AN ABSTRACT OF THE THESIS OF
Gary H. Perdew for the degree of
in
Food Science and Technology
Title:
Doctor of Philosophy
presented on
June 25, 1984
Alterations in Levels and Synthesis of Proteins in Trout
Hepatocytes Due Jo Dietary gyc]opropenoid Fatty Acids
Abstract Approved:
___
~"~
*
^
'Daniel P. Selivonchick
Cyclopropenoid fatty acids (CPFA) are unique compounds that
contain a highly strained and reactive cyclopropene ring structure.
These compounds have been shown to cause a number of toxic effects
in a variety of animals.
Rainbow trout (Salmo gairdneri) have
proven to be particularly sensitive to CPFA.
Studies have revealed
that CPFA are both carcinogenic and cocarcinogenic in rainbow trout.
However, the mechanism(s) of these adverse biological effects are
not understood.
In the present report a series of studies were
performed in order to determine the effect of CPFA on the levels and
synthesis rates of trout hepatocyte proteins.
In the first study, the influence of dietary CPFA on protein
synthesis was measured via the use of amino acid double labeling
experiments in isolated hepatocytes.
Both the microsomal and
cytosolic subcellular fractions were examined in these studies after
seperation by lithium dodecyl sulfate polyacrylamide gel
electrophoresis.
In the cytosolic fraction, the synthesis of
proteins with apparent molecular weights in the range of 68,000 to
74,000 were significantly decreased.
A marked depression in both
the level and synthesis rate of microsomal proteins was observed for
proteins that migrate in the 200,000 to 240,000 relative molecular
mass region in polyacrylamide gels.
These high molecular weight
proteins do not appear to be membrane proteins and one of them has
biotin associated with it.
Using avidin-peroxidase staining, it was
shown that the mass of this protein was reduced in CPFA-fed trout by
80%.
The possible identity of these proteins is discussed.
In the second study, initial attempts were made to use two
dimensional gel electrophoresis to study alterations in individual
liver microsomal polypeptides from trout fed CPFA.
In order to
effectively resolve membrane proteins in the first dimension
(isoelectric focusing) changes in the standard techniques were
needed.
Replacement of the detergent nonidet-40 with
3-[(3-cholamidopropyl)dimethylammonio]-l-propane sulfonate (CHAPS)
in isoelectric focusing of trout liver microsomes have greatly
increased resolution.
These results have allowed effective
resolution of complex polypeptide patterns for comparative purposes.
In the third study, antibodies against 6-napthoflavone-fed
rainbow trout cytochrome P-450 (LNL) were employed to localize the
corresponding polypeptide(s) via protein blotting and immunochemical
staining.
Microsomes isolated from
6-napthoflavone-fed trout
contained only a single polypeptide.
In contrast, control
microsomes contained two distinct polypeptides differing only in
their isoelectric points.
Thus, an additional P-450 isozyme in
rainbow trout was tentatively identified.
CPFA treatment caused a
preferential decrease in only one of the isozymes found in the
control samples.
The presence of concanavalin A binding
glycopolypeptides was determined.
The two P-450 isozymes localized
on control microsomal gels were found to bind concanavalin A,
suggesting that these isozymes are giycoproteins.
Another result of
CPFA treatment was a shift in a closely related group of membrane
glycopolypeptides, labeled gp80, gp82, gpSO,, and gpSZ,.
A
decrease in the mass of gp80 and gp82, and a corresponding increase
in mass of gp80, and gp82, was observed.
Alteration in Levels and Synthesis of Proteins in
Trout Hepatocytes Due to Dietary Cyclopropenoid Fatty Acid
by
Gary H. Perdew
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Completed June 25, 1984
Commencement June 1985
APPROVED:
Professor of F,dod Science and Technology in charge of major
Head of Department of Food Science and Technology
Dean of Grfiduate SchoorfT
Date thesis is presented
June 25, 1984
Typed by Sue Hecht for
Gary H. Perdew
ACKNOWLEDGEMENTS
I wish to thank Drs. Daniel P. Selivonchick and Henry W. Schaup
for their support and guidance in the course of this project.
In
addition, the assistance and advice of Janet Williams, Mark
Einerson, Ted Will, John Casteel, and Jerry Hendricks is gratefully
acknowledged.
A special thanks to Marcia Harris for her help during
various phases of this work.
This investigation was supported by
Grant IROICA 30087-03 from the United States Public Health Service.
CONTRIBUTION OF AUTHORS
Dr. Donald Buhler's contribution to the third manuscript was to
provide the antibodies used to localize the cytochrome P-450 isozymes on
two dimensional electrophoretic gels.
Drs. Daniel Selivonchick and
Henry Schaup served in advisory roles during the course of this work.
TABLE OF CONTENTS
PAGE
I.
Literature Review
Structure and Occurrence of Cyclopropenoid Fatty
Acids
Physiological Effects of CPFA
. Cellular Effects of CPFA
Cocarcinogenic and Carcinogenic Effects of CPFA. . .
Hepatocarcinogenis - The Two Stage Model ......
Effects of Promoters on Gene Expression/Protein
Synthesis
Possible Mechanism(s) of CPFA in Tumor Promotion
and Cocarcinogenesis
II.
Alterations in Levels and Synthesis of Proteins in
Hepatocytes of Rainbow Trout Fed Cyclopropenoid
Fatty Acids
Abstract
Introduction
Materials and Methods
Results
Discussion
III.
The Use of a Zwitterionic Detergent in Two-Dimensional
Gel Electrophoresis of Trout Liver Microsomes. . ...
Abstract
Introduction
Materials and Methods
Results and Discussion
IV.
Alterations in Hepatic Microsomal Protein Levels in
Rainbow Trout Fed Cyclopropenoid Fatty Acids: Analyzed
by Two Dimensional Gel Electrophoresis
Abstract
Introduction
Materials and Methods
Results and Discussion
V.
References
1
2
3
7
9
13
14
16
17
18
19
24
28
47
48
49
50
52
56
57
58
60
62
69
LIST OF FIGURES
FIGURE
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
PAGE
Percent leakage of LDH activity from control and CPFAtreated trout hepatocytes into the incubation media,
as a function of time.
30
Hepatocytes from control and CPFA fed trout were
labeled with [ C]-leucine and [ H]-leucine,
respectively.
32
Plot of Np. values determined from a mixture of
[ C]TControl/[ H]-control supernatant (upper panel)
and [ C]-control/[ H]-CPFA (lower panel) supernatant
after seperation via gradient gel electrophoresis.
34
Hepatocytes from control and CPFA fed trout were
labeled with [ C]-leucine and [ H]-leucine,
respectively.
36
Plot of Np. values determined from a mixture of.
C1 Cj-con^ol/C H]-control (upper panel) and [ C]control/[ H]-CPFA (lower panel) microsomes after
seperation via 7.5%-15% gradient gel electrophoresis
38
After Coomassie-Blue staining, a control (upper panel)
and CPFA (lower panel) microsomal gradient gel was
scanned with a Zeineh soft laser scanning densitometer
interfaced with an Apple computer.
40
Microsomes were subjected to a Tris/water/Tris wash
as described in Materials and Methods. Each fraction
was applied to a 7.5%-15% gradient gel and scanned
with a computer-interfaced soft laser densitometer.
42
Control and CPFA liver microsomes were isolated in
triplicate, electrophoresed and blotted as described
in Material and Methods.
44
IFLDS of trout liver microsomal proteins followed by
silver staining. The gels were run as described under
Materials and Methods.
54
Control liver microsomal IFLDS silver stained gel is
shown with two areas of interest marked with a square
and a circle.
65
Concanavalin A-peroxidase stained blots of A) control
and B) CPFA microsomal IFLDS gels.
67
TABLE
TABLE
1.
PAGE
The Effect of Tris/water/Tris Washing on the
Particulate Membrane Fraction.
46
Alterations in-Levels and Synthesis of Proteins in
Trout Hepatocytes Due to Dietary Cyclopropenoid Fatty Acids
LITERATURE REVIEW
Structure and Occurrence of Cyclopropenoid Fatty Acids
Cyclopropenoid fatty acids (CPFA) are unusual fatty acids,
containing one of the most strained structures in nature.
CPFA are
found in plants in the order Malvales; within this order are
several plants used in the human and animal diet.
The major CPFA
have been characterized in these plants; they are 7-(2-octyl 1
cyclopropenyl) heptanoic acid and 8-(2-octyl 1 cyclopropenyl)
octanoic acid, given the trival names malvalic and sterculic acid,
respectively (1,2).
The biosynthesis of these fatty acids has been
recently reviewed (3).
The chemical structures of sterculic and
malvalic acids are given below.
sterculic acid
CH3(CH2)7C=C(CH2)7C00H
malvalic acid
CH3(CH2)7C=C(CH2)6C00H
CHp
CHp
Due to the steric strain in the cyclopropene ring CPFA are
highly reactive and are capable of undergoing a number of
reactions, including oxidation, addition, polymerization, and
reduction (4).
In order to study CPFA in biological systems
sensitive methods of detection are needed.
Cyclopropene assay
methods have been reviewed by Coleman in 1970 (5).
For example, a
classic detection method for CPFA, the Halpnen test, takes
advantage of the ability of sulfur compounds to add across the
cyclopropene ring (6).
More recently, nuclear magnetic resonance
spectroscopy (NMR), and high pressure liquid chromatography (HPLC)
have been utilized as nondestructive assays for CPFA (7,8).
In the United States cotton (Gossypium hirsutum) is
essentially the only possible source of CPFA in the human diet.
Cottonseed "nuts" have been approved in the U.S. for consumption as
a snack item and for use in baked products.
Crude cottonseed oil
contains from 0.4-0.8% sterculic acid and between 1.1-1.6% malvalic
acids (4).
Fortunately the majority of the CPFA is destroyed by
modern processing methods, although small levels do persist (9,10).
Cottonseed meal contains small amounts of CPFA and is commonly used
in animal feed.
The oil extracted from kapok (Eriodendron
anfractuosum), may contain up to 14% CPFA, and is used as a food
oil in Japan, as well as, other eastern countries (10).
Sterculia
foetida oil contains up to 70% CPFA, and is used as a source of
CPFA for experimentation (11).
Several reviews on cyclopropene
chemistry and occurrence are present in the literature (3,12,13).
Physiological Effects of CPFA
While no known effects have been established in humans, CPFA
exhibits a wide range of adverse physiological alterations in other
animal species (13).
The first characterized effect of CPFA was in
hens fed cottonseed oil, subsequently causing a pink discoloration
in egg whites (14).
The cause of this defect was initially
attributed to a halphen positive compound present in the cottonseed
meal (15).
The pink pigment has been identified as a
iron-conalbumin complex (16).
It is commonly accepted that the
presence of iron in the egg white is caused by CPFA induced
increase in permeability of the egg yolk vitelline membrane
(17,18).
Other CPFA associated changes in laying hens include,
decreased egg hatchability (18), altered fatty acid profiles of egg
yolk lipids (13,19), a delay in the sexual maturity of hens, and
decreased weight gain (20).
An increase in embryo mortality was
also noted in quail fed CPFA (21).
Another species that has been examined for physiological
effects of CPFA is the rat.
For example, Miller eta]_. (22) have
shown that dietary CPFA caused a high incidence of prenatal and
postnatal mortality.
CPFA were also shown to impair reproduction
(23), retard growth rate (24), and at a dietary level of 0.35% were
found to cause widespread hemorrhaging, renal tubule degeneration,
liver necrosis (25), and mitogenic activity in the pancreas and
liver (26,27).
Rainbow trout (Salmo gairdneri) are particularly susceptible
to the effects of CPFA.
Among the alterations seen are necrosis of
hepatocytes, unusual glycogen deposition, the appearance of
"fibers" in the cytoplasm of many cells, fibrotic blood vessels and
ducts, increased liver/body weight ratios, and fatty infiltration
of the liver (28).
Other reports include reduced growth rates
(29), and bile duct hyperplasia (30).
Cellular Effects of CPFA
The most studied effect of CPFA at the cellular level has been
on lipid metabolism.
CPFA fed to rats have been shown to cause a
decrease in the unsaturated/saturated fatty acid ratios of both
liver and adipose tissues (31,32).
Similar alterations in fatty
acyl compositions have been established in rainbow trout (29) and
White Leghorn hens (33).
The cause of this effect has been
attributed directly to an inhibition of fatty acyl desaturase (FAS)
in hen liver (33,34), green algae (35), mice (36), and rat (37).
Sterculic acid was a more effective inhibitor of FAS than malvalic
acid (38).
It has long been accepted that, in animals,
monounsaturated fatty acids such as oleic and palmitoleic acids are
formed predominantly by enzymic desaturation of the corresponding
saturated fatty acids.
Initial reports presented evidence that in
vitro CPFA inhibits fatty acid desaturase via irreversible binding
of the cyclopropene group to the enzyme's sulfhydryl groups
(33,39).
Studies performed by Jeffcoat and Pollard, utilizing rat
3
liver microsomes and H-sterculic acid, revealed that, under
conditions of high A9 desaturase inhibition there was no covalent
binding (40).
They did find, although, that the noncovalent
binding energy of the active form of sterculic acid,
sterculoyl-CoA, to the enzyme is much greater than that of stearoyi
CoA.
These authors concluded that effectiveness in preventing
desaturation of stearic acid must be either in a better fit to the
enzyme or in a polarization interaction dependent on the
cyclopropenoid ring in the vicinity of the C-9 and C-10 atoms of
the fatty acid.
It should be noted that the marked inhibition of
fatty acyl desaturase activity seen in vivo may be caused by an
actual reduction in enzyme content, not by direct inhibition.
Until the amount of fatty acyl desaturase is measured, this
possibility should not be ruled out.
Higher serum cholesterol levels and a greater incidence of
atherosclerosis have been found after feeding diets containing CPFA
to cockerels (41,42), and rabbits (43).
In the rabbit, however,
both liver and plasma cholesterol levels were increased.
In
contrast, cockerel liver cholesterol levels were reduced.
Recently, studies have shown that CPFA decrease the
unsaturated/saturated fatty acid ratio of the fatty acid moiety of
serum cholesterol esters in mice, this may help explain the
elevated serum cholesterol levels by slowing clearance rates (44).
In contrast to other enzymatic activities sn-glycerophosphate
acyltransferase (GPAT) has been shown to be induced with dietary
CPFA at all stages of development in mice (36).
The stimulation of
GPAT activity may be related to the increased amount of fat found
in livers of animals fed a CPFA diet (31).
Lactate, malate,
glucose-6-phosphate, and NADP-1inked isocitrate dehydrogenases were
all shown to undergo significant reduction in activities upon
feeding 200 ppm CPFA (45).
Along with the reduced liver
dehydrogenase activities a concurrent decrease in the overall
protein levels was observed.
Whether or not these alterations in
enzyme activities are due to enzyme levels or enzyme inhibition
remains to be determined.
Many of the physiological effects attributed to CPFA are
believed to be mediated through CPFA induced alterations in
membrane systems.
In addition to the altered fatty acid
composition of membranes, CPFA have been shown to be incorporated
into neutral lipids and phospholipids of rat and rainbow trout
(46,47).
These studies have revealed that the methylene carbon of
the cyclopropene ring was not oxidized, indicating that the
cyclopropene ring remained intact.
14
Upon injection of
C-sterculic acid, and subsequent isolation of hepatic
microsomal lipids, the isotope was associated largely with choline
phospholipids (CP) and ethanolamine phospholipids (EP).
Phospholipase A2 treatment of purified CP and EP showed
14
C-sterculic acid to be preferentially esterified to the
1-position of the glycerol backbone (48).
These results suggest
that CPFA are recognized by the sn-glycerophagtfate/acyltransferase
as saturated fatty acids upon transfer to the glycerol backbone.
In young rats CPFA have been shown to increase erythrocyte
hemolysis rates and reduce the glutathione induction of
mitochondrial swelling.
Both changes were believed to be caused by
altered membrane function (24).
Scarpelli found CPFA to induce
disruptions in the rough endoplasmic reticulum (RER), as determined
from electron micrographs (49).
Scarpelli noted that alterations
in the RER membranes by CPFA are quite similar to those seen in the
rat hepatocyte after exposure to carbon tetrachloride (CClJ.
Changes in this membrane organelle are of particular interest
because the RER is the site of lipid synthesis and membrane
biogenesis.
It is becoming more apparent, through studies of
reconstituted systems, with membrane enzymes that the constituent
phospholipids may play a role in modulating enzyme activity
(50,51).
A number of membrane-bound drug metabolizing enzymes are
affected by CPFA in rainbow trout.
The mixed function oxidase
(MFO) enzymes that have shown a consistent decrease with dietary
CPFA are cytochrome P-450 (52,53,54), NADPH-cytochrome c reductase
(52), ethoxyresorufin- and ethoxycoumarin-O-deethylases (55),
aldrin epoxidase (53), and codeine demethylase (24).
MFO enzymatic
activities that have maintained control levels include
benzo(a)-pyrene monooxygenase (55), cytochrome b5 (54), and the
ability to O-dealkylate phenacetin (49).
Similar effects on MFO
enzymatic activities have been shown in rabbits (56).
Further
studies have revealed both a dose and time dependent decrease in
P-450 in rainbow trout fed diets containing CPFA (54).
Sterculic
acid caused a 45% decrease in rainbow trout, while only a 6%
decrease was observed for malvalic acid (57).
These comparative
data illustrate the overall less toxic response of trout to
malvalic acid.
Cocarcinogenic and Carcinogenic Effects of CPFA
In 1960 an outbreak of liver cancer in hatchery-reared rainbow
trout occurred in the United States and Europe.
The causative
agent in most of these outbreaks was found to be aflatoxin
contaminated cottonseed meal (58).
The CPFA in the cottonseed meal
was subsequently found to enhance the carcinogenic effect of
aflatoxin B, in rainbow trout (59).
In these studies it was
found that trout receiving basal diets containing 4 ppb aflatoxin
B, plus 220 ppm CPFA showed a 90% incidence of hepatic tumors
after six months, while trout receiving 4 ppb aflatoxin B, in
their diet developed only a 20% incidence of liver tumors after 9
months.
In addition, a large increase in the volume of tumor
tissue in fish fed CPFA plus aflatoxin B,, compared to aflatoxin
B, alone, was observed.
Subsequent tumor studies have focused on
8
the synergistic effect of CPFA on the carcinogenic response to
aflatoxin B, metabolites.
Those tested include, aflatoxin M,
(60), aflatoxin Q, (61), and aflatoxicol (62).
The
cocarcinogenic effect of CPFA has also been established with
2-acetylaminofluorine (AAF), a highly characterized carcinogen
(63).
An absence of a synergistic effect between cyclopropenoid
fatty and aflatoxin in rats was initially'reported (64).
In
contrast, rats fed 10 ppb aflatoxin B, plus 220 ppb CPFA showed
an increase from 59% to 70% in tumor incidence when compared to
rats fed 10 ppb aflatoxin alone (65).
Although only a slight
increase was noted, this study leaves open the question of whether
CPFA is cocarcinogenic in mammalian species.
The hepatocarcinogenicity of CPFA has been established in
rainbow trout through a dose-response feeding study (66).
In this
study, it was found that the maximum tumor incidence occurred at 45
ppm and actually decreased at higher doses.
For a more complete
account of cocarcinogenic and carcinogenic tumor studies in rainbow
trout the reader is referred to the review written by Hendricks
(67).
To date little work has been done to establish CPFA as a
classical promoter of carcinogenesis.
One study utilized trout
embryo exposure to aflatoxin B, followed by a diet containing 50
ppm CPFA (68).
Trout receiving the CPFA diet showed as much as a
53% increase in tumor incidence over trout that received the
control diet.
Recently, CPFA has been tested in an in vitro cell
communication assay that has been developed by Trosko et aj_. (69),
9
to screen for potential tumor promoters.
CPFA were shown to
exhibit a linear dose-response, although CPFA were only weakly
positive when compared to phorbol esters in this assay (70).
Hepatocarcinogenesis-The Two Stage Model
In the 1940's the multistage process of skin tumor development
was proposed by Rous (71).
He coined the now classic terms
"initiation" and-"promotion" to describe the process of
carcinogenesis.
In this scheme, initiation represents the
production of potentially tumorigenic cells via exposure to a
carcinogen, and promotion is the completion of neoplastic
transformation as a result of subsequent treatment with a compound
that, by itself, is not capable of producing a carcinogenic
response.
A large body of evidence has established these, concepts
in skin carcinogenesis (72-74).
Not until the last decade has the,
liver, a nonepidermal system, been unequivocally demonstrated to
follow the initiation-promotion concept.
Many compounds have been found to exhibit both carcinogenic
and promotional activity as determined in feeding trials.
Several
chemicals that-do not damage DNA, epigenetic agents, have been
shown to be carcinogenic in prolonged feeding experiments, this
positive response could occur solely by promotion of spontaneous
preneoplastic foci (75).
This observation casts doubt as to
whether epigenetic agents (e.g. CPFA, phenobarbital) are actually
carcinogens.
The sequential feeding of a given carcinogen followed
by another known carcinogen, that enhances the carcinogenicity of
the first treatment, is known as summation, and should not be
considered an "initiation-promotion" experiment (76).
It would
10
seem appropriate at this time to reserve the term "promoter" for
epigenetic compounds.
By examining and comparing the characteristics of various
hepatocarcinogenic promoting agents perhaps a common "requirement"
for promotion can be determined.
Promotors of hepatocarcinogenesis
that have been identified by initiation-promotion experiments
include:
phenobarbital, butylated hydroxytoluene,
2,3,7,8-tetrachlorodibenzo-p-dioxin, choline deficiency,
polychlorinated biphenyl, carbon tetrachloride, and contraceptive
steroids.
The review of Peraino et al_. (77) contains a more
complete listing of known hepatocarcinogenic promoters.
Treatment
with the carcinogen AAF followed by feeding of phenobarbital (PB),
has been the most used initiation-promotion protocol.
of a number of studies (78-82) have indicated:
The results
a) the percentage
of rats with tumors increased with- increasing duration of AAF
feeding; b) shifting rats to dietary PB following AAF exposure
markedly enhanced tumor incidence; c) the tumor incidence was
controlled primarily by the duration of exposure to PB rather than
by the time between AAF and PB treatments; and d) increasing the PB
feeding period after AAF exposure revealed a continual enhancement
of tumor formation over time.
These studies suggest that hepatic
tumorigenesis follows a multi-stage sequence similar to skin
carcinogenesis.
The sequence of PB and AAF exposures is important
in the type of response obtained.
Feeding PB before exposure to
4-dimethylaminoazobenzene actually decreased tumor incidence,
apparently because the PB induced MFO activity favors
detoxification of the carcinogen (83).
Attempts to examine the
11
possible promoting effects of PB on tumorigenesis in tissues other
than liver have indicated a high degree of specificity towards
hepatocarcinogenesis (84,84).
Consequently, it is believed that
the stimulation of liver growth by PB (86) may play a role in its
promotional activity.
More recently, a study examined the effect
of varying the concentration of PB on enhancement of
2-acetylaminofluorene-induced tumorigenesis.
The results indicated
an increase in tumor formation at PB levels less than those
required for growth stimulation (81).
Furthermore, butylated
hydroxytolune (BHT) has been shown to exhibit many of the
promotional characteristics of PB, except BHT does not stimulate
DNA synthesis, indicating a lack of mitogenic activity (80).
Thus,
mitogenic activity does not appear to be required for tumor
promotion, and not all mitogens are capable of enhancing tumor
formation.
Both CC1- exposure and partial hepatectomy have been used as
methods for stimulating hepatocyte proliferation.
When these
treatments were coupled with administration of a carcinogen tumor
formation was enhanced (87,88).
A wide range of hepatotoxic
effects, such as an increase in lipid peroxidation and triglyceride
levels, occur upon treatment with CC1-.
Under these conditions
an increase in cell turnover can be detected.
Nevertheless,
attempts to draw a relationship between hepatoxicity and
enhancement of tumorigenesis have been largely unsuccessful.
For
example, simultaneous CC1 .-carcinogen treatments have been shown
to cause enhancement.
However, substitution of CC1. with a
different hepatotoxin, allyl alcohol, produced no increase in tumor
12
formation (89).
These results suggest that the cocarcinogenic
effect of CC1. is probably masked by the myriad of toxic effects
that occur; again illustrating that the ability to enhance
mitogenic activity is not sufficient to cause tumor promotion.
Preinitiation hepatectomy has become an important part of
recent protocols aimed at analyzing mechanisms of multistage
hepatocarcinogenesis (90).
This procedure improves the sensitivity
of the carcinogenic response, thus permitting the use of subtoxic
initiating dosages of carcinogen.
Preinitiation hepatectomy may be
replaced by CC1. injections (91), but the latter method appears
inadequate in that it simultaneously causes a wide range of toxic
effects.
Thus, the use of preinitiation hepatectomy allows the
study of hepatocarcinogenic processes devoid of the complications
of toxic side effects seen with CC1..
Production of lipotrope deficiency (e.g. choline, methionine,
and folic acid) prior to or following exposure to a carcinogen
increases the tumorigenic response (92,93).
Additionally,
lipotrope deficiency causes profound disruptions in lipoprotein
metabolism, accompanied by fatty infiltration of the liver, leading
to hepatic cirrhosis (94).
Thus the induction of lipotrope
deficiency would appear to stimulate hepatocyte proliferation in
much the same manner as CC1. treatment, through cell injury and
cellular regeneration,
it would be difficult to see how events
related to enhancement of tumorigenesis could be distinguished from
nonspecific biochemical effects.
Nevertheless, studies on
phenotypic alterations induced by promoters are necessary in order
to identify key promotional events.
13
Effects of Promoters on Gene Expression/Protein Synthesis
Very little work has been performed on alterations in levels
and/or synthesis of protein due to hepatocarcinogenic promoters.
In contrast, a number of studies have been completed with phorbol
esters, potent promoters of skin carcinogenesis.
The pomotional
phase of tumor development, appears to involve the expression of
latent or dormant genes, and perhaps the repression of specific
genes (95).
Phorbol esters, as with other promoters, induce a
hyperplasic state that, in itself, causes a number of alterations
to occur.
With this observation in mind, it would seem
advantageous in studies with a tumorigenic promoter to also use as
a control a hyperplasic agent that is not a tumor promoter.
Several investigators have used this approach to study the effect
of the 12-0-tetradecanoyl-phorbol-13-acetate (TPA), a phorbol
ester, on newly synthesized proteins in mouse epidermis (96,97).
The results of these studies showed that changes in protein
profiles induced by TPA are not simply the consequence of
hyperplasia.
A dedifferentiation has been suggested to occur,
based on reports of induction of several new epidermal proteins
characteristic of newborn epidermis after treating adult mouse skin
with TPA (98,99).
Recent studies have further characterized
specific proteins induced by TPA and related tumor promoters
(100-102).
Taken together these reports suggest that tumor
promoters induce a specific program of changes in target cells
rather than a generalized alteration of protein synthesis.
similar approach may be useful in assessing specific protein
alterations due to promoters of hepatocarcinogenesis.
A
14
Possible Mechanism(s) of CPFA in Tumor Promotion and
Cocarcinogenesis
Whether or not CPFA potentiates AFB, carcinogenesis via
enhancement of the initiation or promotional phase has recently
been addressed.
Studies on the effects of CPFA on the metabolic
activation and deactivation of aflatoxin, and the subsequent
ability to bind to DNA have revealed the following:
1) in the Ames
assay (103) the use of an S-9 fraction isolated from CPFA treated
rainbow trout and aflatoxin has shown a reduction in mutagenic
activity when compared to control S-9 fraction (54); 2) hepatocytes
isolated from CPFA treated trout have shown a significant reduction
in AFB, binding to DNA in cell suspensions (104).
These studies
strengthen the idea that the mode of action of CPFA, as a
cocarcinogen, is in the promotional phase.
In a number of cell surface assays, such as cell attachment,
cell-cell metabolic cooperation, and lectin binding, alterations
with phorbol ester treatment have been observed (105,106).
Additionally, changes in membrane fluidity have been demonstrated
after exposure to phorbol esters (107).
It is believed that these
effects play a role in the overall promotion process.
The
incorporation of CPFA into membrane phospholipids may be capable of
perturbating plasma membrane functions, although little data is
available to support this possibility.
An increase in mitogenic
activity has been observed in both rainbow trout hepatocytes (26)
and rat pancreatic acinar cells (27) after dietary exposure to
CPFA.
This increased activity could be due to disruption in the
plasma membrane, an important site of mitotic regulation.
Another
15
possible explanation for enhanced mitogenic activity may be an
increase in cell turnover due to injury.
This would be similar to
the results obtained with CC1« exposure (88).
As discussed earlier, tumor promoters are believed to
potentiate alterations in gene expression that lead to the
manifestation of the cancer phenotype.
Several observations have
been made in rainbow trout concerning alterations in hepatic
proteins with dietary CPFA:
1) the level of microsomal protein is
decreased (52,54); 2) variations in enzyme levels have been noted
(45,54); 3) a decrease in the level of amino acid incorporation
into protein (108); and 4) a marked depression in high molecular
weight microsomal proteins was reported (109).
Further work is
needed to assess the significance of these results in relation to
possible changes in gene expression that may play a role in the
promotion process.
16
Alterations in Levels and Synthesis of Proteins in Hepatocytes of
Rainbow Trout Fed Cyciopropenoid Fatty Acids
Gary H. Perdew ,
2
Henry W. Schaup
and Daniel P. Selivonchick
Department of Food Science
and
Department of Biochemistry and Biophysics
Oregon State University
Corvallis, OR
97331
2
17
ABSTRACT
3
14
Double labeling experiments with [ H]-leucine and [ C]-leucine
were performed to compare isolated hepatocyte protein synthesis rates in
microsomal and cytosolic fractions from rainbow trout reared on a diet
containing cyclopropenoid fatty acids.
Polyacrylamide gel
electrophoresis was used to fractionate the protein mixtures.
In the
cytosolic fraction, the synthesis of proteins with apparent molecular
weights in the range of 68,000 to 74,000 were significantly decreased.
A pronounced depression in both the level and synthesis rate of
microsomal proteins was observed for proteins that migrate in the
200,000-240,000 relative molecular mass region in polyacrylamide gels.
These high molecular weight proteins do not appear to be membrane
proteins and one of them has biotin associated with it.
Using
avidin-peroxidase staining, it was shown that the mass of this protein
was markedly reduced (80%) in experimental fish.
18
INTRODUCTION
Dietary CPFA
have been shown to exhibit a wide range of
effects on hepatic tissues; including histological alterations (49),
a decrease in various cytosolic and microsomai enzymatic activities
(45,54), altered microsomai protein levels (54), and an enhancement
of the hepatocarcinogenicity of aflatoxin 8, (110).
Recent
experiments have been aimed at determing the cocarcinogenic
mechanism(s) of CPFA.
Dietary CPFA have been shown to depress the
cytochrome P-450 dependent microsomai mixed function oxidase (MFO)
system which is believed to be responsible for conversion of AFB,
to the 2,3 epoxide considered the ultimate carcinogen (111).
Hepatocytes isolated from CPFA-fed trout have shown a marked
reduction of AFB, binding to DNA in vitro (104).
These studies
suggest that CPFA work through a post-initiation cocarcinogenic
mechanism.
Recent reports suggest that tumor promotion by
epigenetic agents are mediated through alterations in membrane
composition and function (112).
The phorbol ester
12-0-tetradecanoyl-phorbol-13-acetate, a potent promoter, has been
shown to decrease specific protein levels.
These alterations
perhaps play an important role in tumor promotion in skin (96,98).
Our previous studies have shown alterations in hepatic microsomai
protein levels in rainbow trout fed CPFA (109).
Whether these
protein level changes are due to alterations in protein synthesis is
the focus of this study.
Abbreviations used are:
CPFA, cyclopropenoid fatty acid(s);
AFB,, aflatoxin B,; PBS, phosphate buffered saline.
19
MATERIALS AND METHODS
Hepatocyte suspensions.
Fish were fed ad libitum diets
containing 300 ppm CPFA in the form of methyl sterculate (113) for
9-10 weeks, with appropriate controls being fed a diet without CPFA.
Hepatocytes were isolated from 18-month-old Mt. Shasta strain
rainbow trout (Salmo gairdneri), using collagenase as described
previously (114).
The isolated hepatocytes were washed three times
and resuspended in a buffer containing 2% bovine serum albumin
(Sigma Type V), 139 mM NaCl, 5.37 mM KC1, 0.81 mM MgS04, 1.26 mM
CaCl2, 20 mM glucose, and 12 mM NaH2P04-Na2HP04 at pH 7.2.
In addition, a mixture of 22 amino acids were added to the
incubation buffer at a level five times that used for rat
hepatocytes as described by Selgen (115).
The hepatocytes (50 mg/ml, wet weight) were then incubated at
220C for 3 hours.
An amino acid mixture without leucine was used in
the incubation medium when radiolabeled leucine incorporation into
protein was required.
A 10 ml suspension in a 125 ml flask was
gassed with 95/5 Op/CO^ continuously on a shaker bath set at 70
cycles/min.
One mCi of L-[2,3,4,5-3H]-leucine (110 Ci/mmole), 0.5
mCi of L-[U-14C]-leucine (346 mCi/mmole), or 0.290 umoles of
nonlabeled leucine was added in a volume of 500 yl to the
appropriate flask.
Cell viability was assayed by the lactate
dehydrogenase (LDH) leakage method (116).
On six successive days hepatocytes were isolated from two fish,
and pooled to yield one daily cell preparation.
Three control and
three CPFA hepatocyte preparations were used for protein labeling.
20
3
Each day one flask of hepatocytes was labeled with [ H]-leucine,
and a second flask was incubated with nonradioactive leucine.
In a
third flask, two of the three control cell suspensions were labeled
with [
C]-leucine.
Subcellular fractionation.
After the incubation period each
cell suspension was pelleted at 100 x g for 5 min in a refrigerated
centrifuge.
All subsequent steps were performed at 40C.
The
hepatocytes were resuspended in 4 ml of 0.32 M sucrose, 20 mM tris,
1 mM phenylmethylsulfonylfloride (PMSF), pH 7.4.
Approximately 99%
disruption, as determined by light microscopy, was achieved by
nitrogen cavitation at 750 p.s.i. for 15 min in a nitrogen bomb
(Parr Instrument Company, Moline, 111.).
Nuclei were removed from
the homogenate by centrifugation at 2000 x g for 10 min, the
post-nuclear supernatant was then centrifuged at 10,000 x g for 20
min to remove mitochondria.
The post-mitochondrial supernatant was
centrifuged at 105,000 x g, for 90 min to yield a soluble
supernatant (S) and a microsomal (M) fraction.
The M pellet was
washed with 0.15 M Tris, pH 8.0, and repelleted at 105,000 x g for
90 min.
The S fraction was dialyzed 48 hrs. against several changes
of 0.1 M ammonium bicarbonate, 20 mM B-mercaptoethanol to remove
free radiolabeled leucine.
This fraction was lyophilized and the S
and the M fraction were resuspended in LDS sample buffer as
described (109).
The specific activity (dpm/yg) of protein was
determined from a 10% TCA precipitate heated at 100oC for 15 min
prior to TCA precipitation.
The number of dpm in the protein
precipitate was determined by solubilization in 0.5 ml 0.3 N NaOH,
21
subsequent neutralization with 0.1 ml IN HC1, and addition of 6 ml
of Aquasol (New England Nuclear, Boston, MA).
Protein
concentrations were determined by the Lowry method using bovine
serum albumin as a standard (117).
LDS-polyacrylamide gel electrophoresis.
A 14 x 32 x 1.5 cm,
sucrose stabilized linear 7.5-15% gradient gel, with a 5% stacking
gel, was prepared and run as previously described (118).
However,
the N,N,-methylene-bis-acrylamide concentration was lowered to
0.018%/1% of acrylamide.
Molecular weight standards purchased from
Bio-Rad Laboratories were run in neighboring lanes (109).
The M
and S fractions solubilized in LDS sample buffer were heated for 5
3
min at 100oC prior to application to the gel. The [ H] and
[
C]-labeled samples were mixed at a ratio of 4/1 on a dpm basis
for double-label analysis, while maintaining the total protein mass
applied per lane at 150 yg.
Following electrophoretic seperation of the proteins
individual lanes were sliced into 2.5 mm segments and placed in
scintillation vials along with 0.5 ml of 90% NCS (Amersham
Radiochemical Corp., Arlington Heights, IL).
The samples were
incubated at 370C for 18 hours, before adding 6 ml of scintillation
cocktail containing 0.4% Omnifluor (New England Nuclear, Boston,
MA) in toluene.
The vials were incubated for 48 hours at room
3
14
temperature before analysis for [ H] and [ C] content.
Samples were analyzed in a Beckmann LS 7500 scintillation counter
using a double-labeled program.
Only samples with [
C] and
[ H] content greater than twice background were considered in.the
analysis.
The samples with background substracted were used to
22
calculate normalized ratios via the following equation:
N
Ri
C
= [
14
=
(Csc/Csh>
x
(Cth/Ctc)
3
C]dpm per gel slice; C . = [ H]dpm per gel slice;
3
C., = total [ H]dpm in all the gel slices;
C.
= total [
C]dpm in all the gel slices.
When visualization of electrophoretically seperated proteins
was required, gels were stained with 0.25% Coomassie Blue R-250 in
50% methanol, 10% acetic acid overnight, and destained in 50%
methanol, 10% acetic acid.
The gels were then scanned using a
Zeineh soft laser scanning densitometer (Biomed Instruments, Inc.,
Fullerton, CA) interfaced with an Apple He computer.
Utilizing a
software package (Model ER1P3 Biomed Instrument Inc.) the areas
under the scan were intergrated for comparative purposes.
Protein blotting.
Protein from a 14 x 17 x 1.5 cm., linear
7.5-15% gradient one dimensional gel were electrotransferred to
nitrocellulose (NC) paper essentially as described by Gibson (119).
The electrotransfer was performed in a E-C electroblot unit (EC
Apparatus Corp., St. Petersburg, FL) for 16 hours at a constant
voltage of 8 volts/cm.
Protein was quantitatively transferred off
the polyacrylamide gel, as determined from Coomassie Blue R-250
staining of the gel after transfer.
Following the transfer the
blot was stored at 40C overnight in PBS buffer [0.9% (w/v) NaCl;.
10 mM NapPO-, pH 7.4] containing 3% bovine serum albumin.
The
NC blot was then incubated, with shaking, in 100 ml of PBS buffer
containing 100 yg of avidin-peroxidase conjugate (Sigma Chem.) for
4 hours.
The nitrocellulose blot was subsequently washed and
23
stained as described (120).
Isolation of liver microsomes.
Rainbow trout liver microsomes
were isolated as previously described (109), except the initial
microsomal pellet was washed in 0.15 M Tris-HCl, pH 8.0., and
centrifuged at 105,000 x g for 90 min.
Tris/water/Tris washing procedure.
Tris washed unlabeled
microsomes were resuspended in distilled water at a concentration
of 1 mg/ml, and incubated at 30oC for 15 min.
The ruptured
vesicles were resealed by cooling to 40C, then centrifuged at
105,000 x g for 90 min.
The resulting membrane pellet was washed
in 0.15 M Tris, pH 8.0, and centrifuged at 105,000 x g for 90 min
(all centrifugations were at 40C).
Enzyme assays.
described (121).
NADPH cytochrome-C-reductase was performed as
Glucos£-6-phosphatase was assayed as described by
Swanson (122), except KF and EDTA were added to the reaction
mixture to minimize acid and alkaline phosphatase activities (123).
Inorganic phosphate was determined by Fiske-Subbarow Pi analysis
(124).
RNA determination.
Ribonucleic acid (RNA) was extracted
(125), and measured by the Orcinol method (126).
24
RESULTS
Preliminary studies focused on optimizing the level of
[ H]-leucine incorporation into total hepatocyte protein.
Little
data is available on absolute pool sizes for amino acids in trout
perfused liver or hepatocytes.
In this study, equilibrium amino
acid levels determined for isolated, perfused rat liver were used as
the "normal" values (115).
The normal liver extracellular amino
acid levels present in trout may well be greater than levels seen in
rats, considering the high (70%) protein content of trout diet.
By
increasing the amino acid concentration 5-fold over the rat "normal"
levels a 30% increase in [ H]-leucine incorporation into TCA
precipitable protein was noted..
A 12 mM phosphate buffer (see
Materials and Methods) increased incorporation into protein by
seven-fold, when compared to 100 mM imidazole buffer which was used
previously.
These results are consistent with those reported for
rat hepatocytes (127).
The increased incorporation into protein of
labeled leucine was sufficient to proceed with the analysis of
protein synthesis.
Hepatocyte viability was assessed by lactate dehydrogenase
activity.
As shown in Figure 1, no significant difference in
viability between experimental and control hepatocytes was observed,
with a marginal change over a 3 hour incubation period.
LDS
polyacrylamide gel electrophoresis was used to analyze the proteins
extracted from microsomal (M) and supernatant (S) fractions
recovered from hepatocytes as described in Materials and Methods.
The double-label procedures permit determination of whether or not a
given protein band in CPFA-fed fish is present in similar or
25
different amounts relative to control fish.
The distribution of
radioactivity in the S fraction, shown in Figure 2, was expressed as
% dpm.
In all fractions examined, an excess of [
C] radioactivity
can be identified just ahead of the tracking dye (T.D.).
Labeled
material migrating near the LDS front was previously believed to be
lipids (128,129).
The excess [
C] in this zone is apparently
due at least in part to differential utilization of the uniformly
labeled [
C]-leucine carbon skeleton.
The chloroform phase of a
Folch extraction (130) contained 50% of the total dpm in
rl4
[
CJ-leucine labeled microsomes.
The ratio plots included
values between the top of the running gel and the T.D. only.
NR.
values were calculated as described in Materials and Methods.
If
the rate of protein synthesis for protein(s) in a given zone of the
polyacrylamide gel is comparable between control and experimental
groups then an NR. value of one would be expected.
However, if
the rate for protein synthesis is decreased an increase in the NR.
would be observed.
[
14
NR. values were determined for a mixture of
3
C] and [ H] labeled control supernatant proteins to determine
the precision of the double label method (Figure 3).
The standard
deviation for this control-control mixture was 0.13.
When control-
CPFA samples were analyzed only values which were greater or less
than 1 by 2 standard deviations were considered significant.
A
significant decrease in relative rate of protein synthesis was
observed in the 68-72 Kd Mr region (Region A) of a gradient gel.
Comparison of control and CPFA supernatant Coomassie brilliant blue
stained acrylamide gels via densitometry revealed no significant
difference in relative mass of proteins in Region A.
A control-CPFA
26
microsomal % dpm diagram is shown in Figure 4.
NR. plots of
radiolabeled control-control and control-CPFA microsomal gels are
given in Figure 5.
A decrease in relative synthesis rate of
approximately 50% in the 220-240 Kd Mr region (Region B) was noted;
no other significant consistent differences were determined.
Two
gel fractions in the 45-66 Kd Mr region were found to be
statistically significant in two of the three microsomal
preparations.
Further experimentation will be needed to clarify the
significance of these deviations.
Relative protein mass levels in
Region B were expressed as a percent of total densitometric scan
area (Figure 6) from Coomassie brilliant blue stained gels, a 49%
decrease was observed with CPFA treated trout hepatocytes.
The
post-nuclear fraction was examined on Coomassie brilliant blue
stained gels in order to discount the possibility that the
microsomal protein level alterations were not the result of
differences between experimental and control microsomes created
during the isolation procedure.
Reductions in protein levels seen
in microsomes were also noted in the post-nuclear fraction.
In
order to determine whether the proteins in Region B are membrane or
water soluble proteins, a Tris/water/Tris washing procedure was
performed essentially as described by Glaumann (131).
This washing
procedure removes the water soluble lumenal contents of microsomal
vesicles and proteins that are loosely adhered to the membranes.
The intact microsomes, washed membranes, and water wash were
analyzed for glucose-6-phosphatase, NADPH cytochrome P-450
reductase, protein, and RNA.
These results (Table 1) suggest that
integral and pheripheral membrane proteins are not removed by this
27
washing procedure.
The laser densitometry scans shown in Figure 7
indicate that most of the protein in Region B is water soluble.
We
have found that computer analyzed laser scans to be highly
reproducible from sample to sample.
The molecular weights of the proteins in Region B of the gel
and previously reported effects of CPFA on lipid metabolism
suggested that fatty acid synthetase might well be the protein
affected by CPFA.
possibility.
(132).
However, acetyl CoA carboxylase was also a
Biotin is covalently bound to acetyl CoA carboxylase
Therefore we decided to use avidin, which forms a complex
with biotin, as a probe for the presence of acetyl CoA carboxylase.
Through the use of protein blotting and avidin-peroxidase staining
techniques the major protein in Region B was found to contain
biotin.
The results shown in Figure 8 reveal a decrease of 80% in
avidin binding after feeding a 300 ppm CPFA diet for 6 weeks..
A
linear response in avidin binding was established by loading
increasing amounts of microsomes in series on a gel.
Results, to be
published elsewhere, have shown that between 4-14 weeks on CPFA diet
an approximately 50% decrease in mass is maintained in Region B.
28
DISCUSSION
Double-labeling techniques that measure relative rather than
absolute rates of protein synthesis have been successfully used to
assess abnormalities in genetic diseases (132-134), effects of
phorbol diesters in cultured muscle cells (135), and the sequence of
assembly of ribosomal protein on nascent RNA in _E. coli (136).
Absolute rates of synthesis are meaningful only in very controlled
circumstances since they are sensitive to media composition (137),
feeding frequency and age of the fish.
This report illustrates the
usefulness of isolated hepatocytes to assess alterations in relative
rates of hepatic protein synthesis due to dietary factors, e.g.
CPFA.
One-dimensional LDS-PAGE gradient gels were chosen to separate
the protein mixtures for double-label analysis. . One should keep in
mind that several proteins may coelectrophorese.
This could cause a
masking of a potential deviation in an individual protein's
synthesis rate.
In fact, this would explain the lack of a
detectable decrease in protein levels in Region A of a supernatant
gel even though a decrease in protein synthesis rate was observed.
Other possible limitations in double-labeled one-dimensional gel
analysis have been discussed by Pena et aj_. (129).
Unfortunately,
high molecular weight proteins, such as those in Region B, were not
resolved by isoelectric focusing thus precluding the use of two
dimensional gels to assess changes in the synthesis of high
molecular weight proteins.
In addition, attempts to utilize
two-dimensional gel techniques were not successful due to low
radiospecific acitivity in individual proteins.
29
The protein(s) in Region B show an apparent decrease in
relative synthesis rate and mass by 50%, suggesting that the rate of
synthesis determines the level of protein seen at a given time.
The
large decrease observed with the specific biotin staining technique
indicates that more than one protein is present in Region B.
The
biotin containing protein is reduced by 80%, while comigrating
protein(s) are not reduced, thus yielding a net 50% decrease in mass
as well as synthetic rates of proteins in this region.
The only
class of proteins known to have biotin attached to the polypeptide
are carboxylases.
A survey of published reports indicate that only
acetyl CoA carboxylase would be consistent with the following data:
1) a molecular weight of 230-240 Kd Mr (138); 2) a close association
with the microsomal fraction (139); and 3) the presence of biotin
(132).
Alterations in the rate of synthesis of a acetyl CoA
carboxylase have been shown to play a role in the long term
regulation of de novo fatty acid synthesis (140).
Further work is
needed to firmly establish the identity of the biotin-containing
protein.
Whether or not these qualitative alterations in protein
synthesis are directly related to the cocarcinogenic properties or
the overall toxic effect of CPFA is difficult to determine.
Nevertheless, it is interesting to. note that only depressions in
synthesis rates and levels of proteins were observed.
30
Figure 1. Percent leakage of LDH activity from control (gS:-:) and
CPFA-treated (;££) trout hepatocytes into
the incubation media, as a function of time.
The values
are means obtained from three different cell preparations,
31
•■i'lo';IuX —
C9
iwX n
E
ISI
nm'
5" I
0
■p
--
Q
fc
Tl
i
2
Time (hours)
Figure 1.
3
32
Figure 2. Hepatocytes from control and CPFA fed trout were labeled
with [14C]-leucine (•-•) and [3H]-leucine (OO),
respectively.
Supernatant fractions were isolated and
mixed for LDS-PAGE (see Materials and Methods).
The plot
shows the distribution of the supernatant proteins of the
double-labeled mixture after separation by 7.5%-15%
LDS-PAGE.
Arrows indicate the beginning of the separating
gel and the tracking dye band.
The bar marks Region A,
the region of a statistically significant alteration in
protein synthesis.
33
1Q
"60"
GEL FRACTION
Figure 2.
34
Figure 3. Plot of NR. values determined from a mixture of
[
14
3
C]-control/[ H]-control supernatant (upper panel)
and [
C]-control/[ H]-CPFA (lower panel) supernatant
after separation via gradient gel electrophoresis.
_3
Molecular weight markers,
marke'
Mr x 10 , are given at the
top of the upper panel
35
i
ill
i
;
i i
-2SD
0.6-
JM.01.4_i
i
I
I
i
i
i
'
'
■
'
-2SD
-2SD
2SD
i
20
JO
BO
GEL FRACTION
Fiqure 3.
so
rair
36
Figure 4. Hepatocytes from control and CPFA fed trout were labeled
with [14C]-leucine (•-•) and [3H]-leucine (O-O),
respectively.
LDS-PAGE.
Microsomes were isolated and mixed for
The plot shows the distribution of the
microsomal proteins of the double-labeled mixture after
7.5%-15% LDS-PAGE.
Arrows indicate the beginning of the
separating gel and the tracking dye band.
The bar marks
Region B, the region of statistically significant
alteration in protein synthesis.
37
T
80
GEL FRACTION
Figure 4.
'
100
38
Figure 5. Plot of NR. values determined from a mixture of
[
[
14
3
C]-control/[ H]-control (upper panel) and
14
3
C]-control/[ H]-CPFA (lower panel) microsomes after
separation via 7.5%-15% gradient gel electrophoresis.
_3
Molecular weight markers, Mr x 10 , are given at the
top of the upper panel.
39
0.6:
X
to
2»
<o
in
_
—^
1
LL_L_L_iJl
2SD
£1.0
2SD
1.4
0.6:.
2SD
r-V-V^Af
1.0-
■2SD
1.4"
Eli2.22.6-
'
1
'_
20
L
j
J_
40
60
GEL FRACTION
Figure 5.
i
80
i
i
100
40
Figure 6. After Coomassie-Blue staining, a control (upper panel) and
CPFA (lower panel) microsomal gradient gel was scanned
with a Zeineh soft laser scanning densiometer, interfaced
with an Apple computer.
Region B is shaded in each scan.
The postion of a 200 Kd Mr protein standard is given in
each panel.
-i
to
r
■
■
'
•
'
■■■
J
...
i—,—L.
Detector Response
42
Figure 7.
Microsomes were subjected to a Tris/water/Tris wash as
described in Materials and Methods.
Each fraction was
applied to a 7.5%-15% gradient gel and scanned with a
computer-interfaced soft laser densiometer.
The intact
microsomal, membrane, and water fraction scans are shown
in panels A, B, and C, respectively.
Panel A-1, B-1 and
C-l are computer generated enlargements of regions
enclosed in the dotted-line boxes shown in panels A, B
and C.
The position of a 200 Kd Mr protein standard is
given in each panel.
Region B is shaded in each scan.
el?
Detector
Response
44
Figure 8. Control and CPFA liver microsomes were isolated in
triplicate, electrophoresed and blotted as described in
Materials and Methods.
The blot was stained with
avidin-peroxidase revealing biotin containing proteins.
45
<<<
U- U-U. r-CNJ CO
CLQ.Q. I I I
OOOOOO
Mrx
10"3
200—
116926645
Figures.
46
Table I.
The Effect of Tris/water/Tris Washing
on the Particulate Membrane Fraction
Assay
% Recovery in
Membrane Pellet
Protein
45.0
RNA
30.0
Lipid
96.4
Glucose-6-phosphatase
99.0
NADPH-cytochrome
P-450 reductase
96.0
47
The Use of a Zwitterionic Detergent in Two-Dimensional
3
Gel Electrophoresis of Trout Liver Microsomes
1
2
Gary H. Perdew , Henry W. Schaup , and
Daniel P. Selivonchick
Oregon State University
Department of Food Science
and Technology
and Department of Biochemistry and Biophysics
2
Corvallis, Oregon 97331
Published in Analytical Biochemistry
135, 453-455 (1983)
3
Technical Paper No. 6811, Oregon Agricultural Experiment
Station, Oregon State University, Corvallis, Oregon 97331
48
ABSTRACT
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-l-Propane
sulfonate, a zwitterionic detergent, has been shown to exhibit
superior membrane protein solubilizing characteristics as compared to
nonionic detergents.
Replacement of NP-40 with CHAPS in isoelectric
focusing of rainbow trout liver microsomes has increased resolution
markedly.
The two-dimensional electrophoretic system described will
allow effective resolution of up to 300 yg of crude microsomal
protein.
CHAPS exhibits no effect on the stability or type of pH
gradient when compared to NP-40 during isoelectric focusing in the
presence of urea.
49
INTRODUCTION
CHAPS*, a zwitterionic detergent, has been utilized recently in
various enzyme purification schemes.
Zwitterionic detergents have
proven to be particularly effective at disaggregating hydrophobic
proteins, such as cytochrome P-450 (141).
They offer greater
solubilizing characteristics, have no net charge, and thus should
prove ideal when used in conjunction with urea, for solubilizing
membranes in IFSDS.
Nonionic detergents are typically used in IFSDS.
Streaking of polypeptides in the first dimension of IFSDS gels is
a common occurrence (142-144).
The degree of protein streaking in IF
varies widely, and may lead to masking and cross-contamination of
neighboring polypeptides.
These problems are likely to hinder
analysis of the individual polypeptides.
In this report we
demonstrate the usefulness of CHAPS in preventing the streaking of
trout liver microsomal proteins separated by IF.
*Abbreviations used: CHAPS, (3-[(3-cholamidopropyl)dimethylammonio]-lpropane sulfonate; IFSDS, isoelectric focusing in the first dimension,
followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
in the second dimension; NP-40, Nonidet-40; IF, isoelectric focusing;
LDS, lithium dodecyl sulfate; SDS, sodium dodecyl sulfate.
50
MATERIALS AND METHODS
Chemicals.
All reagents used for isoelectric focusing and
polyacrylamide gel electrophoresis were obtained from Bio-Rad
Laboratories.
CHAPS was obtained from Sigma Chemical Company;
Molecular weight standards were purchased from Bio-Rad, and prepared
for electrophoresis as previously
described (109).
Microsomai Proteins.
Microsomai proteins were isolated from the liver
of Mt. Shasta strain rainbow trout (Salmo gairdneri) as previously
described, and suspended at 10 mg/ml protein in 100 mM sodium
phosphate, pH 6.8, containing 20% (w/v) glycerol.
Protein
concentrations were determined as described by Lowry et al_. (117).
One volume of microsomes was solubilized in two volumes of lysis
buffer (3% CHAPS or NP-40, 1.5% pH 3.0-10 ampholytes, 0.5% pH 5-7
ampholytes, 9.5 M urea, 70 mM dithiothreitol).
The solubilized
microsomes were saturated with urea,
then titrated to pH 4.1 with 3 N HC1.
Isoelectric Focusing and Polyacrylamide Gel Electrophoresis.
Isoelectric focusing was performed according to O'Farrell (145) as
modified by Vlasuk et al_. (146), with the following minor alterations:
(i) A mixture of 1.5% pH 3.0-10 ampholytes, 0.5% pH 5-7 ampholytes,
and 2% NP-40 or CHAPS (w/v) was used in the gel.
(1i) Long (115 mm)
isoelectric focusing gels were formed in 140-mm tubes (3.0 mm i.d.).
(iii) IF gels were forced from the tubes with a pipet bulb directly
51
into 8 ml of LDS sample buffer and equilibrated for 1 h and 50 min.
(iv) Two hundred micrograms of protein was applied to each gel.
A Bio-Rad protein 32-cm-slab cell unit was utilized to run a 14 x
32 x 1.5 cm, sucrose stabilized linear 10-15% gradient gel including a
2-cm stacking gel.
The upper and lower gel buffers were prepared as
previously described (147,148).
In order to avoid localized heating
during electrophoresis, gels were run at 40C.
because of its greater solubility at 40C.
SDS was replaced by LDS
Gel electrophoresis was
performed at a constant current setting of 18 mA for 18 h.
The run
was terminated when the bromphenol dye was 3 cm from the bottom of the
gel.
Silver Staining.
Gels were silver stained by the method of Wray et
a]_. (149), except a solution of 0.05% citric acid and 0.02%
ethanolamine was utilized to stop development (150).
pH Gradient.
IF gels were cut into 5-mm segments and placed in a
sealed tube with 500
JJ!
of deionized water.
Gel segments were allowed
to equilibrate for 2 h, before pH measurements were taken.
52
RESULTS AND DISCUSSION
Isoelectric focusing of trout liver microsomes either by the
method of O'Farrell (145) or Vlausk et a_l_. (146) failed to yield a
polypeptide pattern free of protein streaking.
apparent between pH 4.1 and 5.5 (Fig. 9A).
The problem is most
Also a heavy brown
background near the acidic end of the gel was noted in the second
dimension following silver staining.
Protein streaking may be due to
strong protein-protein or protein-ampholyte interactions.
Zwitterionic detergents have previously been shown to exhibit
disaggregating properties.
Almost no protein streaking can be
observed when CHAPS is substituted for NP-40 in IFLDS and the dark
brown background is eliminated (Fig. 9B).
brown background is currently unknown.
The composition of the
In order to establish that the
apparent increase in resolution is due to a decrease in protein
streaking, concanavalin A-peroxidase stains of a protein blot on
nitrocellulose paper was performed as described elsewhere (151,152).
This data confirmed that CHAPS decreased protein streaking (data
submitted but not published).
IFLDS with a 1/1 (w/w) mixture of NP-40
and CHAPS in the first dimension revealed an intermediate improvement
in resolution.
Though CHAPS is the only zwitterionic detergent we
have examined, it is possible that other sulfobetaines would give
similar results.
The pH gradient was essentially the same, regardless of the
detergent used.
O'Farrell (153) has shown that during equilibrium IF
the basic end of the pH gradient is partially degraded.
Gonemne et
a!. (154) suggested that zwitterionic detergents may increase pH
gradient stability during nondenaturing IF.
A time course study
53
revealed that CHAPS/urea IF exhibited no increase in pH gradient
stability when compared to NP-40/urea IF between 5,600 and 11,000 V-h.
Use of 3.0-mm (i.d.) IF tubes instead of 2.5-mm tubes increases
the total gel volume by 30%, with no apparent decrease in resolution.
We have obtained well-resolved polypeptide patterns when up to 300 yg
of microsomal protein were applied to the gels.
In conclusion, zwitterionic detergents may offer improved
resolution in many IFSDS applications.
This may be due to their
greater solubilizing characteristics and compatibility with
isoelectric focusing.
54
Fig. 9.
IFLDS of trout liver microsomal proteins followed by
silver staining.
The gels were run as described under
Materials and Methods.
was 200 yg.
The sample protein concentration
Molecular weights of standards are given on
left-hand side of each gel.
is labeled above each gel.
The approximate pH gradient
(A) The first dimension was
run in the presence of NP-40.
(B) The first dimension was
run in the presence of CHAPS.
The second dimension in
both (A) and (B) were identical using LDS-polyacrylamide
gel electrophoresis.
§-
g"
:SS-
CD
' .' ••t.iMii;'V>,..
55
56
Alterations in Hepatic Microsomal Protein Levels
in Rainbow Trout Fed Cyclopropenoid Fatty Acids:
Analyzed by Two Dimensional Gel Electrophoresis
1
2
3
Gary H. Perdew , Henry W. Schaup , Donald R. Buhler ,
and Daniel P. Selivonchick
Oregon State University
Department of Food Science , Department of
2
Department of Biochemistry and Biophysics , and
3
Department of Agricultural Chemistry .
Corvallis, Oregon 97331
57
ABSTRACT
Two dimensional gel electrophoresis was used to analyze the
effect of dietary cyclopropenoid fatty acids (CPFA) on hepatic
microsomal polypeptide patterns.
Antibodies against
B-naphthoflavone-fed rainbow trout cytochrome P-450 (LNL) were
employed to localize the corresponding polypeptide(s) via protein
blotting and immunochemical staining.
Microsomes isolated from
6-naphthoflavone-fed trout contained only a single cytochrome P-450
polypeptide.
In contrast, control microsomes contained two distinct
cytochrome P-450 polypeptides differing only in their isoelectric
points.
Thus, an additional P-450 isozyme in rainbow trout was
tentatively identified.
CPFA treatment caused a preferential
decrease in only one of the isozymes found in the control samples.
The presence of concanavalin A binding glycopolypeptides was
determined.
The two P-450 isozymes localized on control microsomal
gels were found to bind concanavalin A, suggesting that these
isozymes are glycoproteins.
Another result of CPFA treatment was a
shift in a closely related group of membrane glycopolypeptides,
labeled gp80, gp82, gp80,, and gp821.
A decrease in the mass of
gp80 and gp82, and a corresponding increase in mass of gp80, and
gpSZ, was observed.
58
INTRODUCTION
CPFA* have been shown to cause a variety of hepatotoxic effects
when included in the diet of different animals.
Among the alterations
that have been noted in rainbow trout are: an increase in the
saturated to unsaturated fatty acid ratio (29); a decrease in several
enzymes involved in xenobiotic metabolism including a depressed level
of cytochrome P-450 (52-55); a decrease in microsomal protein content
(52), an increased mitogenic activity (26); and a marked disruption in
the rough endoplasmic reticulum membrane organization (49).
In previous studies, we have shown that although CPFA did promote
major histological changes, only a few changes in microsomal membrane
proteins were observed (155).
Also of interest, but so far neglected,
is the possibility of a CPFA induced shift in isozymes, resulting in
an overall depression in trout hepatic cytochrome P-450 fraction.
A
shift in isozyme profiles could be equally as significant as an
overall depression in levels of P-450, considering the possible
differing substrate specificities of each isozyme.
The most powerful
technique available for resolving isozymes in a complex membrane
system is the two dimensional gel electrophoresis system, developed by
O'Farrell (145).
♦Abbreviations:
We have improved upon this technique by the
CPFA, cyclopropenoid fatty acid(s); CHAPS,
(3-[(3-cholamidopropyl)diniethylammonio]-l-propane sulfonate; NP-40,
Nonidet-40; IFLDS, isoelectric focusing in the first dimension,
followed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis
in the second dimension; BNF, 6-naphthoflavone.
59
substitution of NP-40 with the zwitterionic detergent, CHAPS.
This
substitution results in greater solubility of membrane proteins in the
first dimension with minimal streaking and an overall enhanced
resolution of individual membrane polypeptides (118).
In the present study, IFLDS has been coupled with immunochemical
and concanavalin A staining techniques in order to detect CPFA induced
alteratio.ns in isozymes.
60
MATERIALS AND METHODS
Dietary Treatments.
Sixteen to eighteen-month old Mt. Shasta
strain rainbow trout (Salmo gairdneri) were fed a semi-purified
casein control diet (156).
Fish were maintained on the CPFA diet,
which contained 300 ppm CPFA in the form of methyl sterculate (113),
for 10 weeks before sampling.
Another group of fish were fed
B-naphthoflavone (500 ppm) in a control diet for 4 weeks.
Microsome Isolation.
Microsomal fractions were isolated from
the livers of 3-5 Mt. Shasta strain rainbow trout (Salmo gairdneri)
as previously described (109), except the microsomal pellets were
washed with 0.15 M Tris-HCl, pH 8.0.
Microsomal pellets were
resuspended and solubilized with CHAPS for isoelectric focusing as
previously described (118).
Protein concentrations were determined
by the method of Lowry et al_. (117), using bovine serum albumin as a
standard.
Two Dimensional Polyacrylamide Gel Electrophoresis.
Reagents
used for isoelectric focusing and polyacrylamide gel electrophoresis
were obtained from Bio-Rad Laboratories, Richmond, CA.
Concanavalin
A (Type III) and peroxidase (Type VI) were purchased from Sigma
Chemical Co., St. Louis, M0.
All other chemicals were reagent grade
or the best grade available.
Isoelectric focusing and
polyacrylamide gel electrophoresis were performed as previously
described (118).
Molecular weight standards were purchased from
Bio-Rad Laboratories.
The pH gradients in the isoelectric focusing
tubes were measured as described elsewhere (118).
Protein Blotting.
Transfer of protein from IFLDS gels to
nitrocellulose was accomplished by the method of Burnette (151).
61
The transfer was performed at 8 volts/cm, constant voltage, for 24
hrs in an EC Electroblot unit (EC Apparatus, St. Petersburg,
FL) at 40C.
Essentially all protein was transferred from the gel as
judged by silver staining the IFLDS gel.
Glycoproteins that bind
concanavalin A were visualized on the nitrocellulose sheet (152).
Control concanavalin A staining was performed to detect nonspecific
staining, by exclusion of metal ions from the concanavalin A
solution and the addition of ImM EDTA.
Concanavalin A binding to
glucose or mannose residues of glycoproteins requires the presence
of metal ions (Mn
, Ca
, Mg
).
Immunochemical staining was
accomplished by the method described by Glass et a_l_. (120).
Rabbit
antibodies to rainbow trout P-450 (LM2) and P-448 (LM4. ) were
prepared as previously described (157).
Silver Staining.
IFLDS gels were silver stained according to
the procedure of Wray et al. (149).
62
RESULTS AND DISCUSSION
Three distinct forms of liver microsomal cytochrome P-450 have
been isolated from BNF-treated rainbow trout.
The three forms were
designated: LM4b (P-448 type), LM4a (P-488 type), and LM2
(P-450 type).
The antibodies raised against these enzymes were used
to localize the individual isozymes in the polypeptide profile of an
IFLDS gel by electrotransfer to nitrocellulose paper and
immunochemical staining.
Because of the complexity of the
polypeptide pattern the blots were then stained with concanavalin
A-peroxidase after immunochemical staining.
This provides reference
points to localize the isozyme on a silver stained gel.
Immunochemically staining a control blot with antibodies against
LMp revealed two polypeptides, these two proteins are circled in
Fig. 10.
In Illustration C the exact location on a control
microsomal gel can be seen.
The two proteins labeled 1 and 2 were
present, as judged by silver staining, in approximately equal
amounts.
Comparable samples from BNF treated trout have a greatly
reduced level of polypeptide 1 and increased amounts of polypeptide
2 (Fig. 10, llustration D).
These data indicate that the isolation
of LNL from BNF treated fish would yield polypeptide 2.
Thus the
presence of a second putative P-450 isozyme in control microsomes
with a similar pi, the same molecular weight, and cross-reactivity
with antibodies to LMp would explain these results.
Dietary
treatment with CPFA caused a preferential depression in polypeptide
1, which in fact could no longer be detected, as shown in Fig. 10,
Illustration E.
Attempts to localize LM.
and LM-. have been
unsuccessful because these isozymes streak across the acidic portion
63
of the isoelectric focusing gel, and are present in low levels in
control microsomes.
Glycoproteins in trout liver microsomes were identified on
blots of IFLDS gels by their affinity for concanavalin A (Fig. 11).
Polypeptides 1 and 2 were shown to bind concanavalin A, thus
suggesting that polypeptides 1 and 2 are glycoproteins.
P-450
isozymes from a variety of sources have been reported to be
glycoproteins (158).
The effect of CPFA treatment on the
glycoprotein profile is shown in Fig. 11, Blot B.
CPFA caused an
almost total depression in polypeptide 1, in relation to the level
of polypeptide 2.
This result is in agreement with data obtained
from silver stained microsomal gels.
Another alteration observed with dietary CPFA is a shift in
polypeptide levels within a family of glycoproteins.
This shift can
be seen both in the silver stained gels. Fig. 10A and 10B, and in
the concanavalin A blots, Fig. 11.
These glycopeptides (gp) are
_3
labeled according to their respective Mr x 10
values. With CPFA
exposure, both a decrease in gp80 and gp82 and an increase in
gp80, and gp821 was observed.
This type of shift within a
related group of glycoproteins have previously been shown in other
systems to be due to changes in the size of and the number of
oligosaccharide moieties (159,160).
Although alterations in this
related group of polypeptides could be the result of other
post-translational modifications such as phosphorylation and
proteolytic cleavage.
We have found the use of concanavalin A-peroxidase stained
blots to be more useful than silver staining to detect mass changes
64
in glycopolypeptides.
The arrow shown in Fig 11B marks a region
where a reduction in protein mass with CPFA treatment is often but
not reproducibly observed.
We are uncertain of the source of this
variation, nevertheless other alterations described here were
consistent over three different generations of fish.
To determine whether or not these glycoproteins were membrane
proteins, a control microsomal preparation was subjected to a
Tris/water/Tris washing procedure which yields a particulate
membrane preparation.
This procedure has been previously shown to
remove the lumenal contents of the microsomal vesicles as well as
loosely adhering proteins (131).
Approximately 55% of the total
trout liver microsomal protein is found in the water wash (155).
The particulate membrane preparation was subjected to IFLDS, and a
silver stained gel revealed the presence of the gp80 and gp82
family.
This observation suggests that this group of
glycopolypeptides are membrane glycoproteins.
Why CPFA would cause
a shift only in one specific group of glycoproteins is currently
under investigation.
65
Figure 10.
Control liver microsomal IFLDS silver stained gel is
shown with two areas of interest marked with a square
and a circle.
Numbers to the left of the gel are
relative molecular mass (Mr).
The approximate pH
gradient in the first dimension is labeled above the
gel.
Illustrations marked A-E, correspond to a selected
portion of the key areas of interest from: A) a control
gel; B) a CPFA gel; C) a control gel; D) a BNF gel; and
E) a CPFA gel.
66
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sp
ep
92K-iL. ^
Figure 10.
yp
8p
67
Figure 11.
Concanavalin A-peroxidase stained blots of A) control
and B) CPFA microsomal IFLDS gels.
The two areas of
interest are marked with a square and a circle, and
correspond to the same areas as in Fig. 10.
B
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