MDCH), genistein, lavendustin A, and
quercitin significantly inhibited the EGF stimulated
phosphorylation. Tryphotin, HDAA, PKC inhibitor and ouabain
did not significantly inhibit the phosphorylation.activity of
RT-HPM. I
Discussion

The results in this report describe a system where the
binding of EGF is accompanied by the alteration of
phosphorylation in RT-HPM. These events suggest that in
rainbow trout, TCDD-induced alteration of EGF receptor
phosphorylation status may have an important role in the
modulation of extracellular signals that regulate cell
function (Cohen, 1983). Of the other hormonally responsive
systems that have been examined in mammals, only the insulin
receptor and its associated biological activity have been
examined in fish. Thus, this is the first report that
examines the EGF receptor and its associated tyrosine kinase
activity in rainbow trout.

In previous studies of the binding of EGF (Newsted and
Giesy, 1991) , we found that RT-HPM membranes bound

12sI-EGF bound/mg protein . This

approximately 250 fmol of
binding value of EGF to RT-HPM is equivalent to EGF binding
values in human fibroblasts but is approximately 100-fold less

than that observed in A-431 cells (Carpenter et al., 1979).

111

Table 7. Effect of protein kinase inhibitors on the EGF

stimulated phosphorylation of RT-HPM.

 

 

Kinase Inhibitor Concentration Kinase Activity
(I‘M) (Pm01/ mg )
Control 47.4 (6.53)
MDHC 50 17.4 (5.13)'
Lavendustin A 50 27.9 (5.74)'
Genistein 50 23.9 (10.4)'
Tyrphotin 50 19.5 (30.1)'
HDAA 50 39.3 (3.96)
pxc Inhibitor 50 40.8 (5.43)
Ouabain 100 64.8 (17.1)
Quercitin 100 4.5 (3.51)’
RCAM 50 10.8 (4.52)‘
CAMP Inhibitor 100 49.6 (10.2)

 

Significant differences from control values determined at

p < 0.05.

112

Binding of 125I-EGF results in the formation of EGF receptor
complexes that is accompanied by an 1.5- to 3-fold stimulation
in the phosphorylation of RT-HPM (Fig 10, A and B). The
increase in EGF-stimulated phosphorylation of RT-HPM was
observed by 2 min at 0 c and 24 C. The lack of difference
between the EGF stimulated phosphorylation rates in fish
membranes at both temperatures is interesting in that the
lower temperature reduced the rate and extent of EGF-
stimulated phosphorylation of mammal membranes (Carpenter et
al., 1979). However, the phosphorylation rate in RT-HPM was
slower than that observed in mammals where an increase in EGF-
stimulated membrane phosphorylation can be detected in less
than 1 min. These results point to a major difference between
cold-temperature adapted.water fish and mammals in regards to
possible membrane composition and its effect on enzyme
function at low temperatures.

EGF concentrations required for maximal and half-maximal
EGF-stimulated phosphorylation_ reaction values were
‘ approximately half the concentration necessary for maximal and
half-maximal binding of EGF to RT-HPM. Furthermore, at 0 C,
the binding of EGF to RT-HPM will have reached a steady state
under standard binding and phosphorylation conditions. From
these data we can conclude that complete saturation of the EGF
receptor sites is required for stimulation of phosphorylation
activity. The finding in RT-HPM is similar to that observed

in A-431 cells where there was a 7-fold difference between

113

half-maximal stimulation of phosphorylation and half-maximal
EGF binding saturation concentration (Carpenter et al. , 1979) .

EGF-stimulated phosphorylation reactions in RT-HPM were
dependent on the presence of Mg” and MnflL This observation
is consistent with data from mammalian studies that show Mg”’
and Mn2+ dependence of the EGF receptor-associated tyrosine
kinase activity (Hunter and Cooper, 1985) . Furthermore,
induction of basal phosphorylation reactions at high an'
concentrations was relatively low compared to the EGF-
stimulated reactions. This result is consistent with data
from mammalian studies that show Mn” inhibit ATPases and other
phosphorylation reactions in EGF-stimulated membranes.

Binding of EGF to RT-HPM was accompanied by the
incorporation of phosphotyrosine into a 180,000 dalton protein
that corresponded to the same molecular weight as that of the
A-431 EGF receptor (Fig. 15). This band was also observed in
westerns conducted with the EGF receptor monoclonal antibody
(AB-4). While a 180,000 dalton band was not evident in
radioactivity incorporated into EGF-stimulated. RT-HPM, a
150,000 daltons was observed in the autoradiographs. The
dichotomy between these analyses may indicate that there was
EGF receptor degradation occurring during our analyses. One
explanation for these results is that the phosphotyrosine-
antibody“may’have recognized other'phosphotyrosine-containing
proteins in our RT-HPM preparation while the AB-4 monoclonal

antibody would have recognized only the glycosylated epitope

114

of the EGF receptor of either intact or partially degraded
receptor. The existance of the 180,000 dalton protein band
in the phosphotyrosine and EGF receptor antibody westerns
supports the receptor degration hypothesis since both 180,000
and 150,000 dalton bands are observed in mammalian EGF
receptor where degradation has occurred (Carpenter, 1987).

The activation of tyrosine kinases is important in cell
proliferation, carcinogenesis and cell differentiation through
mechanisms seeming to involve the control of intracellular
signals and protein trafficking (Schlessinger, 1988) . A major
event in the binding of EGF to its receptor is the
autophosphorylation of tyrosine residues and the
phosphorylation of cellular proteins. These reactions are
carried. out. by' an intrinsic ‘tyrosine kinase located on
cytoplasmic domain of the EGF receptor. Examples of in yixo
EGF receptor tyrosine kinase substrates include: p42: p34-39,
the lipocotins or calpactins: p185”, a protein encoded by the
non proto-oncogene: and phospholipase C-gamma which is linked
to the induction of EGF-mediated phosphatidylinositol turnover
(Velu, 1990) . In our study, we have reported that EGF
stimulated the phosphorylation of various tyrosine kinase
substrates in a manner that is similar to that observed in
mammals . EGF-st imulated trout membranes readily
phosphorylated angiotensin II, [Val°]-angiotensin II and RR-
Src protein while casein and histones were phosphorylated to

a much lesser extent. This phosphorylation pattern has also

115

been described for the rat in a study that compared the
phosphorylation of exogenous substrates by insulin and EGF
(Klein et al., 1985). In EGF-mediated phosphorylations, the
significance of substrate specificity, in yixo or in 11319: in
the physiological mediation of the EGF-mediated mitogenic
signal is unknown at this time in both mammalian systems and
in fish.

To characterize further the EGF-mediated tyrosine
kinase activity in rainbow trout membranes, we used specific
inhibitors of tyrosine kinase activity as well as inhibitors
of other protein kinases. The use of these specific
inhibitors provides a valuable tool for the elucidation of the
role of EGF-mediated events that involve the biological
function of EGF receptor cell proliferation and
differentiation. Results from our study show that EGF-
mediated phosphorylation was inhibited by specific tyrosine
kinase inhibitors. These inhibitors included 4-
hydroxycinnamamide derivatives which are competitive
inhibitors of substrate binding to the EGF receptor-associated
protein tyrosine kinase and Lavendustin A, a derivative from
ngopgonyooo gzioooloyonono, a competitive inhibitor of ATP
binding to the tyrosine kinase. Of the compounds that did
not inhibit EGF stimulated phosphorylation, the lack of
inhibition by HDAA and tyrphotin is puzzling in that both are
potent inhibitors of tyrosine phosphorylation activity; This
was especially true of tryphotin which is a specific inhibitor

116

of EGF-mediated tyrosine kinase activity (Gazit et al.,
1989). As expected, inhibitors of protein kinase C, ATPases
and of cAMP-dependent kinase did not reduce the EGF-stimulated
phosphorylation of RR-Src. Quercitin, a biflavone, is a
nonspecific inhibitor that inhibits most.protein kinases such
A-kinase, C-kinase, tyrosine kinase and casein kinase I and II
(Shiaishi et al., 1987). In our assays, quercitin was a
potent inhibitor of EGF-stimulated tyrosine kinase activity
and protein kinase C activity in RT-HPM. Ouabain, an ATPase
inhibitor, stimulated the phosphorylation of RR-Src in EGF-
stimulated membranes. This result indicates that ATPases may
have a role in mediating the phosphorylation of membrane
components such as the EGF receptor in RT-HPM through
dephosphorylation of membrane protein or through competition
for triphosphate nucleotides such as ATP. The results
observed in these assays are similar to those observed in
mammals where treatment with tyrosine kinase inhibitors reduce
tyrosine kinase activity associated with the EGF-receptor.
The reduction in EGF receptor tyrosine kinase activity has
"been shown to correlate with altered regulation of cell growth
and proliferation (Klein et al., 1985).

We have observed differences between mammals and fish
relative to EGF-stimulated.phosphorylation and EGF-binding in
RT-HPM. Since rainbow trout are poikilotherms and
evolutionarily divergent from mammals, the role that the EGF

receptor might play in cell growth and differentiation is a

117
question that needs further research. The similarity between
the characteristics of binding of EGF and EGF-induced
phosphorylation in mammals and rainbow trout indicates that
many factors that control cell proliferation and

differentiation in mammals may also be present in fish.

118

REFERENCES

Carpenter,G. (1985) . Binding assays for epidermal growth
factor. In Mothodo in Enzmolooy (L. Birnbaumer and B.W.
O'Malley, Eds), Vol 109, pp. 101-115. Academic Press, New
York.

Carpenter,G. (1987) . Receptors for epidermal growth factor
and other polypeptide mitogens. m Box; moonom 56,
881-914.

Carpenter, G. and Cohen, S. (1979). Epidermal growth factor.
Anni Rex; Biogflemo 48, 193-216.

Carpenter,G., King,L., Jr. and Cohen,S. (1979). Rapid
enhancement of protein phosphorylation in A-43l cell
membrane preparations by epidermal growth factor. L
5191; EDEN; 254, 4884-4891.

Cohen,S. (1983) . The epidermal growth factor (EGF) . Canoe]: 51,
1787-1791.

Cohen,S., Carpenter,G., and King,L., Jr. (1980). Epidermal
growth factor-receptor-protein kinase interactions: co-
purification of receptor and epidermal growth factor-
enhanced phosphorylation. L 5.1.9.1.... m 255, 4834-4842.

Gazit,A., Yaish,P., Gilon,C., and Levitzki, A. (1989).
Tyrphostins I: Synthesis and biological activity of
protein tyrosine kinase inhibitors. L Mosh ohm 32,
2344-2352.

Gospodarowicz,D. (1981) . Epidermal and nerve growth factors in

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mammalian development. Ann; BoyL Enyoiolo 43, 251-263.

Gregoriou, M. and Rees, A.R. (1984). Properties of monoclonal
antibody to epidermal growth factor receptor with
implications for the mechanism of action of EGF. The M
L 3, 929-937.

Hunter, T. and Cooper, I.A. (1985). Protein tyrosine kinases.
Ann... 3.91:. bioehem... 54, 897-930.

Klein,H.H. , Freidenberg,G.R. , Cordera,R. , and Olefsky,J.M.
(1985). Substrate specificities of insulin and epidermal
growth factor receptor kinases. Bioonono filQEDXEr Boo;
92m 127, 254-263.

Laemmli, U.K. (1970). Cleavage of structural proteins during
the assembly of the head of bacteriophage T4. m 227,
680-685.

Margolis,B. Lax,I., Kris,R., Dombalagian,M., Honegger,A.M.,
Hocuk,R., Givol,D., Ullkrich,A., and Schlessinger,J.
(1989) . All autophosphorylation sites of epidermal
growth factor (EGF) receptor and HERZ/neu are locted in
their carboxy-terminal tails: identification of a novel
site in EGF receptors. 11.81911.§n§me. 264, 1-667-10671.

Moolenaar,W.H., Bierman,A.J., Tilly,B.C., Verlaan,I.,
Honegger,A.M., Ullrich,A., and Schlessinger,J. (1988).
A point mutation at the ATP-binding site of the EGF-
recpetor abolishes signal transduction. 21129 L 7, 707-
710.

Newsted,J. and Giesy, J. (1991). Characterization of

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epidermal growth factor binding to the hepatic plasma

membrane of rainbow trout (Qnooznynonno MILES) . m
QQERQIai EDQQQIint (in press).

Pike,L. (1983). Assay of growth factor stimulated tyrosine
kinases using synthetic peptide substrates. In Moog in
Enzynolo Vol. 146, pp. 353-362, Peptide Growth Factors,
(D.Barnes and D.A. Sirbasku, Eds), Academic Press, New
York.

Ritvos,O. , Jalkanen,J. , Pekonen, F. , Stenman,U.M. , and Ranta,T.
(1988) . Epidermal growth factor but not insulin-like
growth factor I potentiates adenosine 3',5'-
monophosphate-mediated chorionic gonadotropin secretion
by cultures human choriocarcinoma cells. Enooogino 123,
859-865.

Schlessinger,J. (1988). The epidermal growth factor receptor
as a multifunctional allosteric protein. M92023... 27,
3119-3123.

Schonbrunn,A., Kranshoff,M., Westendorf,J.M., and
Tashjian,A.H., Jr. (1980). Epidermal growth factor and
thyrotropin-releasing hormone act similarly on a clonal
pituitary cell strain. 21.9e111 Biol; 85, 786-797.

Shiraishi,T., Domoto,T., Imai,N., Shimada,Y., and Watanabe,K.
(1987). Specific inhibitors of tyrosine-specific protein
kinase, synthetic 4-hydroxycinnamamide derivatives.
W W 3:1,, m 147, 322-328.

Steel, R.G. and Torrie, J.H. (1980). 211.119.19.195 mom

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in Stotiotioo. 2nd Edition, McGraw-Hill Book Co., New
York.

Towbin,H. , Staehelin,T. , and Gordon,J. (1979) . Electrophoretic
transfer of proteins form polyacrylamide gels to
nitrocellulose sheets: procedure and some applications.
m m m sop non 76, 4350-4354.

Ushiro,H. and Cohen,S. (1986). Identification of
phosphotyrosine as a product of epidermal growth factor-
activated protein kinase in A-431 cell membranes. L
Biol; goon; 255, 8363-8365.

Velu, T.J. (1990). Structure, function and transforming
potential of the epidermal growth factor receptor. Moi;
Qfill; Engggxinolo 70, 205-216.

CHAPTER 3

EFFECT OF 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD)
ON THE EPIDERMAL GROWTH FACTOR RECEPTOR IN
HEPATIC PLASMA MEMBRANES OF

RAINBOW TROUT

INTRODUCTION

Many species of salmon are very sensitive to halogenated
aromatic hydrocarbons (HAHs) such as polychlorinated dibenzo-
p-dioxins (PCDDs), polychlorinated biphenyls (PCBs),
polychlorinated naphthalenes (PCNs), polychlorinated
azobenzenes (PCABs) and.polychlorinated.dibenzofurans (PCDFs)
(Kleeman et al., 1988: Kleinow et al., 1987: Dauble et al.,
1988). Of the HAHs, 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD) is regarded as the most toxic. IRainbow trout are among
the species most sensitive to' TCDD with an 80 day L050 of
10,000 ng/kg body weight (Spitsbergen et al., 1988). TCDD is
found as a contaminant in most Great Lakes fish. Fish taken
from the most highly contaminated areas average about 0.4 ng
TCDD/g body weight (DeVault et al., 1989). Of the HAHs, only
PCBs and PCNs have been manufactured commercially while the
other HAHs are found as by-products and impurities of the
manufacture of other chemical products such as chlorophenols
and PCBs (Bowes et al., 1975; Rappe and Buser, 1980).
Combustion of chlorinated chemicals of industrial and
municipal origin also are a source of HAHs to the Great Lakes
(Ballschmitter et al., 1983: Buser, 1979). As a result of

their use and misuse, HAHs are found throughout the biotic and

123

124
abiotic compartments of most freshwater and marine aquatic
ecosystems. Due to their lack of metabolism, long residence
time in the environment, their lipophilicity and toxic
potency, TCDD as well as the other HAHs pose potentially
serious health effects to many fish populations in the Great
Lakes.

The administration of HAHs to animals elicits a number of
common biologic and toxic responses in a diverse number of
species (Poland and Knutson, 1982: Spitsbergen et al., 1988).
Exposure to TCDD will result in a variety of species- and
tissue-specific effects that include tumor promotion,
teratogenicity, immunotoxicityy hepatotoxicity, epithelial
hyperplasia and enzyme induction (Safe, 1986). The mechanism
of toxic action TCDD and HAH are mediated through an
intracellular receptor, the Ah receptor (Poland and Knutson,
1982: Whitlock, 1987). Toxic effects elicited by TCDD and
HAHs are thought to be a result of the stereospecific,
reversible binding of these compounds to the Ah receptor with
a subsequent alteration of gene expression initiated by the
ligand-receptor complex. Events known to be mediated through
this mechanism include the induction of ornithine
decarboxylase, cytochrome P-450 monooxygenases and glutathione
S-transferases (Safe, 1986). While induction of enzymes is a
though to be a significant event in TCDD exposure, induction
alone is not directly related to TCDD's lethal action since

the most sensitive mammalian species, the guinea pig, does not

125
exhibit significant induction mixed-function monooxygenases at
concentrations of TCDD which are lethal (Poland_and Knutson,
1982) . Furthermore, the broad spectrum of biologic and toxic
responses observed in different tissues of TCDD exposed
animals indicate a more complex mechanism than that of a
simple direct-acting toxicity.

Many of the effects of TCDD are comparable to those
associated with the modulation of several hormone-mediated
responses (Safe, 1986). The fact that TCDD effects are more
pronounced in young, growing animals than those observed in
mature animals has.led to the hypothesis that many of TCDDs
toxic effects may be a result of an alteration of the
developmental process. This hypothesis is supported by
evidence that TCDD and its isomeric analogs cause an
alteration in the proliferation and/or differentiation of
epithelial cells Of the skin, intestine, urinary bladder and
thymus (Greenlee et al. , 1987) . The TCDD-induced effects
noted above are consistent with effects induced by the
alteration of cell surface structure and function such that
there is an alteration of membrane-associated constituents
such as enzymes, receptors, .ion channels and- surface
glycoproteins (Madhukar et al., 1988).

In mammals, the alteration of plasma membrane has been
shown to be one of the features of TCDD-induced toxicity
(Matsumura et al. , 1984) . In M studies have shown that

exposure to TCDD results in a reduction in the levels of

126

insulin, low'density lipoprotein, and epidermal growth factor
activities as well as a reduction of concanavalin A binding
and ATPase activity in the hepatic membranes of the rat. Of
these parameters, the reduction in the binding of EGF was the
most sensitive. The reduction in the binding of EGF was dose-
dependent and compound specific (Madhukar et al. , 1984) .
Along with the reduction in EGF binding, there was an observed
increase in protein kinase activities that are known to be
important in the modulation of the biological potency of the
EGF receptor (Cochet et al., 1984: Downward et al., 1985).
Alteration of EGF binding in.TCDD-treated animals is believed
to be mediated by the Ah receptor since degree of TCDD-induced
reduction of the binding of EGF segregated with the presence
of the Ah locus.

The significance of the relationship between the
alteration in EGF .receptor’ homeostasis and TCDD-induced
lesions is the close similarity of the effects induced by both
TCDD and EGF in target tissues. For example, young mice
treated with either EGF or TCDD induce early eye opening and
'early'tooth.eruption (Cohen.and.Elliot, 1962: Heinberg et al.,
1965: Madhukar et al., 1988). Other similar lesions include
cleft palate formation, proliferation of gastric and
intestinal. mucosa, and fatty infiltration «of ‘the liver.
Finally, both TCDD and EGF cause an increase in the
phosphorylation of the EGF receptor and the subsequent
activation of intrinsic tyrosine kinase activity (Bombick

127
1986). Protein tyrosine kinases are involved in the control
of normal and abnormal cell growth such as development of
embryonic tissues, hepatocarcinogensis, and nonmalignant
hyperplastic cell growth (Gentleman et al., 1984: Hunter and
Cooper, 1985: Olson 1985).

Pathological lesions observed in rainbow trout which have
been treated with TCDD are similar to those observed in
mammals (Miller et al., 1973: McConnell, 1980: Spitsbergen et
al., 1988). As in mammals, lymphomyeloid and epithelial
tissues are the primary sites of TCDD toxicity in fish.
Additional evidence for a similar mode of toxic action for
TCDD between fish and mammals is the induction of mixed-
function oxygenases which are regulated by the Ah receptor
(Kleinow et al., 1987: Vodicnik and Lech,1982). The Ah
receptor has been identified in rainbow trout liver (Heilmann
et al., 1988) and in the rainbow trout hepatoma cell line RTH-
149 (Lorezen and Okey, 1990).

This study was undertaken to examine the effect of TCDD
on EGF binding in the hepatic membranes of rainbow trout.
Specific objectives were to: 1) determine the effect of time
and dose on EGF binding to liver plasma membranes: 2) examine
the effect of TCDD on protein kinase activities and: 3)
examine the relationship between cytochrome P-450

monooxygenase activity and EGF binding in TCDD-treated fish.

128
MATERIALS AND METHODS
Qnonioalo. TCDD was purchased from Accustandard Inc. (New
Haven, CT) . Receptor grade EGF, protein kinase substrates RR-
SRC and AC-MBP, and protein kinase C inhibitor (PKC 19-36)

”SI-EGF

were purchased from Gibco-BRL (Grand Island, NY).
(150—200 pC/pg) and 3°[1]P-ATP (3000 Ci/mmol) were purchased
from NEN (Boston, MA). All other chemicals were purchased
from Sigma (St. Louis, MO) . The anti-EGF-receptor mouse

monoclonal antibody was purchased from Sigma (F4-clone).

Eifih- Sexually immature rainbow trout (onoornynonno
wigs) weighing 50-100g were obtained from Stony Creek Trout

Farm, Grant MI. Fish were held at 12 t 1 C in 1,000 liter
tanks supplied with a continuous flow of oxygenated,
dechlorinated tap water. Fish were fed (silvercup Trout Food)
three times weekly at 1% body weight. The photoperiod was
16:8 hr (light:dark).

Ireotmont of Fish. Fish received a single ip injection
(2 ml/kg) of TCDD dissolved in corn oil at doses indicated in

the figures and tables. Corn oil was used as the vehicle for
all experiments. In general, fish were sampled 7 days post-
injection. For time course studies, fish were sampled 2, 5,
10, 20, and 40 days after injection. At sampling, the fish
were killed and the liver was immediately excised, weighed and

frozen in liquid nitrogen for further in yitzo studies.

 

L /_:1

129

memo Membrane Isoleoion. Rainbow trout hepatic plasma
membranes (RT-HPM) were prepared by the method of Newsted and
Giesy (1991) . Purified membranes were stored in 50 mM Tris-HCl
(pH 7.2) which also contained 0.25 mM sucrose and protease
inhibitors (100 kallikrein units/ml aprotinin: 10 pg/ml
leupeptin and 10 pM E-64) . Aliquants of the membrane
preparations were stored under liquid nitrogen (-196 C).
Protein analysis was by the method of Lowry et al., (1951)

with BSA as the standard.

ESiE flinging nosey. Binding assays were carried out by the
method of Newsted and Giesy (1991) . Results were reported as
fmol 125I-EGF bound/mg protein. Binding curves were fitted and
the equilibrium dissociation constants (k0) and the apparent
maximum binding capacity (Elm) were calculated with the LIGAND
program (McPhearson, 1985) . The K, and 3m were expressed as

nM and fmol of 125I-EGF bound/mg protein, respectively.

Mine Kinase Agony. Tyrosine kinase assays were
conducted in microfuge tubes by a modified method of Ribot et

al. (1984). Reactions were carried out in 60 pl volumes and
contained 50 mM HEPES, pH 7.4, 20 mM Mg”, 5 mM Mn”, 100 pM
vanadate, 5 pM okadaic acid, 0.05% nonidet NP‘40, 50 pM [SZP]
ATP (1000-2000 cpm/pmol) and 20 to 100 pg RT-HPM. To half of
the tubes, 120 pM RR-SRC synthetic peptide was added while the

other half received water. The assay was initiated by the

130
addition of [14”PJATP. After incubation for 30 min at 4 C,
the reaction was stopped by the addition of 150 pl of 5%
trichloroacetic acid and 1.56 mM KHZPOp Subsequently, 10 pl
of bovine serum albumin (BSA) was added to aid in protein
precipitation, and the samples were incubated on ice for 15
min. The precipitated protein was removed by centrifugation
at 5,500xg for 15 min at 4°C. From each tube, two 50 pl
aliquots of supernatant were spotted on phosphocellulose
filters (Whatman P-8l). Phosphocellulose filters were washed
four times in 75 mM phosphoric acid (5 min) and once in
acetone (2 min). , The air dried filters were counted for
radioactivity in 7 ml of Safety Solv. Tyrosine kinase
activity was expressed as the difference between samples with
and without the RR-SRC peptide and normalized for time and

protein concentration.

Bretein Kinese Q Amy. Protein kinase C (PKC) activity

was measured by a modification of the procedure of Bell and
Sargent (1987). To determine the activity of "particulate"
PKC, RT-HPM were extracted in the following manner: RT-HPM
were homogenized in 20 mM Tris, pH 7.5, 0.5 mM EDTA, 0.5 mM
EGTA, 0.5% Triton X-100, and 25 pg aprotinin and leupeptin/ml.
Homogenized membranes were incubated for 30 min at 0 C and
the cellular debris was removed by centrifugation for 2 min in
a microcentrifuge. .Assays were conducted with supernatant in

microfuge tubes in 50 pl total volume. The diluted enzyme

131
preparation (20 pl) was assayed at 10 C for 5 min. The
reaction mixture contained 20 mM HEPES, pH 7.5, 10 mM MgCl, 1
mM DTT, 20 pM vanadate, 50 pM [1-32P1ATP (1000-2000 cpm/pmol) ,
10 pM CaClz, 120 pM Ac-myelin basic protein (Ac-MBP) , 40 pg
diolein and 40 pg phosphatidylserine. Before being added to
the reaction tubes, equal volumes of diolein and
phosphotidylserine in chloroform were combined and the solvent
was removed under nitrogen. The residue was then suspended in
a small volume of 20 mM HEPES, pH 7.4, by sonication for 5 min
at 0 C and the resultant liposome mixture was used in the .
assay. Basal activity was measured in the presence of 10 mM
EGTA instead of CaCl2 and liposomes. The reaction was stopped
by spotting 25 pl onto phosphocellulose paper. The filters
were washed twice in 150 mM phosphoric acid and then twice in
distilled water. The filters were dried and counted in 7 ml
Safety Solv. Protein kinase activity was calculated as the
difference between the sample that contained EGTA sample
without EGTA and expressed as pmol of RP incorporated per min

per mg membrane protein.

WM. Immunoprecipitation of EGF-related
proteins was carried out as outlined by Gill et al. (1984).

Plasma membrane protein (100 pg) was phosphorylated in 11m
using [1-32P1ATP as outlined in Chapter 2 except that RR-SRC
was omitted from the mixture. The phosphorylated membrane

protein was solubilized in 1 ml RIPA buffer (10 mM K2HPO,, 0.15

132
M NaCl, 1% Triton x-100, 1% sodium deoxycholate, 0.01% sodium
dodecyl sulfate and 2 mM EDTA) for 1 hr at 0°C. The
solubilized material was centrifuged at 12,000xg for 10 min
and the supernatant was incubated overnight at 4 C with 10 pl
of mouse anti-EGF receptor monoclonal antibody. The
immunocomplex was precipitated using goat antibody to mouse
immunoglobulin G (IgG) conjugated to W engene
cells (Tachisorb-M, Calbiochem) for 1 h at room temperature
with frequent mixing. Samples were centrifuged (2,000xg for
15 min) and the supernatants removed. Pellets were washed
twice with 1 ml PBS at 0 C followed by centrifugation. The
final pellet was heated for 5 min at 98 C in SDS-Laemmli
buffer and the solution was centrifuged to remove
particulates. The supernatant was precipitated with 10% TCA,

dissolved by 1 M NaOH and counted by liquid scintillation.

Rhosohoamino Asia Analxsis. Proteins from

immunoprecipitation and phosphorylation assays were
precipitated by trichloroacetic acid and the precipitate was
washed twice with chloroform:methanol (2:1) and once with
acetone. The pellet was air dried and hydrolyzed in 6 N HCl
at 110 C for 2 h in a sealed Reacti-Vial. The HCl was removed
under vacuum and the phosphoamino acids analyzed by thin-layer
chromatography by the method of Cooper et al. (1983) . The
amino acids were visualized with ninhydrin spray, and the

radioactivity was measured by scrapping the spots and counting

133

by liquid scintillation.

Mfg M31 Agony. Microsomes from individual fish
livers were prepared from post-treated fish by the method of
Ankely et al.(1987). 7-Ethoxy O-deethylase (EROD) activity
was measured using the fluorometric procedure of Pohl and

Fouts (1980).

W We. All results are expressed as means
i S.D. for each experimental point. Statistical differences

between control and treatment values analyzed by Student's t-
test or by analysis of variance. Comparison among means was
conducted with a posteriori teSt, sum of squares simultaneous
test procedure (SS-STP) (Steel and Torrie, 1980) . Significant

differences were established at the p< 0.05 level.

RESULTS

Effects on E91: Binding

The effect of TCDD exposure to the EGF receptor was
examined in hepatic plasma membranes of rainbow trout. The
effect of a single i.p. injection of TCDD significantly
decreased EGF binding at all tested concentrations by day 10
(Fig. 16). The reduction in the binding of EGF was maximal at
1.0 pg TCDD/kg body weight (b.w.) and remained unaltered at
the 10 pg TCDD/kg b. w. treatment. Probit analysis of the

dose-response data computed the effective TCDDIdose to reduce

Figure 16.

134

Dose-response relationship for the binding of
”SI-EGF and TCDD exposure in RT-HPM. Liver
plasma membrane preparations were made on day
7 post treatment. Binding of EGF to RT-HPM is
reported as fmole EGF bound/ mg protein. Each
sample point represents the mean of 5 fish.
ED50 was calculated by probit analysis.Means

that are significant different from control

(p < 0.05) are designated by *.

135

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136

the binding of EGF (ED50) to be 0.17 pg TCDD/kg body weight.
To study the nature of these changes, Scatchard analyses were
conducted on the membrane preparations (Fig. 17) EGF binding
generally exhibits a biphasic relationship which is indicative
of multiple classes of receptors with differing affinities
(Newsted and Giesy 1991). In the TCDD-treated animals, the
high and low affinity receptors levels were reduced relative
the receptor values seen in RT-HPM from untreated fish.
Scatchard analysis of saturation curves showed that the
reduction in EGF binding was accompanied.without any apparent
change in receptor affinity (K0) of the treated trout
membranes compared to control membrane values (Table 8).

To study whether the in yiyo reduction in EGF binding
wasrelated to general TCDD-induced effects, condition factors
and liver somatic indices were calculated for both untreated
and TCDD-treated fish. Condition factors were calculated as:

CF== final weight/(lengths) x 100% (1)
while the liver somatic index was calculated as:

LSI- (liver weight/ body weight) x 100% I (2)
Statistical analysis demonstrated no significant relationship
between CF and the binding of EGF in TCDD-exposed rainbow

trout (R28 0.15) (Fig. 18). In addition, the correlation

between the TCDD dose and CF was not statistically significant

Figure 17.

137

125I-EGF binding to

Scatchard analysis of
control (0) and TCDD treated (I) RT-HPM. Each
point is the mean of 3 determinations of

different membrane preparations.

138

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measure. I

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139

Table 8. Comparison of EGF binding affinities and binding
capacities in plasma membranes of control and.TCDD-

treated rainbow trout.‘

 

125I-EGF receptor Parameters

 

 

KI)1 Kl32 Bmax‘l Bmaxz
Treatmentb nM nM fmol/mg fmol/mg
Control 0.20 3.9 16.0 245
5.0 0.11 3.7 2.7' 244
10.0 0.12 3.6 0.9' 172'

 

KD and Bmax of the high and low affinity EGF-receptor
binding sites were analyzed by Scatchard analysis from
saturation plots of the binding data.

Injection concentrations were in pg TCDD/kg b.w.

Significantly different at p < 0.05.

 

Figure 18.

140

Comparison (as percentages) of EGF binding,
EROD activity, condition factor and liver
somatic index in TCDD-treated 0(10 pg/kg) and
control rainbow trout. Values are expressed
as % control activities and are represented as
means of 5 determinations. Means within a
response that significantly differ from

control (p <0.05) are designated by *.

141

 

 

     

 

 

 

 

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142
(Ra-0.03) . However, the observed negative correlation between
LSI vs EGF binding was highly significant (R2= 0.937) . The
enlargement of the liver is consistent with previous studies
that show a TCDD-induced hyperplastic response in juvenile
rainbow trout (Spitsbergen et al., 1988).

The induction of cytochrome P-450 monooxygenase activity
(EROD) was used in our study as a biomarker to examine the
relationship between exposure to TCDD and the alteration of
hepatic function in rainbow trout. EROD was chosen since it
has been well established that TCDD induces EROD activity in
rainbow trout at the concentrations used in this study
(Kleinow et al. , 1990) . The seven day EDso for EROD activity
was 0.66 pg TCDD/kg b.w. Induction of EROD activity was
negatively correlated (RZ= 0.608) to EGF binding. EROD
activity was significantly elevated from conctrol levels at
0.1 pg TCDD/kg b.w. while the binding of EGF was significantly
reduced from control levels at 0.01 pg TCDD/kg.

Time course experiments‘were. conducted to examine further
the effect of TCDD on the binding of EGF in rainbow trout
plasma membranes (Fig. 18) . Over the 40 d observation period,
TCDD-treated trout exhibited no significant reduction in or
growth as measured by the condition factor. While overall
growth of the treated trout was unaffected, there was an
increase in weight of the liver, as indexed by LSI, that was
significantly greater than controls values. The increase in

liver weight was first observed at day 2 and persisted

143

throughout the experimental period. The increase in LSI
values were also associated with an increase in EROD activity.
EGF binding was also significantly reduced at day 2 and
remained so for the duration of the study. Binding was
reduced to its lowest point by day 10 (31% below control).
However, by day 20, EGF binding began to return to control
values and by Day 40 was approximately 62.5% of the control
EGF binding.

To test whether the phenomenon of TCDD-induced reduction
in EGF binding could be associated with other toxicants that
may induce general membrane alterations, we examined several
other compounds that were representative of other contaminant
classes (Table 9). Of the compounds tested, only TCDD and
the nonortho-substituted PCBs caused. a reduction. in EGF
binding. The doses chosen for this experiment were great
relative to the TCDD dose but were based on their ability to
induce MFO activity maximally in rainbow trout. No
manifestations of toxicities were observed for TCDD or PCBs
whereas trout treated with DDT and dieldrin exhibited some
toxic symptoms but no mortality.

Tenn arrests on Kinase Aotixitx

TCDD exposure induced alterations in protein kinase
activity of RT-HPM (Fig. 19). Significant elevation of all
kinase activities in treated RT-HPM was observed by day 5 and
reached a maximum by day 20. Kinase activities returned to

control values by day 40. This trend was also observed for

144

 

 

 

Table 9. Effects on in yiyo administration of various
toxicants on 12°I-EGF binding RT-HPM.'

EGF Specific Binding
Toxicant Dose % of Control 1 S.D
Control 100.0 f 33.3
2,3,7,8- TCDD 10 pg/kg 29.4 i 14.7'
3,3',4,4',5-pcn 100 pg/kg - 30.8 i 3.2'
3,3',4,4'-TCB 500 pg/kg 31.5 i 17.5'
2,3,3',4,4'-PCB 4.5 mg/kg 68.7 i 60.3
3,3',4,4',5,5'-HCB 500 pg/kg 31.4 i 20.0'
DDT 10 mg/kg 95.8 i 45.0
Dieldrin 10 mg/kg 91.2 t 32.0
° Chemicals were administered in a single i.p. dose. Corn

oil served as the vehicle.

Significant differences were at p < 0.05.

Fish were sampled on day 7.

Figure 19.

145

Changes in the binding of EGF, protein kinase
C (PKC) and tyrosine kinase (PTK) activities,
and EGF-receptor phosphorylation (EGF-R) as a
function of time after a single i.p. injection
(10 pg TCDD/kg). Results are reported as %
control activities and are represented as
means of at least 3 determinations. The data
was analyzed analysis of variance and
Student's T test. Differences (*) between
control and treatments were determined at p <

0.05.

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147
EGF-receptor phosphorylation where phosphorylation
concentrations were elevated by day 2 and remained elevated
through day 40.

Protein kinase C activity was defined as the portion of
kinase activity stimulated by calcium and phosphatidylserine.
Specificity of the protein kinase C assay in RT-HPM was
established by the use of a specific protein kinase inhibitor
(PKC 19-36) (Hardie 1988). In TCDD-treated rainbow trout,
there was a significant increase in PKC activity by day 5 that
reached a maximum level on day 20. However, by day 40 the
protein kinase C activities had returned back to control
levels. Protein kinase C activity and EGF binding were
negatively related in TCDD-treated RT-HPM. As protein kinase
C activity increased to a maximum by day 5 in the treated
animals, there was a concurrent reduction in EGF binding such
that it reached a minimum 10 d after injection with TCDD.
When PKC activity returned to control values at day 20, there
was an increase in the binding of EGF to control values.

Tyrosine kinase activity in TCDD-treated trout became
significantly elevated by day 2, reached a maximum at day 10
and remained elevated through day 40. Examination of the
increase in protein tyrosine kinase activity showed that it
occurred about 2 days prior to the increase in PKC and the
reduction in EGF binding. Studies have shown that ligand
activated EGF-receptor has an intrinsic tyrosine kinase

activity that is responsible for its biological potency

148
(Carpenter,1983) . Thus, the elevation in tyrosine kinase
activity may be linked to an overexpression of EGF receptor
activity in TCDD-exposed trOut.

To understand better the relationship between changes in
protein kinase C and tyrosine kinase activity in the plasma
membranes of TCDD-treated trout and the alteration in EGF
receptor activity, we examined the phosphorylation of the EGF
receptor. The results of the experiments showed that in TCDD
treated trout, there was a significant increase in the
phosphorylation of the EGF-receptor by day 2. The elevation
in the phosphorylation of the receptor remained relatively
constant throughout the duration of the study (Fig. 19). To
examine further the relationship between the phosphorylation
of the EGF receptor and kinase activity, we measured
phosphoamino acids in immunoprecipitated EGF-receptor (Fig.
20). At all sampling times, there was an increase in
phosphotyrosine and phosphothreonine values of immuno-
precipitated EGF receptor protein in TCDD-treated rainbow
trout. However, unlike phosphotyrosine or phosphothreonine,
phosphoserine values were decreased on day 5 and returned to
control by day 40. The increase in phosphothreonine
corresponds to the increase in PKC activity over the study
period.

TCDD dose-response studies were conducted that showed
relationship between alterations in protein kinase activity

and EGF binding (Fig. 21). TCDD caused a dose-dependent

Figure 20.

149

32P incorporation into phosphoamino acids of
immunoprecipitated EGF-receptor in a time
course study. Trout were given a single i.p.
injection (10 pg TCDD/kg) in corn oil. Data
are shown as to pmol 3°PO‘ incorporated into
immunoprecipitated protein. Each value
represents the mean of 3 determinations.
Results were analyzed by analysis of variance
and by Student's T test. Significant
differences between control and treatments (*)

were detected at p < 0.05. t-test.

(pmole 32-P incorporated)

150

 

 

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Figure 21.

151

Dose-response relationships between TCDD dose,
protein kinase activity and EGF-receptor
phosphorylation in the hepatic plasma
membranes of rainbow trout. Results of
protein kinase C and tyrosine kinase assays
are reported as pmole szPo‘ incorporated per mg
membrane protein and EGF-receptor
phosphorylation was' reported pmol phosphate
incorporated per mg protein of original
starting membrane protein. Each point
represents the mean of 3 determinations.
Differences between control and treatment

values were determined at p < 0.05.

152

 

 

 

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153
increase in protein kinase C and tyrosine kinase activity that
also included an increase in EGF-receptor phosphorylation over
control values. ‘These trends are opposite of that observed in
the EGF binding assays where the increases in PKC (R°=0.997) ,
tyrosine kinase (R2=0.789) and EGF-receptor phosphorylation
(R2= 0.963) were negatively correlated to EGF binding.

The relationship between protein kinase activity and the
EGF-receptor phosphorylation state was examined further in the
dose-response study. Correlation analysis of the results show
that there was a positive correlation between PKC and tyrsoine
kinase activity (R%= 0.715) and PKC and EGF receptor
phosphorylation (Rz=0.609) . These data are consistent with
the hypothesis that kinase activity regulates the
phosphorylation of the EGF-receptor in TCDD toxicity.
Phosphoamino acid analysis of the receptor showed that
phosphotyrosine was increased approximatery 2.5 times over
control levels while phosphoserine and phosphothreonine
increase approximately 2 and 6 times over control values,
respectively, for the dosage regime. Statistical analysis
showed a positive relationship for phosphotyrosine
concentration and protein tyrosine kinase activity as well as
between phosphoserine and phosphothreonine concentrations and
PKC activity. However, no significant correlations were found
between tyrosine kinase activity and EGF-receptor phospho-
serine or phosphothreonine levels nor was there a significant

relationship between PKC and tyrosine kinase activities.

154

DISCUSSION

The EGF receptor is a transmembrane glycoprotein widely
distributed in many tissues (Adamson and Rees 1981). This
receptor is unique among many other receptor types because it
contains an intrinsic tyrosine kinase activity that is
responsible for its biological activity. The EGF receptor has
been shown Ito Zbe involved. in embryonic (development and
cellular repair processes as well as in neoplastic cellular
growth responses (Carpenter 1983: Hunter 1984) . Expression of
the biological activity of the EGF receptor is believed to be
under the control of tyrosine kinases which act to stimulate
the activity of the receptor through the phosphorylation of
tyrosine residues of the receptor. Activity of the EGF
receptor can be reduced by PKC through the phosphorylation of
serine and threonine residues. The phosphorylation of the EGF
receptor by PKC can result in the reduction in the binding of
EGF and in the tyrosine kinase activity associated with the
receptor (Cochet et al., 1984: Downward et al., 1985).
‘Alteration of gene function has been demonstrated in TCDD-
treated female rats where TCDD induced both a reduction of EGF
binding and a parallel decrease in steady-state levels of
uterine EGF receptor mRNA (Astroff et al., 1990) Thus, the
control of cell surface EGF receptor levels and of their
biological potency are under'biochemical and genetic control.

Alteration of either of these mechanisms may result in the

155
alteration of cell processes.

In these studies we have examined the relationship
between TCDD administered in yiyo and alterations of EGF
receptors in the plasma membranes of rainbow trout. These
data agree with results observed in mammals in that TCDD
caused a reduction in the binding of EGF as well as an
increase in protein kinase activity in hepatic plasma
membranes (Matsumura et al. , 1984) . As' seen in mammalian
studies, reduction in EGF binding in rainbow trout hepatic
membranes was accompanied by an increase in tyrosine kinase
activity and the phosphorylation of the EGF receptor.
Although no definite cause-effect can be proven by this data,
the decrease in the binding of EGF may be due in part to
increased tyrosine kinase activity that phosphorylates
tyrosine residues of the EGF receptor. Data to support this
hypothesis is the increased phosphotyrosine concentration of
the EGF receptor in RT-HPM of TCDD-exposed rainbow trout.
However, while the reduction of EGF binding was correlated to
the increase in tyrosine kinase activity and the increase in
EGF receptor phosphorylation, there was a less significant
relationship between tyrosine kinase activity and EGF receptor
phosphorylation. This suggests that other kinases may be
involved in the regulation of EGF binding.

Other classes of toxic agents have been shown to reduce
EGF binding in a manner similar to that observed in TCDD

exposures. Examples of these agents include oncogenic

156

retroviruses such as v-src (Erikson et 1a., 1981), hormones
such as thyroid hormone T3 (Hayden and Severson 1983), and
tumor promoters such as phorbol esters (Lee and Weinstein
1979). While the mode of action of these agents are not
similar, many of their effects are believed to be mediated
through protein kinase C. An example would be 12-0-
tetradecanoyl-phorbol-13-acetate (TPA), where the activation
of protein kinase C results in the subsequent phosphorylation
of serine and threonine residues and results in the down
regulation of EGF receptors (Lee and Weinstein 1978).
Increased PKC activity is also related to the alteration of
the phosphoinositol cycle, calciummmobilization, induction of
ornithine decarboxylase, enhancement of glucose transport and
the stimulation of mitogenic activity (Takai et al., 1985).

In this study, we observed a dose and time dependent
increase in PKC that was associated with a reduction in the
binding of EGF in RT-HPM. The increase in PKC activity was
also correlated. with an increase in EGF receptor
phosphorylation an in EGF receptor phosphothreonine
concentration. The observed increase in protein kinase C
activity occcured at approximately the sampling time as that
observed for tyrosine kinase activity (Fig. 19) . This
differs from observation of TCDD-exposed mammals where
induction of tyrosine kinase activity preceeds the induction
of protein kinase C (Bombick, 1986). Thus, in rainbow trout,

the activation of protein kinase C and tyrosine kinase by TCDD

157

may be mediated through separate biochemical mechanisms that
result in the phosphorylation of the EGF receptor and the
subsequent reduction in the binding of EGF in RT-HPM. Our
results support this hypothesis since the increase in EGF
receptor phosphoamino acid concentration is related to
elevations in PKC and tyrosine kinase activities in TCDD-
exposed RT-HPM.

Chemical class and toxicant structure were closely
associated to their ability to reduced the binding of EGF in
RT-HPM and to their ability to induce cytochrome P-450 in
rainbow trout (Kleinow et al., 1987). DDT, dieldrin and PCB
105 did not significantly reduce EGF binding in our study nor
were they potent inducers of cytochrome P-450 monooxygenase
activity in rainbow trout microsomes. This finding supports
the hypothesis that TCDD toxicity is mediated through the Ah
receptor and that those species with low levels of activated,
nuclear Ah receptors are less sensitive to TCDD-induced toxic
effects (Whitlock, 1987). The significance of the
relationship between Ah responsiveness and TCDD toxicity in
rainbow trout is unknown since there is a lack of information
on the biochemical and physiological characteristics Ah
receptor in rainbow trout. However, TCDD induces similar
enzymatic effects in rainbow trout such as mixed function
oxygenase induction and glutathione S-transferase induction
that are known to mediated through the Ah receptor in mammals.

The similarity in enzymatic response implies a common mode of

158
toxic action between mammals and rainbow trout exposed to
TCDD.

In conclusion, we have shown that a single dose of TCDD
results in a significant alteration in plasma membrane
function. In particular, there is a decrease in EGF binding
accompanied by increases in various protein kinase activities.
While the significance of these effects are still unknown in
fish, it is important to note the strong comparison between
these changes and those seen in mammalian studies. However,
one must be careful in relating the alterations of EGF
binding and phosphorylation to in yiyo lesions and symptoms
since the role of the EGF-receptor and kinase activity still

has to be established in rainbow trout.

159

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Gentleman, S . , Martensson,T.M. , Digiovanna,J.'1‘_. , and
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Hunter,T. (1984). The proteins of oncogenes. figii American

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differences in 2 , 3 , 7 , 8-tetrachlorodibenzo-p-dioxin
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Kleinow,K.M. , Melancon,M.J. , and Lech,J.J. (1987) .
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Madhukar,B.V., Brewster,D.W., and Matsumura,F. (1984) . Effects

Of 1J1 2.119 administered 2,3,7,8-tetrachlorodibenzo-p-

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dioxin on receptor binding of EGF in the hepatic membrane
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Madhukar,B.V., Ebner,K., Matsumura,F., Bombick,D.W.,
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165

epidermal growth factor binding to its hepatic plasma

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9311.. Comer... mm (in press) -
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147-174.

CHAPTER 4

THE EFFECTS OF TCDD ON EPIDERMAL GROWTH FACTOR
BINDING AND PROTEIN KINASE ACTIVITY IN THE RAINBOW TROUT

CELL LINE, RTH-149

INTRODUCTION

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most
toxic congener of a larger group of structurally similar
compounds, the halogenated aromatic hydrocarbons (HAHs) . Due
to their toxicity and persistence in the environment, these
compounds represent a serious concern for human health and
impact aquatic species. Several fish species have been shown
to be sensitive to TCDD and other HAHs such as polychlorinated
biphenyls (PCBs) , polychlorinatd dibenzofurans (PCDFs) and
polychlorinated dibenzodioxins (PCDDs) (Kleeman et al., 1986:
Kleinow et al., 1987: Kleeman et al., 1988). Of the fish
species tested, rainbow trout has been shown to be among the
most sensitive with a 80 day LD50 of 10 pg TCDD/kg, body
weight (Spitsbergen et al. , 1988) . While these compounds are
extremely toxic to fish and may have a significant impact on
fish populations in various aquatic environments (Walker et
al. , 1991) , little is known about the mechanisms by which fish
recognize and respond to HAHs.

In mammals, TCDD-induced toxicity, as well as enzyme
induction, are mediated through the binding of this compound
to an intracellular protein, the Ah receptor (Poland and
Knutson, 1982: Whitlock, 1987) . It is through this mechanism

that TCDD regulates a variety of biochemical responses in

168

169

target tissues. Many of these responses are associated with
changes in cell proliferation and altered patterns of
differentiation (Hudson et al., 1985). Examples of these
effects seen in both fish and mammal species, include dermal
hyperkeratinization (fin necrosis in fish), teratogenicity,
lymphoid involution, immunotoxicity and.carcinogenesis (Safe,
1986: Spitsbergen et al., 1988a, 1988b).

The proliferation of many cell types in culture are
regulated by biochemical mediators which include polypeptide
growth factors such as epidermal growth factor (EGF)
(Schlessinger et al., 1982: Carpenter, 1987: Velu, 1990). I
The biological activity of EGF is mediated through a
transmembrane receptor that has a ligand-dependent tyrosine
kinase (activity (Schlessinger, 1988). The IEGF receptor
activity is regulated by the down regulation of receptors and
internalization of the receptors from the cell surface. The
decrease in EGF binding has also been observed in cells
exposed to polycyclic aromatic hydrocarbons (Lee and
Weinstein, 1978) , other growth factors such as platelet
'derived growth factor (PDGF) (Bowen-Pope et al., 1983) and
phorbol esters such as TPA (Karenlampi et al., 1983).
Xenobiotics such as TCDD have also been shown to reduce EGF
binding to hepatic plasma membranes (Madhukar et al., 1984).
This reduction was accompanied by an increase in cellular
protein kinase activity which included the EGF-associated

tyrosine kinase (Madhukar et al., 1988).

170

Previously, we have shown that rainbow trout have hepatic
EGF receptors that are associated with an intrinsic tyrosine
kinase activity (Newsted and Giesy, 1991). Furthermore, we
have shown that in yiyg TCDD exposure of rainbow trout results
in a significant decrease in EGF binding in the liver that is
accompanied by the elevation of protein kinase C and tyrosine
protein kinase activities (Chapter 3). TCDD also causes an
increase in the phosphothreonine and phosphotyrosine content
of the EGF receptor that is associated with the increase in
EGF receptor associated tyrosine kinase activity. However, we
were not able to correlate the alteration in the binding of
EGF to other cellular alterations that are known to be
regulated by the EGF receptor (Carpenter, 1987). Thus, there
is need to further examine the relationship between TCDD-
induced alteration of EGF receptor ligand binding and tyrosine
kinase activity with changes in cell growth and
differentiation.

In this study we report on the effect of TCDD on EGF
binding' and. protein Ikinase activity' in a :rainbow' trout
hepatoma cell line (RTE-149) and how alterations in these
parameters relate to TCDD-induced alterations in nucleic acid
synthesis and cell proliferation. Our null hypothesis was:
"TCDD does not cause an alteration in the binding of EGF or
protein kinase activity that is associated with changes to
cell growth or proliferation”. This cell line was selected as

a model system because it is induced by TCDD, and the

171
cytosolic Ah receptor has been identified (Lorenzen and Okey,
1990). This allowed us to make certain assumptions relative
to the Ah model of TCDD toxicity as it pertains to the

modulation of binding of EGF in rainbow trout.

METHODS

genitals

Epidermal growth factor was purchased from 081 (Lake
Placid, NY). [3H1-thymidine, [1-321ATP and [mm-EGF were
purchased from Dupont/NEN Research Products (Bannockburn, IL).
All cell culture materials including fetal bovine serum and
protein kinase substrates, RR-Src and Ac-MBP, were purchased
from GIBCO-BRL (Grand Island, NY). All other chemicals were
from Sigma (St. Louis, MO).
9.9.11 9:131:19

The RTH-149 cell line employed in this study was derived
from an aflatoxin-induced hepatoma in an adult rainbow trout
(Lannan et al., 1984) . Cultures were obtained from the
American Type Culture Collection (Rockville, MD). The cells
were grown as a monolayer in minimum essential medium (MEM)
(Eagle, modified) with Earle's salts, glutamine, bicarbonate,
50 ug/ml gentamicin and supplemented with 10% fetal bovine
serum. Cells were maintained in a normal atmosphere at 22 C
and the media was changed every third day. At confluence,
cells were harvested by trypsinization. Cells were washed in

cold phosphate-buffered saline [2.5 mM KCl, 1.5 mM KHZPO“ 1.5

172

mM NaCl, 8 mM Nazl-IPO‘, 1 mM EDTA] to remove trypsin from the
cells prior to platting or reseeding of flasks.
9.9.11 151mm: Isolation

All procedures were conducted at 0 C. Washed cells were
allowed to swell for 15 min in a hypotonic buffer [10 mM
HEPES, pH 7.4, 1 mM MgC12] containing 100 mM leupeptin, 0.3
trypsin inhibitor units of aprotinin per ml, 0.5 mM E-64
(Calbiochem) and 1 mM sodium vanadate. Cells were broken by
25 strokes in a Dounce homogenizer with a tight fitting
pestle. NaCl (5 M) was then added to a final concentration of
0.15 M. The homogenate was then centrifuged at 800xg to
collect a nuclear fraction ( P1) . The postnuclear supernatant
was then centrifuged for 1 hour at 40,000xg to form a membrane
fraction (P2) and cytosolic fraction (81). The P2 fraction
was washed once with homogenization buffer and stored at -80
C for analysis. Protein concentrations were determined by the
method of Bradford ( 1976) using bovine serum albumin (BSA) as
the standard.

9211mm

Cells were seeded into 12 well plates (Falcon) at an
initial density of 2.2 x105 cells/well and incubated
overnight. They were then washed with PBS, and the medium was
replaced with culture medium supplemented with 1% fetal bovine
serum (FBS) . After 24 h, EGF or TCDD was added at the

appropriate concentration and cells were incubated for the

times indicated. Medium was changed after 48 h. Cell number

173
was determined in duplicate using a hemocytometer.
W 91 DNA mama;

DNA synthesis was estimated by the incorporation of
[3H]thymidine (90 Ci/mmol) into trichloroacetic acid-insoluble
material. Subconfluent cultures in 24-well plates (Falcon),
were made quiescent by a 48 h incubation in a serum free
medium. Cultures were then exposed to TCDD for 24 h.
[BHJThymidine (1 pCi/ml) was added to each well and the
cultures were incubated for 4 h room temperature. Monolayers
were washed 4 times with 1 ml ice-cold PBS, 2 times with 1 ml
10% trichloroacetic acid (10 min each) and once with ethanol.
The cells were solubilized with 300 pl of 0.5 M NaOH and the
radioactivity was measured by liquid scintillation counting.
The [SHJthymidine incorporation was normalized to cell
protein.

EGF: 511151183 55211::

Binding studies were conducted as a modified procedure of
Newsted and Giesy (1991). RTH-149 P2 membranes (50 pg) were
incubated in binding buffer [20 mM Hepes, pH 7.2, 180 mM NaCl,
1 mM DTT, 0.1% BSA] containing 10 mM leupeptin, 0.3 trypsin
inhibitory units of aprotinin /ml and 20 pM E-64 with varying
concentrations of 125I-EGF‘ ( 1.1 pCi/nmol) ranging from 0.05 to
10 nM. After 2 hr at 18 C, the reactions were stopped by the
addition of 3 ml of ice cold buffer and filtered through GF/F
filters (Whatman) . The filters were washed 3 times with

buffer and counted in a Packard auto-gamma counter. Non-

174

specific binding was defined as binding in the presence of a
750-fold excess of unlabeled EGF at each [‘z’IJEGF
concentration. EGF receptor numbers and affinities were
estimated from plots by fitting the data with the LIGAND
program as modified by McPherson (1985).
Maine Kinase been

Tyrosine kinase assays were conducted in microfuge tubes
by a modified method of Ribot et al. (1984). Reactions were
carried out in 60 pl volumes and contained 50 mM HEPES, pH
7.4, 20 mM “92+, 5 mM Mn”, 100 pM vanadate, 5 pM okadaic acid,
0.05% nonidet NP40, 50 pH [1329]”? (1000-2000 cpm/pmol) and
20 to 100 pg membrane protein. To half of the tubes, 120 pM
RR-SRC synthetic peptide was added while the other half
received water. The assay was initiated by the addition of
[1-32PJATP. After incubation for 30 min at 4°C, the reaction
was stopped by the addition of 150 pl of 5% trichloroacetic
acid and 1.56 mM KHZPO“ Subsequently 10 pl of bovine serum
albumin (BSA) was added to aid in protein precipitation and
the samples were incubated on ice for 15 min. The
-precipitated protein was removed by centrifugation.at 5,500xg
for 15 min at 4°C. From each tube, two 50 pl samples of
supernatant.were spotted.on.phosphocellulose filters (Whatman
P-81). Phosphocellulose filters were washed four times in 75
mM phosphoric acid (5 min) and once in acetone (2 min).
Radioactivity of air dried filters was quantified for in 7 ml

of Universol (ICN, Irvine, CA). Tyrosine kinase activity was

 

175

expressed as the difference between samples with and without
the RR-SRC peptide and normalized for time and protein
concentration.
mtein Kinase 9 been

Protein kinase C activity (PKC) was measured by a
modification of the procedure of Bell and Sargent (1987) . To
determine the activity of "particulate" PKC, plasma membranes
were extracted in the following manner: Membranes were
homogenized in 20 mM Tris, pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA,
0.5% triton x-loo, and 25 pg aprotinin and leupeptin/ml.
Homogenized membranes were incubated for 30 min at 0 C and the
cellular debris was removed by centrifugation for 2 min in a
microcentrifuge. Assays were conducted with supernatant in
microfuge tubes in 50 pl total volume. The diluted enzyme
preparation (20 pl) was assayed at 10 C for 5 min. Each assay
contained 20 mM HEPES, pH 7.5, 10 mM MgCl, 1 mM DTT, 20 pM
vanadate, 50 pM [1-321ATP (1000-2000 cpm/pmol), 10 pM CaClz,
120 pM Ac-myelin basic protein (Ac-MBP) , 40 pg diolein and 40
pg phosphatidylserine. Before being added to the reaction
tubes, equal volumes of diolein and phosphotidylserine in
chloroform were combined and the solvent was removed under
nitrogen. The residue was suspended in a small volume of 20
mM HEPES, pH 7.4, by sonication for 5 min at 0 C and the
resultant liposome mixture was used in the assay. Basal
activity was measured in the presence of 10 mM EGTA instead of

CaClz, and liposomes. The reaction was stopped by spotting 25

176
pl onto phosphocellulose paper. The filters were washed twice
in 150 mM phosphoric acid and then twice in distilled water.
The filters were dried and counted in 7 ml Safety Solv.
Protein kinase activity was calculated as the difference

32

between the EGTA treatment and expressed as pmol of P

incorporated per min per mg membrane protein.
Wham

Immunoprecipitation of EGF-related proteins was carried
out as outlined by Gill et al. (1984). Plasma membrane
protein (100 pg) was phosphorylated in m using [1-32PJATP
as outlined previously except that RR-SRC was omitted from the
mixture. The phosphorylated membrane protein was solubilized
in 1 ml RIPA buffer (10 Mm KZHPO“ 0.15 M NaCl, 1% Triton X-
100, 1% sodium deoxycholate, 0.01% sodium dodecyl sulfate and
2 Mm EDTA) for 1 hr at 0 C. The solubilized material was
centrifuged at 12,0009 for 10 min and the supernatant was
incubated overnight at 4 C with 10 pl of mouse anti-EGF
receptor monoclonal antibody. The immunocomplex was
precipitated by using goat antibody to mouse immunoglobulin G
(IgG) conjugated to W m cells (Tachisorb-M,
Calbiochem) for one hour at room temperature with, frequent
mixing. Samples were centrifuged (2,000xg for 15 min) and the
supernatants removed. Pellets were washed twice with 1 ml PBS
at 0 C' followed by centrifugation. The final pellet was
heated (98 C for 5 min) in SDS Laemmli sample buffer and

centrifuged. The supernatant was precipitated with 10% TCA,

177
dissolved by 1 M NaOH and counted by liquid scintillation.
W1 Anglia:

The data were analyzed by analysis of variance planned
comparisons of the means. Relationships between measured
parameters were investigated by Pearsons pairwise
correlations. The level of significance to detect differences

between treatments was p < 0.05.

RESULTS

119.031.83.122 91 128.5 mm and £911 emu DY 191212

Proliferation of RTH-l49 cells low serum conditions was
increased by exposure to TCDD at concentrations ranging from
3.0 to 150 pM (Fig. 22). At concentrations below 3 pM, cells
remained viable, as judged by trypan blue exclusion, but no
statistical increase in cell number was observed. The TCDD-
induced increase in cell number was similar to that observed
in cells grown in MEM supplemented with 10% FBS. No
cytotoxicity was observed at any of the TCDD concentrations
used in this study. The incorporation of’[3H]thymidine into
TCA-precipitated material was dose-dependent with half-maximal
stimulation occurring at about 3.0 pM (Fig. 23). The greatest
stimulation of [SHJthymidine uptake occurred at 150 pM. The
dose-dependent increase in [3H]thymidine incorporation
corresponded to the observed increase in cell proliferation
over the same concentration range. There was a positive

correlation between protein normalized [SHJthymidine uptake

Figure 22.

178

Effect of TCDD on the stimulation of cell
proliferation of RTE-149 cells. Triplicate
subconfluent monolayers of RTE-149 cells were
incubated with the serum poor medium and
varying concentrations of TCDD. Percent of
control values are given, and significant
differences (p < 0.05) between control and

treated cell values are denoted with *.

179

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180

Effect of TCDD on the stimulation of
[3H]thymidine incorporated in RTH-l49 cells.
Triplicate confluent RTE-149 monolayers were
incubated with [3H1thymidine in a serum-poor
medium for 4 h after a 24 h incubation in the
presence of TCDD.- Percent of control values
are given, and significant differences (p <
0.05) between control and TCDD-treated cells

are denoted with *.

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182
and cell proliferation (R2:- 0.951). The increase in
[3H1thymidine uptake and cell proliferation exhibited the same
trends for the duration of the experiments.
Effect of TCDD 911 £52 hissing

Treatment of RTH-l49 cells with TCDD resulted in a
concentration-dependent decrease in specific binding of EGF
(Fig. 24). The EC50 for inhibition of EGF binding was 27.5
pM. The diminished EGF binding in cultures treated with TCDD
was not due to loss of cells from the dish, as was measured by
total protein, or to a reduction in cell viability as measured
by trypan blue exclusion. Binding of EGF was significantly
inhibited within 6 h post-treatment and reached a maximal
inhibition by 72 h that remained unaltered until 96 h (Fig.
25). The effect of TCDD on the magnitude of EGF binding was
persistent. Binding remained reduced (53%) for at least 7 d
after the removal of TCDD from the growth medium. The
reduction of EGF binding was not due to a loss of TCDD from
the culture medium or to a depletion of serum since the
reduction in EGF binding at 72 h did not differ from 96 h even
though medium and TCDD was replenished at 72 h.

The binding in the control cultures was curvilinear,
whereas in the TCDD-treated cultures there was a progressive
loss in linearity of the Scatchard analysis (Fig. 26).
Analysis of the control cultures by LIGAND indicated the
presence of two EGF binding sites with differing affinities
for 125I-EGF. The binding capacity of the high affinity site

Figure 24.

183

Concentration-dependent inhibition of EGF-
binding to RTE-149 by TCDD. Confluent
cultures of RTH-149 cells were treated with
isooctane (0.05%) or TCDD (0.3 to 150 Pm) for
24 h prior to analysis of EGF-specific binding
at 18 C for 2 h. The data shown represents
means of triplicate determinations on
duplicate. cultures and. are expressed. as a
percentage of the isooctane control.
Significant differences (p < 0.05) between
control and TCDD-treated cells are indicated

with *.

184

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185

Time-dependent inhibition of binding of EGF in
TCDD-treated RTE-149 cells. Confluent
monolayers of RTE-149 cultures were treated
with isooctane (0.05% v/v) or TCDD (150 pM)
for the indicated times. The data shown
represents means of triplicate
determinationson duplicate cultures and are
expressed as a percentage of the isooctane
control. Significant differences (p < 0.05)
between control and treated cells are

indicated with *.'

 

 

 

 

 

 

 

 

 

 

 

     

 

 

 

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187

Scatchard analysis of EGF binding data:
Confluent cultures of RTE-149 cells were
treated with 0.05% (v/v) isooctane (O) or 150
pM TCDD (I) for 24 h prior to the isolation of
the cell membranes. Membranes were incubated

RSI-EGF in

with increasing concentrations of
the presence or absence of 750-fold excess
unlabeled EGF. EGF-specific binding was
measured as outlined in Methods. Each point
is the mean of triplicate determinations of

duplicate cultures.

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189

(Kb =1.5 x 10°10M) was 10.5 fmole/mg protein while the binding
capacity (Bu) for the low affinity site (K°= 4.0 x 109) was
153 fmole/mg protein. Cells treated with TCDD exhibited fewer
high affinity binding sites (Ba-x: 0.5 fmole/mg) and only a
small decrease in low affinity binding sites (Bm= 145
fmole/mg). EGF binding affinity (1(0) of low affinity and
high affinity binding sites of the TCDD-treated cells were
unchanged from control values. The reduction in EGF binding
in TCDD-treated RTH-149 cells was negatively correlated to the
increase in cell number (R2= 0.987) and to the increase in
thymidine uptake (R2= 0.957) . These results indicate that
TCDD itself may be potentially mitogenic in RTE-149 cells by
altering EGF receptor homeostasis.
m 22 12129 9.0 2min Liam 593mm

Protein kinase activities were significantly greater in
RTE-149 cells treated with TCDD for 24 h (Fig. 27). TCDD
exposure resulted in approximately a 4-fold increase in
protein kinase C activity. The time-dependent increase
corresponded to the reduction of EGF binding in the cells
'(Fig. 25). Protein kinase C activity was greater after 12 h
and then returned to control values by 96 h (Fig. 27) .
Induction of protein kinase C activity was dose-dependent with
an EC50 of about 23 pM (Fig. 28). This value agrees closely
with the EC50 value for the reduction in EGF binding.
Correlation analysis showed a significant negative correlation

(R2:- 0.835) between EGF binding and protein kinase C activity.

Figure 27.

190

Effect of TCDD on protein kinase C (PKC)
activity, tyrosine kinase (PTK) activity and
EGF receptor (EGF-R) phosphorylation in
confluent RTH-149 cells. Cells were treated
with 0.05% (v/v) isooctane or 150 pM TCDD and
incubated in serum poor medium for 24 h. Cell
membranes were isolated at the indicated times
and the protein kinase activity was determined
as outlined in Methods. Each point represents
the mean of triplicate determinations of
duplicate cultures. Significant differences
(p < 0.05) between control and TCDD-treated

cell values are denoted with *.

C (PKC)
vity am!
:ion in
treated
TCDD and

11. Cell

191

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 28.

192

Effect of TCDD-dose on protein kinase C (PKC)
activity, tyrosine kinase (PTK) activity and
EGF receptor (EGF-R) phosphorylation in
confluent RTH-l49 cells. Cells were treated
with 0.05% (v/v) isooctane or TCDD at the
indicated doses incubated for 24 h. Cell
membranes were isolated and protein kinase
assays were conducted as outlined in Methods.
Each point represents the mean of triplicate
determinations of duplicate cultures and are
expressed as a percent of the control.
Significant differences (p < 0.05) between

control and treated cells are denoted with *.

193

 

 

 

 

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194
The observed increase in protein kinase C activity was also
correlated with [3H]thymidine uptake (R2: 0.901) and increase
in cell number (R2= 0.794) .

Protein tyrosine kinase activity, in TCDD treated RTH-149
cells, was maximally elevated by 6 h (Fig. 27) . By 24 h post-
treatment, tyrosine kinase activity had started to return
control values, but remained elevated through 96 h. As was
observed for protein kinase C activity, tyrosine kinase
activity was positively correlated with TCDD concentration
(Fig. 28) . Activity reached a maximum at 3.0 pM TCDD and
remained unaltered up to 150 pM TCDD. The increase in
tyrosine kinase activity was significantly correlated with the
reduction of EGF binding (Rz- 0.672) but was poorly correlated
with the increase in protein kinase C activity (Rza 0.218).
The lack of relationship between tyrosine kinase activity and
protein kinase C is not surprising in that both protein
kinases are activated through separate mechanisms.
Correlations between protein tyrosine kinase and [3H]thymidine
(R2= 0.726) and cell number (R2=0.583) were also significant.

Immunoprecipitation of phosphorylated RTH-149 cell
membranes showed a time-related increase in the radioactivity
incorporated into EGF receptor immunoreactive protein (Fig.
27). The increase in receptor phosphorylation was initially
observed at 12 h and remained elevated through the 96 h period
of the study. The increase in EGF receptor phosphorylation

occurred approximately 6 h after the initial increase in

 

195

protein kinase C and tyrosine kinase activities. As was seen
in protein kinase activity, the phosphorylation of the EGF
receptor increased in a dose-dependent manner (Fig. 28) .
Increased phosphorylation of the EGF receptor was first
observed at 1.5 pM TCDD and reached a maximum at 15 pM.
However, at 150 pM, there was a decrease in the level of
phosphorylation. Some of this decrease may be attributed to
the inhibition of EGF binding (77%) which occurred at this
TCDD concentration (Fig. 24).

The dose-response relationship between protein kinase C
activity and EGF receptor phosphorylation was not significant
(R2== 0.147) in TCDD-treated RTE-149 cells . However, EGF
receptor phosphorylation values and protein tyrosine kinase
activity was positively correlated (R23 0.752) .

DISCUSSION

TCDD modulates the binding of EGF to the rainbow trout
hepatoma cell line, RTE-149. A loss of high affinity EGF
binding sites following treatment with TCDD was observed (Fig.
26) . This loss was both time- and dose-dependent and
correlated with altered biological responses such as greater
synthesis of DNA (Fig. 23). In RTE-149 cells, a significant
reduction in EGF binding occured at a lesser concentrations of
TCDD (27.5 pM) than that required to cause an induction of
aryl hydrocarbon hydroxylase (AHH) activity (500 pM) (Lorenzen
and Okey, 1990). The association between TCDD concentration

and the observed effects supports the hypothesis that the

 

 

196
reduction in binding of EGF is in part mediated by the Ah
receptor (Ivanovic and Weinstein, 1982). The Ah receptor has
recently been detected in this cell line. RTE-149 cells have
fewer Ah sites (about 20 fmol/mg cytosolic protein) than most
mammalian species but have sites of higher affinity, K5~1 nM
(Lorenzen and Okey, 1990).

The reduction in the binding of EGF binding caused by
TCDD in RTE-149 cells was similar to that observed in (SCC-
12F) human keratinocyte cells. Exposure of SCC-12F cells to
TCDD resulted in a dose-dependent reduction of binding of EGF
that was also similar in scope to the reported dissociation
constant for binding of TCDD to the Ah receptor of mouse liver
(Poland and Knutson, 1982). In RTH-l49 cells, the EC50 for
the reduction of binding of EGF was 30-fold greater than the
apparent dissociation constant of the RTH-149 Ah receptor.
This indicates that while the Ah receptor may have a role in
the modulation of EGF binding, some of the reduction in
binding may be caused by other cellular mechanisms.

The TCDD-induced reduction in binding of EGF capacity can
be regulated by two different mechanisms. The first mechanism
involves altered gene expression of the EGF receptor where
down regulation of the receptor is mediated by the alteration
in protein synthesis. The alteration of EGF receptor gene
expression has been observed in rat uterus where TCDD has been
shown to inhibit the increase in EGF receptor binding activity
by 178-estradiol through reduced mRNA transcription (Astroff

197

et al., 1990). The second mechanism by which TCDD may down
regulate EGF receptor binding activity is through the
activation of protein kinases that can either activate or
inactivate the receptor with the subsequent internalization of
the receptor from the cell surface (Carpenter, 1987 ) . Studies
of TCDD-exposed rats and guinea pigs have shown protein kinase
C and protein tyrosine kinase activities were correlated with
the down regulation of both high and low affinity EGF receptor
sites in hepatic plasma membranes (Madhukar et al., 1988).
The second mechanism is consistent with findings from our
study.

In 1119 studies of rainbow trout have shown that TCDD
induced morphological changes are consistent with altered
epithelial differentiation and proliferation of target tissues
(Spitsbergen et al., 1988) . Similarly, the TCDD-induced
alteration in rainbow trout liver morphology is consistent
with liver hyperplasia and fatty infiltration which are also
induced by EGF. Rainbow trout given a single i.p. dose of
TCDD exhibit a dose- and time-dependent decrease in hepatic
EGF binding that was correlated with the increase in protein
kinase C activity ( see Chapter 3). The increase in protein
kinase C was also observed in TCDD treated RTE-149 cells. As
was observed in rainbow trout hepatic plasma membranes,
protein kinase C activity was correlated in a time- and dose-
dependent manner with the reduction in EGF binding. The

increase in protein kinase C was also significantly correlated

 

 

198

with an elevation in [3H] thymidine uptake which is indicative
of altered cell proliferation. Protein kinase C is a calcium-
and phospholipid-dependent protein kinase that is a key enzyme
for intracellular signal induction and the control of cell
proliferation and mitogenesis (Nishizuka, 1988). Protein
kinase C has also been identified as a major cellular receptor
for the potent skin-tumor promoter, 12-0-tetradecanoylphorbol-
l3-acetate (TPA) (Lee and Weinstein, 1978) . TPA-stimulated
protein kinase C is thought to phosphorylate serine and
threonine residues cf critical target proteins which may
directly or indirectly regulate the expression of specific
genes associated with tumor promotion and cell growth. In A-
431 cells, the activation of protein kinase C has been shown
to decrease EGF binding via phosphorylation of serine and
threonine residues on the EGF receptor (Downward et al. ,
1985). Thus, elevation of protein kinase C activity in RTH-
149 cells and the subsequent induction of cell proliferation
and [1H] thymidine uptake may have significant implications
relative to altered plasma membrane homeostasis as it relates
to TCDD-induced toxicity (Matsumura et al., 1984).

Tyrosine protein kinase activity was also elevated in
TCDD treated RTE-149 cells (Fig. 28) . The increase in
tyrosine kinase activity was significantly correlated with the
reduction of EGF binding which may indicate activation of the
receptor with subsequent internalization and the loss of

receptors from the cell surface (Velu, 1990). Studies have

199

suggested that the tyrosine kinase activity of the EGF
receptor is essential for signal transduction, normal receptor
"trafficking" , stimulation of DNA synthesis and transformation
(Moolenaar et al. , 1988) . Significant correlations were
observed between tyrosine kinase activity and cell growth (R2-
0.683) and [3H] thymidine uptake (Rz- 0.826) over the dose
range used in this study. Similar results were also seen in
the in yin rainbow trout study where significant correlations
were observed between protein tyrosine kinase activity and the
reduction in EGF binding. The significance of this finding is
that there was a positive correlation between the increase in
tyrosine kinase activity and receptor phosphorylation levels.
The increase in EGF-receptor phosphorylation may indicate the
activation of the EGF receptor in both TCDD-treated rainbow
trout hepatic plasma membranes and RTE-149 cells. However,
the dose-response relationship between cell number and EGF
receptor phosphorylation were not statistically significant
and may indicate that other regulatory mechanisms are involved
in the cell growth. One such mechanism may be TCDD-induced
'stimulation of protein kinase C activity that has been shown
to be related to increased cell proliferation and
differentiation (Nishizuka, 1988).

In conclusion, our previous findings indicate that
altered binding of EGF in TCDD-treated rainbow trout is
related to changes in protein kinase activity. Data from the

current study corroborates these findings in the rainbow trout

 

 

200
cell line, RTH-149. We have also shown that TCDD alters cell
growth and.[;H]thymidine uptake in a time-and dose-dependent
manner that correlates with increases in protein kinase
activity. These effects are similar to those observed in
TCDD-treated.mammals and may indicate a similar mode of toxic
action between fish and mammals. These results argue that
TCDD may exert multiple effects on the EGF-induced
proliferative pathway, one of which may involve an alteration
of protein kinase C activity and of EGF receptor associated

and other cell surface tyrosine kinases.

201

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Kleinow,K.M, Melancon,M.J. , and Lech,J.J. (1987) .
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Lee,L-S., and Weinstein,I.B. (1978). Tumor promoting phorbol
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Lorenzen,A. , and Okey,A. B. (1990) . Detection and
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Brewster,D.W., and kawamoto,T. (1988). 2,3,7,8-
tetrachlorodibenzo-p-dioxin causes an increase in protein
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Matsumura,F., Brewster,D.W., Madhukar,B.V., and Bombick,D.W.
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and Yarden, Y. (1982). Regulation of cell proliferation

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206

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207

SUMMARY

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent
environmental toxicant that has been shown to affect many fish
species at low environmental concentrations. Evaluation of
the possible effects of TCDD on fish populations is difficult
in that TCDD exists in the environment in a complex mixture of
structuraly similar compounds. Thus, methods to monitor the
effects of TCDD and TCDD-like compound on fish should be
sensitive and specific and be based on the mode of toxic
action of these compounds. While fish are extremely
sensitive to the effects of TCDD, little or nothing is known
about the mechanisms of toxicity of TCDD in these species.
The results presented in this report are the first to examine
the role of the epidermal growth factor receptor (EGF-R) in
the toxicity of TCDD to fish.

Since little was known about EGF-R in fish, we conducted
several studies to characterize the binding and the
biochemistry of this receptor in rainbow trout. Our results
show that in rainbow trout, EGF-binding was peptide specific,
saturable, reversible, and of high affinity. Binding of EGF
to rainbow trout membranes caused an increase in a tyrosine
kinase activity that phosphorylated tyrosine residues of
endogenous polypeptide substrates and of the EGF receptor like

protein. These observations are similar to those seen in

208

mammalian tissues treated with EGF. However, care must be
taken when extrapolating the data generated in these studies
to that of the possible biological action of the EGF-R in
other species of fiSh. Before these extrapolations can be
made, research need to be conducted to examine the role of EGF
in fish. Future research should examine how EGF alters the
regulation of normal cell growth and differentiation and the
development of early life stages. One of the more sensitive
measures of EGF effects is the early eyelid opening in newborn
mice. Analogous studies should be conducted in fish. Efforts
should also be made to isolate both the EGF-receptor and its
ligand in rainbow trout.

Many of the toxic effects induced by TCDD are mediated
through the alteration of membrane receptors. Studies
conducted on. mammals to characterize changes in plasma
membrane homeostasis have shown that alterations in EGF
receptor binding are a sensitive measure of TCDD exposure. We
have shown that a similar mechanism may be operating in fish.
As was seen in mammals, TCDD reduced EGF binding in hepatic
membranes in a time- and dose-dependent manner. The reduction
in binding was associated with an increase in protein kinase
C and protein tyrosine kinase activities. While these
activities correlated with other biological measures of
toxicity (cytochrome P-450 induction), actual documentation of
hepatic lesions was not made during these studies. The lack

of information makes it difficult to interpret the alterations

209
of EGF-binding and protein kinase activities with
toxicological lesions known to occur at the concentrations
used in this study. Studies with the RTH-149 rainbow trout
hepatoma cell line demonstrated that alterations in EGF
binding and protein kinase activities were correlated with
changes in cell proliferation and DNA synthesis. These data
provide some evidence for alterations in EGF receptor activity
as it relates to TCDD toxicity. However, evidence for the in
yiyo role of the EGF-receptor in TCDD toxicity is needed
before this biomarker of TCDD toxicity can be used in any

monitoring program.

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