United States
Environmental Protection
Agency
Environmental Research
Laboratory
Gulf Breeze PL 32561
Research and Development
EPA-600/S3-82-091 Mar. 1983
Project Summary
Development of a
Carcinogen Assay System
Utilizing Estuarine Fishes
B. J. Martin
The overall objective of this research
was the development of closed
systems previously devised in our
laboratory to assay the effects of
chemical carcinogens on marine
teleosts. The results include the
following:
The LC-50 (96 hrs) for benzidine
dihydrochloride (BEN) with respect to
Cyprinodon variegatus (sheepshead
minnow) was determined to be 64
ppm.
Exposure of C. variegatus weekly to
contaminations of 1 ppm BEN
resulted in some individuals
developing liver lesions at 25-29
weeks. The livers of these individuals
contained large fibrotic regions within
which a proliferation of various types
of tubular profiles can be observed.
Exposure of early C. variegatus
embryos to BEN at various concent ra-
tions produced abnormalities at con-
centrations of 50 ppm and above.
Anomalies in the order of frequency of
occurrence were tubed heart
syndrome with distended pericardia,
poor circulation, sparse distribution of
melanophores around yolk, inability to
hatch, abnormal head morphology,
seoliosis, and faint RBC pigmentation.
Acute toxicity concentrations were
established for benzo(a)pyrene (BaP),
BEN, and diethylnitrosamine with
respect to a cell line from Archosargus
probatocephalus (the sheepshead).
Long-term exposures provided
evidence that BaP and BEN have
mutagenic effects on this cell line.
A dechorionation technique was
developed to better observe detailed
cellular and subcellular activities
during early embryonic development
of C. variegatus. Employment of this
technique to observe inverted
blastoderms provided evidence that
the ectodermal cells that cover the
yolk travel from the superficial
blastoderm via a pathway along the
blastoderm floor.
Detailed studies of the gross and
histologic structure of the digestive
tract and histologic studies of the
peripheral blood cells of C. variegatus
were conducted.
Three novel techniques were
developed to study the effects of
carcinogens on C. variegatus at the
cellular level: an aseptic embryo tech-
nique that provides the opportunity to
study embryos in a sterile environ-
ment; an embryo-primary cell culture
technique that incorporates, in one
system, characteristics of both intact
embryos and primary cell cultures;
and a primary hepatocyte cell culture
technique that will be employed to
study the effects of carcinogens on
teleost hepatocytes.
In order to study the immune system
of C. variegatus. standard
immunological techniques were
miniaturized. Serum electrophoresis
disclosed considerable variation
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between BEN-exposed and unexposed
fish, and the presence of antibody-
forming cells in spleen suspensions
from C. variegatus immunized with
human type O erythrocytes was dem-
onstrated by a modified immune ro-
sette procedure.
This Project Summary was develop-
ed by EPA's Environmental Research
Laboratory. Gulf Breeze, FL. to an-
nounce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
Introduction
Murine assay systems continue to be
the mainstay for testing chemical
carcinogens; however, there is a
recognized need for alternate systems.
Since existing systems are not
particularly amenable to assessment of
the aquatic environment, an
environment that is experiencing an
ever increasing quantity and a variety of
potentially dangerous pollutants, it is
imperative that we develop valid
carcinogen test systems appropriate for
this environment.
Teleost fishes are obvious
experimental candidates. In a previous
study of the effects of certain
carcinogenic compounds that are
components of petroleum products, we
developed systems for long-term
exposures of C. variegatus (the
sheepshead minnow) at inland
laboratories (1). This species was
selected because it is common to the
Gulf Region, its biological character-
istics seemed ideal, and it had already
been employed extensively in
toxicological assays. This initial project
demonstrated that C. variegatus was a
suitable model for testing chronic
exposure and provided evidence that
the C. variegatus assay system, when
fully developed, could be employed
successfully in carcinogen assays.
Therefore, the principal objective of
the present project was the
comprehensive development of the C.
variegatus assay system as a tool for the
biological assessment of suspect
carcinogens in the aquatic environment.
An additional goal of the project was to
conduct a search for biochemical or
cellular changes that might indicate the
usefulness of this species as an early
warning detector of teratogenic,
mutagenic, or carcinogenic substances
in the estuarine environment.
Results
Benzidine Exposure
In these experiments, C. variegatus
were maintained in water contaminated
weekly with benzidine dihydrochloride
(BEN) at 1 ppm. In one of these
experiments, in which 50 fish were
exposed for approximately 11 months,
four individuals developed histopatho-
logically similar lesions within a one-
month period (at 25-29 weeks).
The livers of the fish with the lesions
contained large regions in which the
typical parenchyma had been displaced
by randomly arranged collagenous
tissue within which there were
numerous zones that contained a prolif-
eration of different sizes and types of
tubules. The time of occurrence of these
lesions suggest a five- to six-month
latency period.
Benzidine Exposures of Embryos
Exposures of early C. variegatus em-
bryos to BEN conducted at concentra-
tions ranging from 5 - 500 ppm produced
abnormalities only at BEN concentra-
tions of 50 ppm and higher. During this
project about 600 C. variegatus early
embryos were exposed to BEN at 50
ppm or higher. Only about 13% of these
embryos developed normally beyond the
hatching stage. Thus, about 85% of the
embryos exhibited some type of develop-
mental anomaly.
When these data are compared to
those from control experiments in
which 70% of the 300 embryos
developed normally beyond the
hatching stage, the detrimental effects
of exposure to BEN at these concentra-
tions are quite obvious.
Anomalies observed in embryos
exposed to BEN at 50 ppm or higher
were: 1) tubed heart syndrome with
distended pericardia, 2) poor circulation,
3) sparse distribution of melanophores
around yolk, 4) inability to hatch, 5)
abnormal head morphology, 6)scoliosis,
and 7) faint RBC pigmentation.
Anomalies 1 through 4 occurred most
frequently and anomalies 2, 3, and 4
occurred only if embryos were exposed
after somite and lens development.
Interestingly enough, some of the
exposed embryos survived up to 30 days
and yet did not hatch.
SHF-1 Cell Culture Exposures
Benzo(a)pyrene (BaP), benzidine, and
diethylnitrosamme (DENA) were all
found to be acutely toxic to a cell line
(SHF-1) from the sheepshead,
Archosargus probatocephalus (2). BaP
was acutely toxic at 2.0 ug per ml of
growth medium, BEN at 0.1-0.2 mg per
ml, and DENA at 2.0 mg per ml. In cells
exposed to subacute concentrations,
toxicity was evidenced by cell
vacuolization and a general stress
response of SHF-1 cells. Even at low
concentrations, BaP produced this
response. SHF-1 cells were chronically
exposed to subacute concentrations of
each compound through as many as five
subcultivations. After subcultivation,
the time required for the cells to become
confluent gradually increased with each
passage. Foci of multilayered cells
(approximately 1 mm in diameter)
exhibiting a lack of contact inhibition
were observed in several cell cultures
exposed to low concentrations of BaP
and BEN.
Exposure to BaP in the amounts of
100 ng per ml, 50 ng per ml, 40 ng per
ml, 30 ng per ml, and 20 ng per ml were
carried through three passages (28-30).
During passage 28, the cells appeared
unaffected and were subcultured within
the normal seven-day period. In
passage 29, exposure was continued in
some cultures and discontinued in
others. During passage 29, multilayered
foci appeared in a few cultures: those
exposed to 100 ng BaP per ml in both
passages 28 and 29, those exposed to
40 ng BaP per ml in both passages 28
and 29, and those exposed to 40 ng per
ml in passage 28 only. Passage 29
reached confluency in the normal
amount of time (seven days). In passage
30, multilayered foci appeared in essen-
tially all cultures except those exposed
to 40 ng BaP per ml and 20 ng BaP per
ml in passage 28 only. All of the experi-
mental cultures of passage 30 failed to
reach confluency.
Cultures at passage 30 were exposed
to 0.6 ug BEN per ml, 0.5 ug BEN per ml,
0.4 ug BEN per ml, 0.3 ug BEN per ml,
0.2 ug BEN per ml, and 0.1 ug BEN per
ml. These cells were subcultured
through passages 31 and 32 using the
same scheme as outlined for BaP
exposures. Multilayered foci appeared
only in the cultures of passage 32 which •
had been exposed to 0.4 ug BEN per ml
in all three passages (30, 31, and 32).
Foci of multilayered cells were not
observed in any DENA exposures and
did not appear in BaP or BEN exposures
after passage 33.
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Early Embryogenesis
Because of the nature of the chorion,
it was necessary to develop a technique
for the observation of excised blasto-
derms in order to observe the details of
cellular and subcellular activities that
occur during embryogenesis.
The following are key observations
resulting from the use of this technique
to extend our knowledge of teleost
embryology.
The mechanism or mechanisms that
initiate the polar concentration of
protoplasm have been in question for
years. In the current study, this
concentration of protoplasm was never
observed in an egg in which the
subsequent cellular cleavage did not
occur. Therefore, it appears that the
penetration of the micropyle by a
spermatozoon is at least partially
responsible for initiating this
phenomenon in C. variegatus.
Wedge-like structures were observed
on two adjacent cells of the four-cell
stage. These wedges likely function in
the rapid elongation of the cells in one
plane, causing the eight-cell stage to be
oval in shape. The wedges are no longer
apparent by the rounded thirty-two-cell
stage.
Early in epiboly, superficial
blastoderm cells move onto the yolk and
establish a leading edge which
surrounds it: eventually the entire yolk
becomes covered by superficial ecto-
dermal cells of the blastoderm. The
exact source of these cells has been a
long-standing question in teleost
embryology. The technique of excision
and inversion of blastoderms used in
this study permitted observations which
provide evidence that the ectodermal
cells covering the yolk come from the
superficial blastoderm. Observation of
an inverted blastoderm reveals a
channel passing along the floor of the
blastoderm and providing a cellular
pathway to its edge. Our observations
indicate that somatic ectodermal,
endodermal, and mesodermal cells
likely pass out of this channel and along
the pathway mentioned.
Gross and Histological
Anatomy of the Digestive Tract
The histology of the digestive tract of
some species of cyprinodontid fishes
has been studied. However, since a
detailed study of C. variegatus was not
available, a thorough study was con-
ducted. The study provides the baseline
data needed for this project. The diges-
tive tract of C. variegatus is similar in
gross features to that of other cyprino-
dontid fishes, being composed of an
esophagus, intestine, and rectum. The
RLG (relative length of the gut) of this
species is 2.8, which classifies it as an
omnivore.
Peripheral Blood Cell
Morphology
This study was undertaken because
of the obvious importance of hematolog-
ical data in the diagnosis of diseases,
and the fact that inadequate data about
teleost peripheral blood cell morphology
could be found in the literature.
In addition to establishing the
morphology of the formed elements of
the peripheral blood of C. variegatus in
routine fixed preparations, we
developed in vitro techniques to study
the dynamics of these blood cells in live
preparations.
The following is a brief description of
the cell types observed:
Erythrocytes—In fresh preparations,
the definitive erythrocyte is biconvex
and ellipsoid with a centrally located
oval nucleus. The cytoplasm is some-
what opaque when a minimal exposure
to air has occurred. Nuclei are visible in
fresh preparations but not prominent
due to the partial masking effect of the
cytoplasmic hemoglobin. In Romanowsky-
stained preparations, the homogenous
cytoplasm of erythrocytes is eosinophilic
and opaque, staining a pale brownish
pink. The staining properties of the
cytoplasm are seen to be similar to
those of mammalian red blood cells
stained in the same manner. The mean
number of erythrocytes in females is
2.64 x 106/cu mm and in males the
mean number is 3.12 x 106/cu mm.
Erythroid Cells—In the peripheral
hematocrit, several formed elements
can be observed which apparently
either give rise to or are derived from
erythrocytes: 1) The erythroblast. an
immature erythrocyte. 2) The
erythroplastid, an anucleate,
membrane-enclosed volume of
cytoplasm derived from a definitive
erythrocyte. Erythroplastids are
spherical, ovoid, or teardrop shaped. 3)
The senile erythrocytes. In the literature
these are referred to as "basket" or
"smudge" cells. They are seen in
various stages of degeneration in fixed
smears.
Acidophilic Granulocytes—In stained
smears, eosinophilic granulocytes are
the only type of mature granulocytes
found in the peripheral blood of C.
variegatus. Immature cells vary accord-
ing to the staining properties of the
granules and the stage of the nuclei.
Nondefinitive cells are basophilic, baso-
philic with some acidophilic properties,
or completely acidophilic.
Nongranular Leucocytes—Three mor-
phologically distinct nongranular leuco-
cytes were observed. They have been
classified into two separate groups,
thromboid cells and lymphoid cells. The
thromboid cells are subclassified into
"lone nucleus" forms and "extended"
forms. The nuclei of the "lone nucleus"
cells are round to ovoid with little or no
visible cytoplasm surrounding the
nuclei. In stained smears, these nuclei
are a deeply basophilic magenta to dark
purplish blue. The cytoplasm when ob-
served is pink. In fresh preparations, the
nuclei are opaque with little or no visible
protoplasm. These cells are often found
in clusters with long, thin pseudopodia-
like structures forming extended branch-
ing networks among the cells. The "lone
nucleus" form of thromboid cells is
numerous in fish, if they have been
unduly stressed before or during blood
collection or if the blood is not quickly
heparinized or fixed. The "extended"
forms of thromboid cells are either oval
or they have one or two spiked poles. In
strained preparations, their nuclei are
magenta and usually show a pointed or
somewhat rounded indentation. These
cells were designated "spindle cells" in
the earlier literature because of their
characteristically elongated shape.
The lymphoid cells are usually slightly
larger than the "lone nucleus" forms of
thromboid cells but resemble them to
the extent that reliable differential
thromboid-lymphoid cell counts could
not be made. A narrow rim of cytoplasm
is seen surrounding the nucleus of
these cells in stained and fresh
preparations.
Aseptic Embryo Technique
Our initial experiments indicated that
C. variegatus embryos could be main-
tained for up to 18 days under a variety
of aseptic conditions. Efforts are under
way to extend this time until the fish can
be maintained aseptically to the adult
stage.
The aseptic embryo technique
provides an opportunity to study the
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effects of a contaminant on the
organism during its highly susceptible
embryonic period entirely free of any
influence by bacteria or other
organisms normally present. Thus, the
system should be particularly valuable
in carcinogen studies related to enzyme
induction and the pathways by which
procarcinogens are metabolized into
active carcinogenic agents.
Embryo-Primary Cell Culture
Technique
With 75-80% efficiency, embryos in
this system become attached to the
surface of Linbro wells within 2-3 days
and a mixed population of cells begins
migrating from the attached region of
the embryo. As cell migration continues,
the attached region of the embryo
becomes progressively disorganized.
Three morphological types of migrating
cells are commonly observed: f ibroblast-
like cells, pigmented cells, and ovoid-
shaped cells. The pigmented cells often
develop long extended processes that
may interconnect. Frequently, these
pigmented cells form rather extensive
interconnecting networks.
Since the embryo-primary cell culture
technique blends the use of an aseptic
embryo with cell cultures into one
system, it is possible to simultaneously
observe the effects of a specific
carcinogen on a relatively intact
organism, and primary cell cultures from
that same organism.
Primary Hepatocyte Cell Culture
Technique
Since primary hepatocyte cultures
can be expected to be more genetically
similar to the cells of the intact organism
than an established cell line, data
derived from C. variegatus primary
hepatocyte cultures should be more
directly comparable to the whole animal
system. Once the methods for maintain-
ing these primary hepatocyte cultures
have been optimized and adequately
standardized, it should be relatively easy
toconduct assays using numerous dupli-
cates with positive and negative con-
trols. In addition to its value in extending
the usefulness of C. variegatus as an
assay system, the system may have
advantages over mammalian systems
currently in use. For example, the
systems can be operated more econom-
ically because the cultures can be
maintained at room temperature, thus
avoiding the use of expensive incubators
with gas-controlled environments.
Immunologies! Studies
In order to study the humoral immune
response of any organism, serum must
be easily obtainable in reasonable
volumes, which is not a problem in
larger fish where blood may be obtained
in milliliter amounts via heart puncture
or caudectomy. The blood volumes avail-
able from C. variegatus. however, are
measured in microliters. It has been
necessary, therefore, to miniaturize or
modify standard techniques and to
develop a bleeding procedure that sig-
nificantly enhances blood recovery from
•these organisms.
Serum electrophoresis was performed
to provide baseline data for serum
immunoelectrophoresis as well as for
its own intrinsic value for comparison of
normal and carcinogen-exposed fish.
Densitometer scans of duplicate runs
showed good reproducibility. Figure 1
compares the results of densitometer
scans of serum from normal C. varie-
gatus and from fish exposed to 1 ppm
BEN for seven weeks. There are obvious
differences between the serum profiles
of the normal and BEN-exposed fish.
The normal serum shows eight peaks,
whereas the serum from the BEN-
exposed fish shows only five peaks, with
some components evidently present
only in very weak concentrations or
missing altogether. Sera from different
normal individuals showed some vari-
ation but were always similar, producing
seven to nine peaks in densitometer
scans, with most producing eight. Sera
from BEN-exposed fish, on the other
hand, showed wide variations in serum
profiles, both in size and number of
peaks.
a.
Immune rosettes were successfully
produced and identified in spleen cell
suspensions from C. variegatus.
Complete "halos" of erythrocytes were
frequently observed with no leucocyte
evident inside. Careful examination of
these preparations led totheconclusion
that the mounting or staining procedure
often caused the destruction of the
rosette-forming cells, which are
evidently rather fragile. Destruction of
such cells within a complete ring of
erythrocytes left only the ring with little
or no evidence of the rosette-forming
cell itself.
Before bacteriophage neutralization
assays were performed with serum
from fish immunized with MS2 phage,
preliminary experiments were
conducted with serum from normal,
non-immune fish and from non-
immune fish exposed to 1 ppm BEN for
seven weeks. These experiments were
efforts to determine if any natural
neutralizing substances were present.
Table 1 presents partial results. Serum
from normal fish had no significant
effect on the virus liter, as may be seen
by comparing number of plaque-forming
units (PFU) in normal serum-exposed
phage inoculum with the number in
phage inoculum not exposed to serum.
In contrast, significant reduction of
PFU/inoculum was observed if the
inoculum was exposed to serum from
BEN-exposed fish. At a serum dilution of
1/32 to 1/256 or higher, the plaque
reduction became less marked but still
significant. The results suggest that
there are at least two components of
serum from the benzidine-exposed fish
which produce plaque titer. One
b.
\
Figure 1. Comparison of serum electrophoresis profiles of (a) normal serum of C.
variegatus, fb) serum of C. variegatus exposed to 1 ppm benzidine for 7
weeks. Fastest migrating peak is at right of profiles.
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Table 1. Effects of Serum from Normal and Benzidine-Exposed Cyprinodon variegatus on the Titer of MS2 Bacteriophage
Source of Serum
1/2
1/4
Serum Dilution
1/8 1/16 1/32 1/64 1/128 1/256 No Serum
Benzidine-treated fish
Normal fish
Phage control (no serum used)
65*
153
65
159
62
132
89
158
113
154
106
152
110
153
117
169
141
* Numbers represent number of plaque-forming units fPFU). A dilution of phage stock was used which should have produced
approximately 150 PFU/inoculum volume.
dropped below the minimal
concentration for effect at a dilution of
1 /16 to 1 /32 and the other still exerted
an effect at a dilution of at least 1 /256.
Recommendations
This project demonstrates the
feasibility of employing small estuarine
teleosts in laboratory assays of suspect
carcinogens. The relatively low cost of
maintaining such fish and their short
latency period of only five to six months
give this assay an economic advantage
over traditional murine bioassay
systems. Experience with C. variegatus
indicates that a requirement for success
with such systems, however, is that the
fish must be parasite-free and healthy,
and their health must be carefully
maintained during the course of the
exposures. Thus, feral fish brought into
the laboratory must be treated for
ectoparasites and in excellent health
prior to their use in experiments.
Furthermore, all experiments must be
designed and conducted in a manner
that insures the constant maintenance
of a high-quality environment.
The toxicity problems encountered in
this project indicate that one should
always determine the LC-50 of a
compound as a point of reference before
making decisions concerning
concentrations to be used in long-term
chronic exposures.
The repeatable induction of a liver
lesion by weekly 1 ppm contaminations
of the water with BEN demonstrates the
usefulness of this mode of exposure.
Thus, it is recommended that this type of
experiment be continued until the
lesion is completely characterized, its
incidence determined, and its latency
period accurately established.
Additionally, experiments with other
known mammalian carcinogens should
be conducted to provide comparative
data relative to this system.
The variety of anomalies resulting
from the exposure of early C. variegatus
embryos to BEN indicates that this
system is likely to be an effective tool in
the study of carcinogenesis and
teratogenesis. In addition to the
continued study of these BEN-induced
anomalies, exposures of this type with a
number of other known mammalian
carcinogens should be conducted.
The occurrence of multilayered foci in
SHF-1 cell cultures exposed to both BaP
and BEN indicates that teleost cell
cultures, like mammalian cell cultures,
can be employed in this type test. The
effectiveness of this system is
enhanced by the fact that such
exposures are less costly than
exposures of mammalian cells.
The three cellular techniques (aseptic
embryo, embryo-primary cell culture,
and primary hepatocyte cell culture) are
all innovative techniques that are likely
to be developed into useful, rapid test
methods for carcinogenesis; therefore,
their continued development is
recommended.
Our miniaturization and adaptation of
a variety of standard immunological
techniques provide the tools needed to
conduct sophisticated studies of the
immune system of C. variegatus. Thus,
it is now possible to use this species in
the study of the fascinating relationship
between carcinogenesis and the
immune system. The significant effects
of low-level chronic exposure to BEN on
the serum profiles of C. variegatus are a
preliminary indication of the value of
these techniques; therefore, it is
recommended that considerable effort
be devoted to studies of the immune
systems of normal and carcinogen-
exposed C.variegatus.
References
1. Martin, B. J. Effects of petroleum
compounds on estuarine fishes.
U.S. Environmental Protection
Agency. Ecol. Res. Series.
Cincinnati, Ohio. EPA-600/3-80-
019. 1980.
2. Law, W. M., R. D. Ellender, J. H.
Wharton, and B. L Middlebrooks.
Fish cell culture: properties of a
cell line from the sheepshead,
Archosargus probatocephalus. J.
Fish. Res. Board Can. 35.470-
473. 1978.
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B. J. Martin is with the University of Southern Mississippi, Hattiesburg. MS 39406.
J. A. Couch is the EPA Project Officer (see below).
The complete report, entitled "Development of a Carcinogen Assay System
Utilizing Estuarine Fishes," (Order No. PB83-136 333; Cost: $ 10.00, subject to
change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Protect Officer can be contacted at:
Environmental Research Laboratory
U.S. Environmental Protection Agency
Sabine Island
Gulf Breeze, FL 32561
•&U. S. GOVERNMENT PRINTING OFFICE: 1983/659-095/587
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United States Center for Environmental Research Fees Paid
Environmental Protection Information Environmental
Agency Cincinnati OH 45268 Protection
Agency
EPA 335
Official Business
Penalty for Private Use $300
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