United States
Environmental Protect.on EPA-600/9-83-005
4>EPA Research and
Development
EFFECTS OF CARCINOGENS, MUTAGENS,
AND TERATOGENS ON NONHUMAN
SPECIES (AQUATIC ANIMALS )
FOURTH ANNUAL REPORT
NCI/EPA COLLABORATIVE PROGRAM
Prepared for
National Cancer Institute
Office of Pesticides
and Toxic Substances
Prepared by
Environmental Research
Laboratory
Gulf Breeze FL 32561
-------
DISCLAIMER
This report has been reviewed by the U.S. Environmental Protection Agency,
and approved for publication. Approval does not signify that the contents
necessarily reflect the views and policies of the U.S. Environmental
Protection Agency, nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.
-------
Fourth Annual Report
NCI/EPA Collaborative Program
Fiscal Year 1982 (October 1981 - September 30, 1982)
Project 3
EFFECTS OF CARCINOGENS, MUTAGENS,
AND TERATOGENS ON NONHUMAN
SPECIES (AQUATIC ANIMALS).
by
John A. Ccuch, Ph.D., Coordinator, Pathobiologist
W. Peter Schoor, Ph.D. Biochemist
Will Davis, Ph.D., Biologist
and
Lee Courtney, Research Toxicologist
U.S. EPA, ERL, GULF BREEZE, FLORIDA
-------
TABLE OF CONTENTS
II. Abstract 2
III. Introduction 4
IV. Objectives 5
V. Methodological Approaches 5
VI. Major Findings and Results 7
VII. Significance to Biomedical Research and
Program needs of NCI and EPA 13
VIII. Future Plans 14
IX. Date Contract Initiated and
Period of Contract Planned 14
X. Contractors Project Director and
Project Officers 14
XI. Progress Reports on Specific Extramural
Cooperative Agreements 14
XII. Publications Resulting from Program 39
-------
EFFECTS OF CARCINOGENS, MUTAGENS, AND TERATOGENS
ON NON-HUMAN SPECIES-AQUATIC ANIMALS
John A. Couch, Coordinator; W. Peter Schoor, Biochemist;
Will Davis, Biologist; Lee Courtney, Research Toxicologist
United States Environmental Protection Agency
Gulf Breeze, Florida 32561
Aquatic systems and organisms are under both laboratory and field study in
order to develop indicator, screening, and modeling capabilities for detection
and evaluation of risks of carcinogens, mutagens, and teratogens. Studies
include both Gulf Breeze laboratory projects and complementary, extramural
projects. In the fourth year of the program, several advances were made in the
development of laboratory and field carcinogen assays, utilizing fishes such as
the sheepshead minnow (liver lesions via benzidine and aflatoxin exposures),
Menidia peninsulae (liver lesions via aflatoxin exposures), and freshwater cat
fish (papillomatous-like lesions via chlorinated effluent exposures). Emphasis
is still placed on the development and utilization of critical life stage
exposures (e.g., embryo and newly hatched fry exposures) in order to expedite
carcinogen tests and minimize time required for tumorogenic responses.
Preneoplastic hepatic lesion development in Menidia at 12 weeks suggests
promise for this species and exposure method. A novel approach has shown that
tiger salamanders may be good biochemical and histologic indicators of the
presence of certain carcinogens (polycyclic aromatic hydrocarbons - PAH's).
Skin and liver tissues of the salamanders revealed induced enzyme activity (MFO
system) following exposure to the PAH, perylene. Considerable field monitoring
work on mollusks and carcinogenic PAH's along the coast of Oregon has revealed
a positive correlation between prevalence of cellular proliferation disorders
in shellfish and higher concentrations of certain PAH's in natural water.
Emphasis in biochemistry for the last year has been directed mostly toward
the elucidation of the metabolism of the mixed-function oxidases in marine
organisms. From continuing work on the mullet (Mugil cephalus), we have found
that benzo(a)pyrene (B(a)P) is converted mainly to 3-hydroxybenzo(a)pyrene,
9-hydroxybenzo(a)pyrene and 4,5-, 7,8-, 9,10-dihydrodiols of BaP, in j_n vitro
systems containing liver microsomes from Aroclor 1254-treated mullet. Other
metabolites such as diones of B(a)P and probably tri-and tetraols have also
been produced. We have also developed a procedure for the epoxide hydro!ase
enzyme system in order to determine the role of this enzyme on the metabolism
of the polyaromatic hydrocarbons. Our studies show that UDP-glucuronosyl
transferase and sulfotransferase were significantly increased in livers of rats
given phenobarbital (PB), phenolphthalein, phenanthrene, BaP or 3-MC orally for
10 to 14 days. The injection of PB or 3-MC into rats, sea catfish or mullet on
long-term schedule resulted in increase in liver size, and an increase in
UDP-glucuronosyl transferase in livers of all three animal species. We have
observed that the liver microsomal fraction of the killifish, Fundulus
qrandis, has a high in vitro activity when injected with a single dose of 3-MC,
converting B(a)P to 1-OH, 3-OH, 5-OH, 6-OH, 7-OH, (t) 9,10-diol, (t)
-------
7,8-diol, 1,6-, 3,6-, and 6, 12 diones, and 4,5-oxide. There may be an optimal
concentration of MADPH, and this concentration may have a significant effect on
the products of the in vitro reaction, quantitatively as well as
qualitatively. These studies continue to reveal that fish have metabolic
pathways similar to mammals for disposition of certain carcinogens. A brief
study was conducted to determine possible interactions between the 9-OH BaP and
salmon sperm DNA. The interaction between salmon DNA and 9-OH BaP was shown by
relative fluroescence studies. The binding was evidenced by an eight-fold
reduction in relative fluorescence yield. We are developing methods to
determine the possible binding of proximal carcinogenic metabolites to DNA's of
selected aquatic animals.
-------
III. Introduction
A major problem faced by the National Cancer Institute and the
Environmental Protection Agency is that of aiding in determining the fate and
effects of carcinogenic pollutants in the larger environment. One way is to
study wildlife populations that are widespread, but which live in environments
where exposure to ambient pollutants is certain. The use of wildlife
populations -as surrogates for human populations may be considered to be a novel
expansion or logical extension of the use of laboratory animals and animal
models of human diseases as alternatives to the use of human subjects. The
sharing of biologic characteristics by phylogenetically diverse species makes
certain comparative approaches possible.
Problems arise, however, when such factors as selection of sensitive,
indicative species, geographically adequate populations, and representative
segments of the air, land, water biosphere are considered. In this regard,
Office of Research and Development laboratories, such as Gulf Breeze
Environmental Research Laboratory, have exemplary, pilot research programs that
are investigating the use of aquatic animal species as indicators of
carcinogens in the environment. The Gulf Breeze pilot research program has
been under way since August, 1978 and is supported jointly by the Office of
Research and Development (EPA) and the National Cancer Institute through an
interagency agreement. The Gulf Breeze studies are based on the premise that
the aquatic portion of the biosphere (water, biota, and sediment) is the
ultimate "sink" for the runoff, fallout, and discharge of most toxic
pollutants. In addition, animals living in the relatively efficient solvent,
water, are more intimately exposed (total exposure through body surfaces,
giI Is, alimentary tracts) than are species living in terrestrial or air
environments. Aquatic species are also less likely to escape a dissolved or
carried pollutant.
At Gulf Breeze, researchers have been studying species of fish and
shellfish along the Northern Gulf of Mexico (Florida, Alabama, Mississippi) in
order to determine which are good indicators of the role of carcinogenic agents
in the environment. Selected species of fish have been used in both long-term
and short-term laboratory exposures to determine their specific tissue,
cellular, and biochemical responses to known chemical carcinogens which may
occur in the environment. In addition, a significant number of cooperative
agreements with principle investigators from around the nation support an
extramural complemental and supplemental effort in the identification of
aquatic species and systems that may serve as early warning mechanisms.
-------
IV. Summarized Objectives
Objective 1:
Determine fate and effects of carcinogens, mutagens, and teratogens in
aquatic species (individuals; populations).
Objective 2:
Determine role of aquatic species and systems in actual or potential
exposure of man to carcinogens, mutagens, or teratogens (metabolism,
accumulators).
Objective 3:
Develop aquatic species as bioindicator, sentinel, and model systems for
use in study of risks of carcinogens, mutagens, and teratogens in the
general environment (sensitive species and systems).
V. Methodological Approaches
We have underway both in-house and complementary extramural research
projects. The overall project is divided into major disciplinary approaches:
1) Pathobiology and 2) Biochemistry. Therefore, methods outlined below and
results in the next section will be reported under these two complementary,
disciplinary headings. Detailed results of each cooperative agreement in
progress during FY 82 will be included at the end of this report. The
disciplinary area of each cooperative agreement will be identified by project
officer (Couch - pathobiology; Schoor - biochemistry).
1. Pathobiological Methods
Fish carcinogen assay system, toxicology, and histopathology of
induced lesions.
At Gulf Breeze, major emphasis was concentrated on short-term
critical life-stage carcinogen exposures of several estuarine fish
species. Cyprinodon variegatus (Sheepshead minnow), Menidia
peninsulae (Silverside), and Rivulus marmoratus were exposed to the
overt carcinogen aflatoxin B^~. This compound, a natural product of
the metabolic activity of fungi (genus Aspergillus), has been shown to
be of high toxicity and carcinogenicity in mammalian studies
(including humans) and work with freshwater fish species. Selection
of aflatoxin Bi as a test compound was partly based on this high
hazard background, to test marine species with a proven and potent
carcinogen. As such, special precautions and methods were needed to
insure safety.
Exposures were conducted under static conditions within a glove
box. Test organisms were exposed in 3.Si chambers containing 1.5z
seawater, toxicant, and carrier, then rinsed and transfered to clean
holding facilities for development and observation. A detailed
description of the test systems utilized is now in press (Courtney and
Couch, 1982). Carrier controls were run under identical conditions
except for toxicant. Species, test concentrations, exposure duration,
carrier, and life-stage tested varied. 96 hour embryos and 120 hour
newly hatched larvae of _C. variegatus were exposed for 1 hour to
-------
200 ug/i aflatoxin 8} using dimethylsulfoxide (DMSO) as a carrier.
£. variegatus, R_. marmoratus, and M_. peninsulae were exposed to 1000
ug/i aflatoxin 8-j_ (using DMSO and triethylene glycol as carriers)
for exposure durations ranging from 2 to 43.5 hours. Holding and
observation continues on surviving test organisms.
In extramural efforts, Hendrix at Oregon is continuing
histopathologic evaluation of tissues from rainbow trout (Salmg
gairdnerl) exposed for 18 months to dietary benzo(a)pyrene (BAP: 1000
ppm), Aroclor 1260 (PCB: 500 ppm), and toxaphene (50 ppm). Trout
embryo exposures to methyl azoxymethanol acetate (MAMA) and benzidine
dihydoro chloride (BDHC) were conducted and an experiment to produce a
neuroblastoma neoplasm in coho salmon and Shasta strain rainbow trout
was initiated. Martin, at the University of Southern Mississippi, is
examining specimens from over thirty adult and embryo C_. variegatus
benzidine exposures. Additional work has been done with C_. variegatus
involving aseptic embryo techniques. Cell culture techniques have
been investigated using a Fundulus species and C_. variegatus.
Grizzle, at Auburn University, is continuing laboratory and field
experiments to examine causation and development of oral papillomas
found on feral black bullheads (Ictalurus melas) from the final
oxidation pond of a Tuskegee, Alabama sewage treatment plant.
Rose and Anderson are involved in a two part research project
involving tiger salamanders (Ambystoma tigrinum) which have shown an
unusually high prevalence of dermal neoplasia. Rose is studying the
histopathological effects of perylene on the species and Anderson is
studying metabolic activation and/or detoxification of perylene and
B(a)P by tissue enzymes of _A. tigrinum.
Mix, at Oregon State University, is continuing a long-term field
study of mulluscs in Oregon coastal waters and expanding laboratory
research. Major emphasis this year include 1) baseline measurements
of arsenic, cadmium, and nickel levels; 2) methods development for
trace levels of polynuclear aromatic hydrocarbons (PNAH) in seawater;
3) developing a crustacean (barnacle) bioassay system; 4) studying
possible viral associations with Mytijus edulis neoplastic disorders;
and 5) evaluating data previously collected on neoplastic disorders in
M_. edul is.
2. Biochemical Methods
Induction Studies:
Aquatic species such as mullet, killifish, flounder, sea catfish and
others have been and will continue to be exposed to inducers of
microsomal mixed-function oxygenase (MFO) activity either by
intraperitoneal injection or direct exposure in seawater. Since
periods up to one year might be necessary to induce MFO activity by
water exposure, the direct injection of inducer is used at the start
of the investigations in order to optimize other parameters such as
metabolite and conjugation reactions. The long-term, low exposure in
seawater will follow. [Schoor, Melius (CR809493), Strength
(CR809673)].
-------
Metabolite Identification:
Metabolites from the MFO reactions will be identified using high
pressure liquid chromatography coupled with fluorescence detection and
confirmed by stopped-flow fluorescence scanning. Metabolite standards
for benzo(a)pyrene [(BaP)] have been obtained from the Illinois
Institute of Technology through the courtesy of NCI. All the phenolic
compounds have been chromatographed and their excitation and emission
spectra have been obtained in appropriate solvents. All spectra will
be stored on discs for later data manipulation.
Conjugation and Excretion Studies:
Rats are being used to make a series of conjugation products in vivo
by injection of 14C-labelled BaP. They will include glucuronides,
glutathiones, and sulfates. Their occurrence in fish will then be
ascertained by comparison to the standards produced in the rat. This
will be helpful in determining the final dispositon of a
carcinogen like BaP within the animal and in what forms the parent
compound is finally passed back into the seawater.
VI. Major Findings and Progress
As noted earlier under Methodological Approaches, results and findings of
studies to date are reported under the two disciplinary areas of Pathobioloqy
and Biochemistry with extramural cooperative agreement reports at the end of
this report.
1. Pathobiology
Table I summarizes the carcinogen assay/induction studies
undertaken by the Gulf Breeze staff and investigators working under
cooperative agreements. This work represents an investigative effort
encompassing a wide range of organisms and several selected
carcinogens and suspect-carcinogens. In addition to the data
generated, and possibly of even greater value, is the methodology
developed (both in exposure systems and support technology).
The following represents scientific results and accomplishments of
the Gulf Breeze staff and associated investigators during FY 82.
At Gulf Breeze, the static exposure system utilized in the
aflatoxin studies has proven to be an efficient method for short-term
exposures using high hazard compounds. Each species tested performed
well and the use of various life-stages presented no problems. We
feel this system provides a safe and inexpensive method of high hazard
single short-term or pulse exposures for a wide range of life-stages
and species.
Data generated to date include some basic toxicity data with
regard to aflatoxin and the three species tested,and two lesions
related to the exposures. Gross necropsy and histological examination
of C_. variegatus exposed to 200 yg/i aflatoxin B^ for 1 hr. have
revealed a benign hepatic polyp on several specimens. Exact nature of
the lesions is still under investigation and a large number
-------
SPECIES
OYSTER
OYSTER
SHEEPSHEAD
MINNOW
SHEEPSHEAD
MINNOW
SHEEPSHEAD
MINNOW
RIVULUS
SILVERSIDE
SILVER PERCH
CELL LINE
RAINBOW
TROUT
TIGER
SALAMANDER
AGENT
3-Methyl-
Cholanthrene
Benzo(a)pyrene
Triflural in-
(Herbicide)
Aflatoxin BI
Benzidine
,
Aflatoxin 5\
Aflatoxin BI
Asbestos-
(Crocidolite)
Benzo(a)pyrene
Perylene
EXPOSURE
Flowing seawater,
1-5 yg/z for one
year.
Flowing seawater,
1-5 pg/z for one
year.
Flowing seawater,
5 ug/i, 19 months.
Embyro-fry static
200 yg/£ for one
hour held for 18
months.
Recirculated
seawater, 1 ppm,
for 166 days.
Fry xposure
static, 1 ppm,
2 hours, 8 hours.
Fry exposure,
static 1 ppm, 2
hours, 8 hours.
Held 12 weeks.
Asbestos in
culture medium.
1000 ppm, diet,
12 month feeding.
Injection i.p.
RESULTS
Incipient,
perivascular
blood cell
pro! iferation.
No tissue
response.
Focal , benign,
vertebral
growths-non-
neoplastic.
Hepatic polyps
(on surface of
livers). Benign
neopl asm.
*Prol iferatai ve
lesions in
liver; ductile
pro! iferation;
cholangioma;
adenofibrosis;
oval cell
hyperplasia.
No tumor
response yet.
*
Early altered
foci in livers;
enlarged cells;
preneoplastic.
Nodule formation
in culture around
asbestos fibers.
Hepatocellular
Carcinoma.
*
Hepatic tumor.
j_
umors or sus-
pect neoplasms
actually induced
-------
of specimens still remain in holding. In addition, a suspect
pre-neoplastic lesion has been noted in histologic sections of M_.
peninsulae exposed as fry for 2 hrs. to 1000 ug/i aflatoxin (using
both DMSO and TEG as carriers). The lesions, noted in specimens
examined 12 weeks post-exposure, consist of foci of enlarged hepatic
cells. Additional specimens from the exposure are being held for
continued observation and future histologic examination. No lesions
have been noted in those Rivulus examined to date.
During FY 82 a final report summarizing a two-year field
biomonitoring study (Aug. 1978 to Aug. 1980) was completed. This is
now available in manuscript form entitled "A Prospective Study of
Infectious and Noninfectious Diseases in Fishes and Shellfishes in
Relationship to Pollutant Activity in Three Gulf Coast Estuaries".
The main issue of the study was that certain diseases of fishes and
shellfishes (including neoplasia) from coastal populations have been
suggested to be related to, caused by, or enhanced by pollutant
activity. Considerable data have been published from which inferences
have been made that fishes and shellfishes inhabiting contaminated
waters are at higher disease risk than those in cleaner environments.
Most of these studies from which data derived were retrospective,
epizootiological efforts, initiated after the fact because certain
fish diseases were increasingly observed or because fish kills were
noted or had recurred frequently in specific limited bodies of water.
Prospective studies of estuaries without prior knowledge of disease
prevalence to determine possibly previously undetected frequencies and
relationships of diseases and pollutant residues in fishes and
shellfishes have been rare. Therefore, a need existed to determine
prospectively if populations of fishes and shellfishes at higher
disease risks related to pollutant activity could be identified with
standard sampling, pathological, and analytical methods.
The study of three northern Gulf Coast estuaries, Pensacola and
Escambia Bays in Northwest Florida, Mobile Bay, Alabama, and
Pascagoula Harbor in Mississippi Sound, Mississippi, was undertaken in
August, 1978. The specific goals of this prospective study were to:
1) determine and compare relative contamination by select pollutants
on specific sites in and among the three estuarine areas, 2) determine
frequencies of known or new diseases, including neoplasms, in
shellfish (oysters) and fishes at these sites among the estuaries, and
3) to examine critically any relationships between disease frequency
and pollutant contamination among the selected estuarine sites in
order to assess the role of pollutant activity in influencing disease
prevalences in fish and shellfish populations in coastal regions
characterized by varying degrees of human pollutant activity. For
each of the three estuaries, three different kinds of samples
(oysters, fishes and sediments) were collected quarterly for chemical
analysis of pollutant residues from August, 1979 through August, 1980.
One offshore control station was sampled for prevalence comparison to
the estuarine stations. Monthly samples of fishes, and oysters were
collected for disease analyses and diagnoses from one or more specific
sites in the three estuarine systems from August, 1978 through August,
1980 (25 Month Period).
-------
Results of this study suggest that though far from being pristine,
the estuaries studied were not severely unhealthy environments at the
time that the biota were sampled. However, because predictions have
been made that the Gulf Coastal Plain of the southeastern U.S. will
probably be the fastest growing region in the nation in the next
decade, this study and others provide base lines and points of
departure and comparison for studies that should be made,
periodically, on the health of coastal biota in relation to increasing
impact of population and industrial growth.
In extramural projects, rainbow trout exposed to dietary
benzo(a)pyrene (100 ppm) have developed hepatocellular carcinoma at
24% incidence level (see Hendricks attached cooperative agreement
report no. CR809344-010). Aroclor 1260 and toxaphene exposures have
yet to reveal neoplasms; however, tissue examination is not complete.
Methylazoxymethanol acetate (MAMA) and benzidine dihydrochloride
exposures have been completed, however, all necropsy samples have not
yet been processed. Preliminary indications are that MAMA is
carcinogenic to trout. Mechanical problems have hindered progress on
attempted neuroblastoma induction.
Martin (see attached report no. CR809347) has produced liver
lesions, probably adenofibrotic or neoplastic, in C_. variegatus
exposed to 1, 5, and 10 ppm benzidine dihydrochloride (BDHC). Several
experiments are still in progress and characterization of the BDHC
produced lesion is still underway. In BDHC exposed C_. variegatus
embryos (50 to 500 ppm exposure concentrations), 85% exhibited
developmental anomalies compared to a 0.02% rate of anomalies in
controls. Aseptic C_. variegatus techniques have developed to the
point of successful maintenance of embryos for 60 days or more. Four
C_. variegatus cell lines have been developed including three
fibroblastic cell lines, and a striated muscle cell line, to our
knowledge, the first such line developed from fish tissue. In
immunological studies, banding pattern differences have been noted
between BDHC-exposed and non-exposed C_. variegatus and the
characterization of these differences is being pursued. Additional
studies using immunoelectrophoresis, competitive enzyme immunoassay
and bacteriophage neutralization show much promise in exploring the
processes occurring in BDHC exposed C_. variegatus.
Grizzle (see attached report no. CR809336-010) has produced
lesions in caged black bullheads (Ictalurus melas) confined to a
chlorinated sewage effluent pond. The lesions consisted of oral
mucosa hyperplasia, developing after 2 months, and papilloma-1ike
lesions in the mouth fornix, developing during the second year. These
lesions were similar to those found on black bullheads naturally
occurring in the pond. Transmission experiments and ultrastructural
studies failed to indicate a viral etiology for the papillomas.
Induction of glucuronosyl transferase in caged channel catfish within
the pond and Ames tests with the pond water indicate the presence of a
chemical possibly related to the Ictalurus melas papillomas.
The work of Rose and Anderson (see attached report no. CR807740)
includes the development of a radioisotopic method to quantify B(a)P
and perylene (PI metabolism in Ambystoma tigrinum. Hepatic metabolism
of ^C-BaP and 3H-P was studied in normal and induced A.
tigrinum. Uninduced animals produced about 0.2 total C-BaP
metaboi ites/mg protein/30 min. Methylcholanthrene was indicated
10
-------
as a AAH inducer. Metabolism of 3H-P was found greater than that of
14C-BaP, inducers had little effect on metabolism compared to
noninduced Ambystoma. Carbon 14-BaP metabolite profiles of induced
and non-induced animals were compared and it was found that some
inducers, including P, produced a metabolite shift away from the more
potentially carcinogenic compounds. This indicates a possible
protective action by inducers, such as P, for subsequent BaP exposure,
similar action is frequently reported in higher animals. The results
of mutagenicity testing indicate that Ambystoma is quite resistant to
PAH carcinogenesis and suggests that aromatic amines may be more
appropriate model carcinogens. Histopathological lesions were found
in several salamanders injected with perylene, one, a liver cell
neoplasm. Final evaluation of this phase of the study is not yet
complete.
The research project of Mix, at Oregon State University, (See
attached report no. CR808000-01-0) has produced much data related to
shellfish populations along the Oregon coast and valuable
investigative methodology. Baseline measurements of arsenic, cadmium,
and nickel were determined for two species of mussels, and it was
determined that the mussels would be excellent monitors of these
metals in freshwater (Margaritifera marqaritifera) and marine (Mytilus
edulis) environments. The evaluation of a long term study (1976-1981)
of pro!iferative disorders in M_. edulis has continued and produced
much data relating to occurrence, geographical distribution,
prevalence, seasonality, and nistopathology. Extensive investigation,
employing a variety of techniques and methods, failed to reveal the
presence of any RNA tumor virus in M.. edulis exhibiting neoplastic
disorders. Additionally, methods for PNAH determination in seawater
and a crustacean bioassay system utilizing Pollicipis polymerus were
investigated. The bioassay system exhibits great potential for
testing environmental carcinogens and mutagens.
Studies of teratogenic responses have arisen from a broader
research program formerly supported by EPA Energy Effects funding
(R805469). The objective of the investigation was to develop an
experimental test with marine animals suitable for assessing the
by-products of chlorination arising from biofouling control and
disinfection of coastal waters. An additional task was requested by
EPA to test specific compounds. Phthalate tests revealed significant
increase in endematous cardiac malformations in embryos, as well as
abnormal development of vertebral and other skeletal elements among
surviving fishes.
A colony of the fish Rivulus marmoratus was transferred from Bears
Bluff Field Station, S.C. and the College of Charleston to Gulf Breeze
EPA laboratories in 1981. With the previous studies as a foundation,
we have been concentrating on refinement of screening tests for
teratogenic effects. Since Rivulus marmoratus is a self-fertilizing
hermaphrodite, it provides natural clones which allow data tracking on
individuals (temporal variations), as well as lineages and clones and
representatives of wild populations. Our research designs incorporate
all stages of gametogenesis as well; as post-fertilization
11
-------
developmental stages.
This strategy is noteworthy inasmuch as during initial screening
one does not know the breadth of potential "windows of vulnerability"
to teratogenic effects. Thaiidomide, for example, is teratogenically
dangerous to the embryo for only very brief periods. Rodent tests
were run for several years before they "modelled" established human
effects.
The number of potential responses of Rivulus are broad and
apparently clearly distinguishable. Classical teratogenic studies ,
used fishes, and focused mainly on expressed gametes just prior to, or
soon after fertilization. The ability to "scan" broader periods of
gametogenesis, and have the relative economy of fish are among the
obvious advantages of this species. Studies presently underway will
refine testing procedures, and compare results from n-dibutyl
phthalate, thalidomide, and hexazinone (a herbicide). Other reports
presented at the NCI (Koenig, et al.) Bethesda meeting report general
biology and tumorgenic responses.
2. Biochemistry
Induction of transferases (UDPGA and sulfate) was observed in
mullet and in channel catfish. The rate of conjugation was confirmed
by enzyme assay with conjugation using p-nitrophenol as substrate.
This was determined spectrophotometrically and by TLC separation of
the respective conjugates of p-nitrophenol. Metabolites formed by MFO
in microsomes derived from both control and induced animals were
separated by HPLC and identified. Microsomes incubated with both BaP
and UDGPA enabled us to ascertain the extent of conjugation in the
presence of the oxidation system.
In addition to finding BaP metabolites already reported, we
believe we have found some "cis" diols as well as "trans" diols. The
evidence is based on agreement of retention times of standards and
unknown metabolites. Nanogram amounts of the "cis" diols have not yet
allowed more positive identification. In order to establish a kinetic
sequence for the metabolites, we have slowed the MFO reaction at 0°C;
however considerable activity remains at that temperature.
MFO activity has been studied in the sea catfish, Aries felis.
collected from coastal waters in N-W Florida. The oxidase activity
was induced with 3-MC injections and tissue/body mass ratios, hepatic
microsomal protein components and in vitro BaP metablism were all
studied. The effect of 3-MC was measured over a 5-40 mg 3-MC/kg body
mass range for 7 days and at a 20 mg 3-MC/kg body mass dose level for
1, 3, 5, and 7 day periods. Liver/body and kidney/body mass ratios
increased with increasing 3-MC dose levels and treatment period.
Cytochrome P-450, epoxide hydratase and cytochrome b5 reductase all
increased significantly with increasing doses of 3-MC up to 40 mg
3-MC/kg body mass and with increasing time periods. A linear response
was obtained in the Ames test with S. typhimurium TA 98 up to 3-MC
dose level for 20 mg. In the Ames test at a 20 mg 3-MC dose level, we
obtained a linear response over a period of 7 days. The hepatic
12
-------
microsomal fraction from the 3-MC induced sea catfish metabolized BaP
to the usual metabolites including the 3-and 9- phenols, 4,5-, 7,8-
and 9,10-dihydrodiols and quinones. Metabolite production was
enhanced by butylated hydroxyanisole and inhibited by 6-aminochrysene,
adenosine-21-phosphate and l,2-epoxy-3,3,3-trichloropropane.
VII. Significance to Biomedical Research and
Program Needs of NCI and EPA
The following contributions to the needs of NCI and EPA were made during
FY 82:
1. The responsiveness of a number of species to environmental
carcinogens has been demonstrated. The sheepshead minnows have
developed proliferative liver lesions (oval cell hyperplasia, possibly
adenofibrosis) resulting from benzidine exposures and benign hepatic
polyps related to aflatoxin exposure. A pre-neoplastic condition, also
related to aflatoxin exposure, has been produced in Menidia livers.
Finally, rainbow trout developed overt hepatocellular carcinoma
following treatment with benzo(a)pyrene. All three species have high
potential to be used as routine carcinogen assay organisms.
Additionally, the black bullhead has been successfully used to
demonstrate a cause and effect relationship to an existing pollution
situation. Laboratory and field responsiveness have shown it to be a
good indicator organism. Finally, the tiger salamander (Ambystoma sp.)
is particularly interesting as an assay or indicator organism because of
its cutaneous and liver enzyme responses to the PAH, perylene. The fact
that this species has shown cutaneous tumor development in contaminated
environments heightens its significance as a possible indicator
species.
2. Results from the Field biomonitoring study (Report entitled: "A
Prospective Study of Infectious and Non-infectious Diseases in Fishes
and Shellfishes in Relationship to Pollutant Activity in Three Gulf
Coasts Estuaries") indicate the following: First, a low tumor epizootic
in fishes suggests low risks to environmental carcinogens at locations
and times sampled. Therefore, future studies should be retrospective in
regard to neoplasia as an indicator of carcinogen contaminants; i.e.
studies should be based on an a priori indication of tumors in certain
species at certain sites. Secondly, findings indicate that oysters may
be more sensitive indicators of environmental contamination and
especially may provide a more representative response to focal
contamination in coastal regions. Future studies should include careful
monitoring for tissue and physiologic changes indicative of chemical
induced stress in oysters. Furthermore, several diseases of oysters and
fish were detected during the study. At least two of these, a
rickettsia found in oyster digestive gland cells and ichthyosporidosis
from several fish species, are important in terms of possible impact
related to effects on biota and human health. These diseases and their
role in the Gulf coast environment should be investigated further.
Finally, the overall study suggests that, while being far from pristine,
the study area was not a severely unhealthy environment at the time of
investigation. This study and others now provide a base line for
13
-------
future comparison. Studies should be periodically made to monitor the
impact of population and industrial growth along this valuable coastal
region, a region predicted to be one of the fastest growing areas of the
country during the next decade.
3. Biochemical and correlated structural responses of fish liver systems
seem to be relatively similar to responses of mammals to certain
carcinogens. Recent results show that the mixed function oxidases are
induced in fish as well as the conjugating enzymes that aid in the
detoxification and excretion of compounds such as B(a)P. This permits
future comparative studies to determine if biochemical methods may be
incorporated in early warning or sentinel monitoring projects with fish.
The biochemical studies suggest that fish may serve as animal models in
carcinogen (preneoplasia) studies to complement mammalian studies.
VIII. Proposed Course - Future Plans
1. The NCI/EPA collaborative project officially concludes October 1982
and no new projects will be undertaken. During FY83, efforts will be
made toward concluding research projects already underway at Gulf Breeze
and by investigators under cooperative agreements. Major emphasis will
be directed to preparing final reports (due October 1983) and collating
reprints produced as a result of the research period.
2. An in-house study, at Gulf Breeze, will be conducted on the effects of
nltrosamines on fish species using test systems developed during the
NCI/EPA research program. The study will focus on endpoints of tumor
induction and possible other effects (histopathological, physiological,
etc.).
IX. Date Contract Initiated and Period of Contract Planned
Initiated Expiration Date
October 1978 September 30, 1982
X. Contractors Project Director
Dr. Henry F. Enos, Laboratory Director
Gulf Breeze, ERL, EPA
Project Officers for NCI or EPA
NCI: Dr. Herman Kraybill
EPA/ORD: Dr. Wayne Galbraith
Prinicipal Investigator: Dr. John A. Couch, Gulf Breeze, ERL, EPA
XI. Cooperative Agreements Funded - Progress Reports FY 82
Title: Rainbow Trout: A Model for Environmental Carcinogenesis
Prinicipal Investigator: Jerry D. Hendricks
Cooperative Agreement Number: CR-809344-010
Project Officer: John Couch
14
-------
Introduction
Work is continuing on the histopathological evaluation of tissues from
fish exposed for 18 months to dietary benzo(a)pyrene (BAP), Aroclor 1260 and
toxaphene. The data from the BAP dietary study and the BAP intraperitoneal
injection study is complete and a manuscript is in the final stages of
preparation.
Trout embryo exposures to methyl azoxymethanol acetate (MAMA) and
benzidine dihydrochloride (BDHC) were conducted in December, 1981 and the
finger!ings are now 8 months of age. An attempt to produce a rare
neuroblastoma neoplasm in coho salmon fry was unsuccessful but will be
conducted again during the next year.
Current Progress
Dietary exposure of trout finger!ings to 1000 ppm BAP for 18 months
resulted in a 24% incidence of liver neoplasms at 18 months (table 2). Average
body weight for these fish at 18 months was 364 g (table 1). Average
conversion of dry diet to fish weight for rainbow trout is 1:1 indicating that
the average fish would have consumed 364 g of dry diet and consumed 364 mg of
BAP. In another experiment, we injected, IP, a group of 50 trout with 1 mg BAP
in .4 ml of propylene glycol monthly for 12 months and held them an additional
6 months before killing and examination. The total dose in this case was only
12 mg/fish but the incidence of hepatic tumors was 50% (table 3). This may
indicate that BAP is a more potent carcinogen to trout than dietary exposure
demonstrates. It is possible that much of the highly water insoluble BAP is
not absorbed from the trout gut, making the effective dose much lower than what
was put in the diet.,
Gross examination did not reveal any neoplasms in fish fed either Aroclor
1260 (500 ppm) or toxaphene (50 ppm) for 18 months, however, a histological
examination of all tissues is befng conducted.
The first objective of the new project was to determine the effects of two
potential carcinogens on rainbow trout embryos as a continuing effort to assess
the suitability of rainbow trout as indicators of environmental carcinogenesis.
We are interested in determining the carcinogenicity of two rodent carcinogens,
methylazoxymethanol acetate (MAMA) and benzidine dihydrochloride (BDHC), in
rainbow trout embryos as well as the effects of prior exposure of trout embryos
to the environmental contaminant, Aroclor 1254 (PCB), on the carcinogenicity of
MAMA and BDHC.
The 3 year-old female brood fish were removed from the usual OMP brood
ration and placed on our semi purified test diet containing 200 ppm Aroclor 1254
two months prior to spawning. Two months later these fish were each given two
injections of commercially prepared salmon pituitaries and induced to spawn.
Eggs from seven fish were available on the same spawning date and were pooled
to provide the source of PCB-exposed embryos for subsequent embryo exposure.
Eggs from the same number of OMP-control brood females hormonally induced to
spawn on the same day were used for comparative control embryo exposures. Egg
samples from both sources taken on the day of spawning, day of carcinogen
exposure (day 21 of incubation) and periodically thereafter were prepared for
PCB analysis to follow PCB depuration. On day 21 of incubation, 12 groups of
200 viable embryos from both the PCB and control groups were exposed for 24
hours to MAMA and BDHC at the 0.1, 1, 10, 100 and 1,000 ppm levels. Both MAMA
and BDHC were lethal to trout embryos at the 1,000 ppm level for 24 hours.
15
-------
Table 1
Body weight, liver weight/body weight ratios and mortalities, within
groups of trout sampled at 6, 12, and 18-months of diet exposure to benzo(a Jpyrene.
Liver weight 10Q
Sample time Diet Body weight (g)a Mort Body weight
CD 50 ± 10 (23) 17 1.13 ± 0.13 (23)
6 months CD + 1000 ppm BP 41 ± 10 (29) 11 1.20 ± 0.12 (29)
CD 195 ± 49 (32) 8 0.73 ± 0.11 (32)
12 months CD + 1000 ppm BP 158 ± 43 (33) 7 0.83 ± 0.15 (29)b
CD 425 ± 153 (109) 11 0.66 ± 0.16 (109)
18 months CD + 1000 ppm BP 364 ± 125 (Til) 9 0.69 ± 0.17 (89)
aData presented as mean ± SD (no. of fish used 1n determining mean represents pooling of
duplicate tanks).
n used 1n determining mean does not correspond to n used for computing mean body weight, I.e.
data is not biased by Including weights of livers with large tumors.
-------
Table 2
Histologlcal Incidences of liver tumors 1n trout sampled following
12 and 18 months of diet exposure to benzo(a)pyrene.
Sample time
12 months
Diet
CD
CD + 1000 ppm BP
Foci of basophll
0/32
4/33
- 1
1c cells0
0%
2%
Carcinomas
0/32 - 0%
1/33 - 3%
CD 0/109 - 0% 0/109 - 0%
18 months
CD + 1000 ppm BP 5/111 - 4.5% 22/111 - 20*
alncludes fish having only basophillc foci.
Data presented as no. of fish with at least one tumor/total no. of fish sampled.
-------
Table 3
Hlstological Incidences of tumors 1n trout sampled 6 months
after 12 monthly Intraperitoneal inoculations of benzo(a )pyrene (BP) In propylene glycol (PG)
Exp. group
PG controls
PG + BP
Tumor type
Carcinoma
Carcinoma
Basophilic foci
Flbrosarcoma
Papllloma
Organ
Liver
Liver
Liver
Liver
Swlmbladder
Incidence
1/27 - 4%
13/28 - 46%
1/28 - 4%
1/28 - 4%
1/28 - 4%
co
Data presented as no. of fish with at least one tumor/total no. of fish.
Includes fish having only basophillc foci.
-------
The 100 ppm MAMA dose was also highly toxic, resulting in high mortalities.
After hatching and swimup, 100 fry from the control and three highest tolerated
dose levels, (0.1, 1, 10 ppm for MAMA and 1, 10, 100 ppm for BDHC) were kept
and are being fed our semipurified control diet for the 12-18 month tumor
development period. A 20 fish sample will be taken 9 months after treatment
(September, 1982) to assess tumor development. If tumors are present the
experiment will be terminated at 12 months. If not, another 20 fish will be
killed at 12 months and a decision made at this time on whether to terminate
the experiment or continue for another 6 months. A few fish survived the 100
ppm MAMA treatment and were kept to determine tumor development. These fish
grew very poorly, average weight was only 3.3 g after 8 months, and mortalities
were frequent so the remaining 15 fish (PCB and control groups combined) were
killed. All fish, but one from the control group, had grossly observable liver
tumors. This information gives a preliminary indication that MAMA is
carcinogenic to trout. Dose response and the effects of PCB will be determined
at later samples.
The second objective in our proposal was to reenact the conditions which
apparently led to an inadvertent epizootic of neuroblastoma in juvenile coho
salmon reared in chlorinated-dechlorinated river water, the intent being to
reproduce this tumor with subsequent investigation of the causative factors.
The experiment was begun as proposed and involved exposure of coho salmon and
Shasta strain rainbow trout embryos and sac fry to chlorinated-dechlorinated
(sodium thiosulfate) McKenzie River water brought into our laboratory.
However, an unfortunate mechanical failure of our water recirculating pump
resulted in complete loss of the coho salmon fry. The trout, having reached
the swimup stage, had been moved elsewhere and were not in the closed system
when the failure occurred. Consequently, the trout are being monitored for
tumor development as proposed. The experiment using both coho salmon and
rainbow trout will be repeated during the second year of this cooperative
project.
Title: Teleost Carcinogen Assay Systems
Principal Investigator: B. J. Martin, University of Southern Mississippi
Cooperative Agreement Number: CR809347
Project Officer: John A. Couch
During the year we have conducted or have in progress over thirty (30)
whole organism experiments exposing £. variegatus to benzidine dihydrochloride
(BEN). These experiments represent our efforts to reproduce and more
accurately characterize the proliferative liver lesions previously induced by
long-term exposure of C. variegatus to BEN.
1) Benzidine Exposures. C_. variegatus. To date, we have histologically
examined the livers from 44 fish that have been exposed to 1 ppm BEN for at
least 200 days and the lesion was evident in 5 individuals. In an
experiment in which C. variegatus are being exposed to 5 ppm BEN, 4 fish
have been examined hTstologically and the liver lesion was evident in 2
individuals. One of these was sacrificed after 62 days of exposure, and
the other after 132 days. Seven fish have been examined from an experiment
involving exposure to BEN at 10 ppm and one individual had the lesion.
Also, some £. variegatus that survived 96-hour exposure at high
concentrations to BEN during LD-50 experiments were maintained in the
19
-------
laboratory and the liver lesions were evident in one of the 2 individuals
examined histologically. Since a number of the experiments from which
these data are derived are still in progress, we feel that we will be able
to accomplish our goal of characterizing the lesion and more accurately
determining its incidence and latency period by the end of the project
period.
2) Benzidine Exposures. £. variegatus Embryos. Embryos were exposed to BEN at
concentrations ranging from 5 to 500 ppm, and effects were observed at
concentrations of 50 ppm or higher. To date, ca. 600 embryos have been
exposed to BEN at 50 ppm or higher and only 15% developed normally beyond
the hatching stage. In our 300 control embryos, 77% developed normally
beyond the hatching stage and only 0.02% exhibited abnormalities;
therefore, the detrimental effects of BEN at these concentrations are
rather obvious.
Approximately 85% of the embryos exposed to BEN at 50 ppm or higher
exhibited anomalies. These anomalies, in order of frequency of occurrence,
are: 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, 7) Faint RBC
'pigmentation.
Efforts are in progress to examine these anomalies histologically, and
experiments are planned to study the effects of BEN on the early
post-hatching stage of development.
3) Aseptic Embryo Technique. Our main efforts have been to find a suitable
food regime and environmental conditions to maintain healthy £. variegatus
fry for an amount of time sufficient to study the effects of exposure to
carcinogens in a sterile environment. The foods we have tried were
ethylene oxide-treated Mono Lake Flakes, autoclaved Tetramin, autoclaved
Artemia eggs, and chlorox-treated Artemia eggs. We also conducted
experiments with reduced quantities of antibiotics and no antibiotics in an
effort to determine the effects of antibiotics on mortality.
Chlorox-treated Artemia eggs seem to have provided the best food regime
since more animals remained healthy when fed this diet. Also, fry
consistently lived longer when maintined in'an antibiotic-free sterile
environment; however, as one might expect, the preparations without
antibiotics are much more likely to become contaminated. Thus, it was
possible to consistently maintain healthy embryos on chlorox-treated
Artemia eggs in an antibiotic-free sterile environment for 60+ days.
Obvious problems associated with the use of Artemia are that they are
another living organism in the system and, since as yet we have no method
of excluding the non-viable Artemia from the system, the unhatched eggs and
uneaten Artemia accumulate in the preparation. This problem can be
somewhat ameliorated by underfeeding and more frequent water changes.
4) C. variegatus Cell Line Development. We have developed four new cell lines
Trom C. variegatus: A large cell fibroblastic line (through passage 8), a
20
-------
small cell fibroblastic line that forms cellular aggregates in culture
(through passage 10), a small cell fibroblastic line that does not
aggregate in culture (through passage 11), and a striated muscle cell line
(through passage 12). The striated muscle cell line is particularly
interesting since, to our knowledge, it is the first striated muscle cell
line to be developed from fish tissue.
We plan to use all four of these cell lines in toxicity and cardnogenicity
studies during the coming project year.
5) Primary Hepatocyte Cell Culture Technique. Because of our initial success
in developing primary hepatocyte cell cultures from Fundulus. we felt the
development of such cultures from £. varlegatus would not be difficult.
However, after a year of effort in this regard with very little success, we
have decided to work again with Fundulus. Our recent emphasis has been the
determination of media and environment conditions that will provide healthy
and morphologically differentiated cells. Although the cells do not appear
completely differentiated with respect to morphology, we are conducting
tests to determine their state of biochemical differentiation. If these
cells prove to be sufficiently differentiated to be considered "functional
hepatocytes," we will begin to use them in carcinogen assays.
6) Immunological Studies. Agarose and polyacrylamide serum electrophoresis
has disclosed differences in the banding patterns of BEN-exposed and
non-exposed Cyprinodon varlegatus. Densitometer scans of the agarose
electrophoresis preparations indicate that both the number and size of the
peaks from BEN-exposed fish differ from non-exposed individuals. PAGE
(polyacrylamide gel electrophoresis) provides a much better resolution of
the serum differences, and they are clearly visable with the naked eye.
When the Bio-Rad silver stain is used, we can detect significant
differences by using a 1:50 dilution of only lug of serum; thus, allowing
the conservation of serum from each individual fish for additional tests.
By immunoelectrophoresis (Graber-Wil1iams), we are able to demonstrate
differences in precipitation arcs generated from exposed and non-exposed
fish. This technique is not quantitative, but it demonstrates that our
antiserum is sufficiently potent for use in crossed and tandem crossed
rocket procedures.
A competitive enzyme immunoassay has been developed and employed
successfully- with non-fish antibody. We are presently miniaturizing the
procedure so that it can be used with the yl quantities of blood available
from C_. variegatus.
A bacteriophage neutralization procedure has been adapted for use in the
study of the ability of £. variegatus to generate neutralizing factors.
Results show a consistently higher level of neutralization in BEN-exposed
fish (fish are immunized with MS-2 phage).
21
-------
Title: Causes of Paplllomas on Fish Exposed to Chlorinated Sewage Effluent
Principal Investigator:John M. Grizzle
Cooperative Agreement Number: CR809336010
Project Officer: William Davis
Laboratory and field experiments are in progress to determine the cause of
oral papillomas on black bullheads in the final oxidation pond of the Tuskegee,
Alabama, sewage treatment plant. Wild, adult black bullheads in the final
oxidation pond had a 73% prevalence of oral papillomas between December 1979
and July 1980. A gill-net sample of black bullheads from this pond on 28 May
1982 had an oral papillona prevalence of 57%. The occurrence of tumors in this
recent sample indicated a continuation of the original, tumor inducing
conditions.
Exposure of fish to water in the final oxidation pond. Black bullheads
(Ictalurus melas), brown bullheads (Ictalurus nebulosus), yellow bullheads
(Ictalurus natal is), and channel catfish (Ictalurus punctatus) were placed into
cages in the final oxidation pond of the Tuskegee, Alabama, sewage treatment
plant. Control fish were kept in cages in an earthen pond at the Auburn
University Agricultural Experiment Station. The approximately 1-nr cages
were allowed to rest on the pond bottom (sinking cages) or suspended off of the
bottom by floats (floating cages). Cages were placed at three locations in the
final oxidation pond: near the inlet (inlet A), approximately 60 meters from
the inlet (inlet B), or near the pond outlet (outlet). Most cages were stocked
with 50 fish. Table 1 summarized all cage exposures including the percentage
of fish with grossly visible, oral mucosa hyperplasis or papilloma-like
lesions.
The oral lesions on fish confined to cages in the final oxidation pond
were in the same location occupied by oral papillomas in wild black bullheads
from this pond. The hyperplasic lesions were most prevalent during the spring
and regressed during the summer. Black bullheads had a higher prevalence of
oral musoca hyperplasia than the other species. Lesion prevalence was higher
in black bullheads stocked during FY 81 than in those stocked the following
year after similar lengths of exposure. Incidence of hyperplastic lesions was
also higher in black bullheads near the outlet than in those near the inlet,
but low fish survival near the inlet could have influenced this result. No
difference was found between fish in floating and sinking cages.
During their second year of exposure to the final oxidation pond, some of
the black bullheads developed oral lesions that grossly resembled papillomas.
These lesions were in the same locations in the mouth fornix as the
hyperplastic lesions developing during the first year of exposure. The
appearance of the lesions that developed during the second year is described in
a later section of this report.
Lesions on caged fish. Lesions in both fornices of the mouth were examined
from a 175-mm total length black bullhead confined for 537 days to a sinking
cage near the outlet of the final oxidation pond. Grossly, these lesions
appeared similar to papillomas on wild black bullheads in this pond and to the
lesions on other caged black bullheads with a similar length of exposure. The
surface dimensions of the lesions were approximately 8 mm by 5 mm, and each
22
-------
Table 1. Sumrary of exposures of caged fish to the Tuskegee, Alabama, final oxidation pond.
Species
Black bullheads
Yellow bullheads
Brown bullheads
Channel catfish
Stocking Dates
21 Oct 80
23 Feb 81
29 Oct 81
9 Apr 82
12 May 81
29 Oct 81
16 Jan 81
29 Oct 81
Days of exposure
(as of 14 Jun 82)
603
476
226
66
398
228
514
231
Maximum percentage
with oral lesions
95
50
18
0
0
0
25
0
Maximum percentage with
paplllcraa-like lesions
8U
50
0
0
0
0
U
0
Maxinun percent
of control fish
with oral lesions
4
4
0
0
14
0
8.3
0
CM
-------
lesion was raised approximately 1.5 mm above the surrounding oral mucosa. In
paraffin sections of the lesion, the maximum thickness of the connective tissue
between the epidermis and mucosa was 1.5 mm compared to a normal thickness of
approximately 0.2 mm. The maximum thickness of the mucosal portion of the
sectioned lesion was 1.2 mm compared to a normal thickness of approximately 0.2
mm. The connective tissue component of the lesion extended from the hyperemic
submucosa into the epithelial portion of the lesion, but the connective and
epithelial tissues was less intermingled than in larger oral papillomas of wild
bullheads. These lesions were different than the hyperplasic lesions appearing
soon after exposure because these papilloma-like lesions were larger, there was
more folding of the lesion surface, and the connective tissue was more
extensive and intermingled with the epithelial tissue than in the hyperplastic
lesions.
Ultrastructure of papillomas in black bullheads. The oral papilloma of wild
black bullheads from the final oxidation pond has epithelial cells resembling
normal oral mucosa cells and a central connective tissue column that was
continuous with the submucosa. Near the tumor surface there were mucous cells,
and plasma membranes of the epithelial cells adhered closely with those of
adjacent cells. Desmosomes occurred less frequently between the cells than in
normal mucosa. Mitochondria, endoplasmic reticulum, and ribosomes were
concentrated in the peripheral cytoplasm of the epithlial cells. These surface
cells often contained glycogen particles among the tonofilaments. The mid to
near basal portion of the papilloma consisted of stellate epithelial cells
having more tonofilaments, fewer organelles in the cytoplasm, and more
intercellular space than epithelial cells near the surface. Desmosomes
occurred more frequently between the cells in the basal area than near the
surface. Alarm substances cells'occurred in the mid portion of the epithelium
and were surrounded by stellate cells. Numerous lymphocytes were present in
the basal region of the tumor. The basal epithelial cells contained numerous
mitochondria, endoplasmic reticulum, and ribosomes in the cytoplasm. Plasma
membrances adhered to the adjacent cells by desmosomes. Intercellular space
occurred less frequently in the basal region than in the middle portion of the
tumor.
Mutagenicity of the final oxidation pond water. Extracts of the pond water
were tested for mutagenicity with the Ames test (Ames et al., 1975, Mutat. Res.
31:347). An acid fraction was prepared by acidifying the water to pH 1.0,
extracting three times with 200 ml of 25% ether and 75% hexane per liter of
water, and then distilling to remove the bulk of the solvents. The last 20 ml
were evaporated under a stream of nitrogen. A basic fraction was prepared in
the same manner except that the water was adjusted to pH 12-13 before
extraction. Both fractions were tested with and without S-9 (Aroclor-induced
rat liver enzymes). Benzo(a)pyrene was used as a positive control mutagen and
was tested with and without S-9.
The acid fraction was mutagenic to Salmonella typhimurium tester strains
TA98 and TA100 if S-9 was present, and there was a positive dose response (Fig.
1 and 2). The mutagenicity of the acid fraction was higher on some dates than
on others (Fig.3) and may be seasonal. The mutagenicity of the water_at the
inlet and outlet was compared on two dates: there was no difference in
February 1982, but mutagenicity was higher at the inlet in March 1982 (Fig.
24
-------
JOO -
4CO i
§200
100 ••
AC:OIC -.3-9
AC:OIC
LO LS to
to
Tc
10
Figure 1. Response of Salmonella typhimurium strain TA 98 in Che Ames test to
an acid organic solvent extract of the final oxidation pond water.
The water was collected at the pond outlet on 15 July 1981.
JOO •-
OJ
Figure 2. Response of Salmonella
strain TA 100 in the Ames t«st to
an acid organic solvent extract of the final oxidation pond water.
The water was collected at the pond outlet on 29 June 1981. The
results for cwo trials are given on this graph.
25
-------
JAN a MX; st SOT a ocr at NOV & rta at MAA az
fcesponse of Salmooella typhimurium strain TA 100 «„ PK A
« add organic solvent extract of the finlt ilY , ^S CeSt t0
The number of revertanc coloniL f oxidation pond water.
colonies on aedLawith the Stract STSl,- ^ """*•* °f
medium vith extract onl7 S 9 aiQUS Che
S.8+
"CSNTSO.
ua-f.«
i occ a i ra « i
OAft
k. Glucurososyl craasferaae accivlcy la liver caicrosomes of chapel
cacfish confined to cages ta the final oxidation pond dr a control
pond.
26
-------
3). The number of revertant colonies on plates with acid fraction but without
S-9 was similar to the number of colonies on negative control plates (DMSO or
S-9 only); therefore, the mutagenicity of the samples in Fig. 3 was expressed
as the difference in the number of revertant colonies on plates with and
without S-9. The basic fraction was not mutagenic when tested with Salmonella
typhimurium strains TA98 and TA100.
Hepatic enzyme induction. UDP-glucuronosyl transferase and sulfotransferase
activities in livers of black bullheads, brown bullheads, and channel catfish
from cages in the final oxidation pond were measured and compared to control
fish. Dr. D. R. Strength and colleagues determined the enzyme activity of
these fish as part of a separate EPA cooperative agreement. Glucuronosyl
transferase activity in exposed fish was generally higher than in control fish
with channel catfish having the greatest induction response (Figs. 4-6). The
difference between sulfotransferase activity in exposed and control fish was
not consistent. The induction of glucuronosyl transferase in fish confined to
the final oxidation pond indicated exposure to a toxicant. Similar induction
occurs in rats and fish given oral or intraperitoneal doses of carcinogens.
Analysis of water from the sewage pond. Between September 1981 and May 1982,
water leaving the final oxidation pond had 0.1 to 1.0 mg/1 ammonia-nitrogen
(average 0.55 mg/1) and a pH between 5.1 and 7.0. During this same period,
total residual chlorine of water leaving the chlorine contact chamber before
entering the final oxidation pond was 0.2 to 1.0 mg/1 (average 0.53 mg/1). The
residual chlorine concentration in the effluent has been lower during the
experimental studies in this pond than during 1979 when the papillomas were
first found in the wild black bullheads.
Water samples from the inlet to the final oxidation pond were collected on
22 January 1982 and 3 March 1982. The Environmental Health Administration
Laboratory, Montgomery, Alabama, examined the samples with gas chromatography.
Total volatiles, herbicides, pesticides, and PCB's were not detectable.
Transmission of papillpmas by cell-free tumor homogenate. Two attempts were
made to transmit papillomas by injecting homogenized, filtered papillomas into
the mouth fornix of healthy black bullheads. The first group of fish was
adults and was observed for 14 months after injection. The second attempt was
with one-year-old fish that were observed for 11 months after injection. No
signs of papillomas or hyperplastic lesions were found in any of the injected
fish.
Embryo Exposures. Brown bullhead embryos were exposed to aflatoxin B^ (0.5
or 1.0 mg/i) or a concentrate of the final oxidation pond water. Controls were
exposed to uncontaminated pond water or to the solvents used in the chemical
exposures (dimethylsulfoxide and ethanol). Groups of 200 embryos were exposed
to each treatment for one hour on the fourth day after fertilization. Embryos
were kept in a closed, recirculating system and after hatching, were
transferred to 3-meter diameter plastic pools. Fish were sampled after 3 and 6
months, and all remaining fish were fixed after 8 months. These fish are
currently being examined. No evidence of tumor induction has been found.
27
-------
3). The number of revertant colonies on plates with acid fraction but without
S-9 was similar to the number of colonies on negative control plates (DMSO or
S-9 only); therefore, the mutagenicity of the samples in Fig. 3 was expressed
as the difference in the number of revertant colonies on plates with and
without S-9. The basic fraction was not mutagenic when tested with Salmonella
typhimurium strains TA98 and TA100.
Hepatic enzyme induction. UDP-glucuronsyl transferase and sulfotransferase
activities in livers of black bullheads, brown bullheads, and channel catfish
from cages in the final oxidation pond were measured and compared to control
fish. Or. D. R. Strength and colleagues determined the enzyme activity of
these fish as part of a separate EPA cooperative agreement. Glucuronosyl
transferase activity in exposed fish was generally higher than in control fish
with channel catfish having the greatest induction response (Fig. 4-6). The
difference between sulfotransferase activity in exposed and control fish was
not consistent. The induction of glucuronosyl transferase in fish confined to
the final oxidation pond indicated exposure to a toxicant. Similar induction
occurs in rats and fish given oral or intraperitoneal doses of carcinogens.
Analysis of water from the sewage pond. Between September 1981 and May 1982,
water leaving the final oxidation pond had 0.1 to 1.0 mg/1 ammonia-nitrogen
(average 0.55 mg/1) and a pH between 5.1 and 7.0. During this same period,
total residual chlorine of water leaving the chlorine contact chamber before
entering the final oxidation pond was 0.2 to 1.0 mg/1 (average 0.53 mg/1). The
residual chlorine concentration in the effluent has been lower during the
experimental studies in this pond than during 1979 when the papillomas were
first found in the wild black bullheads.
Water samples from the inlet to the final oxidation pond were collected on
22 January 1982 and 3 March 1982. The Environmental Health Administration
Laboratory, Montgomery, Alabama, examined the samples with gas chromatography.
Total volatiles, herbicides, pesticides, and PCB's were not detectable.
Transmission of papilloms by cell-free tumor homogenate. Two attempts were
made to transmit papillomas by injecting homogenized, filtered papillomas into
the mouth fornix of healthy black bullheads. The first group of fish was
adults and was observed for 14 months after injection. The second attempt was
with one-year-old fish that were observed for 11 months after injection. No
signs of papillomas or hyperplastic lesions were found in any of the injected
fish.
Embryo Exposures. Brown bullhead embryos were exposed to aflatoxin B. (0.5
or 1.0 mg/1) or a concentrate of the final oxidation pond water. Controls were
exposed to uncontaminated pond water or to the solvents used in the chemical
exposures (dimethylsulfoxide and ethanol). Groups of 200 embryos were exposed
to each treatment for one hour on the fourth day after fertilization. Embryos
were kept in a closed, recirculating system and after hatching, were
transferred to 3-meter diameter, plastic pools. Fish were sampled after 3 and
6 months and all remaining fish were fixed after 8 months. These fish are
currently being examined. No evidence of tumor induction has been found.
28
-------
Laboratory exposures of black bullheads to sediment extracts. One-year-old
black bullheads were acclimated to static-water, 40-liter aquaria, one fish per
aquarium. Sediment from the final oxidation pond was collected near the inlet
on 14 June 1982. Samples were extracted for organic compounds with methylene
chloride and acetone according to the EPA protocol for extraction of priority
pollutants from sediments.
Two types of skin exposure to the sediment extract will be used:
subcutaneous injection and topical application. The sediment extract was
injected on 30 July 1982, and the fish will be observed for at least one year.
The topical applications will begin during August 1982.
Title: Perylene as a Possible Environmental Carcinogen: Hydrocarbon
Metabolism and Mutagenesis in Ambystoma tigrinum
Principal Investigator: Robert S. Anderson and Francis L. Rose
Coopertive Agreement Number: CR807740
Project Officer: John A. Couch
The unusually high prevalence of various forms of dermal neoplasia in a
population of tiger salamanders (A. tigrinum) inhabiting a sewage lagoon whose
water and sediment are unusually rich in perylene has prompted this study of
the possible role that this polycyclic aromatic hydrocarbon (PAH) might have in
the etiology of these lesions. The work is divided into two portions: 1) the
histopathological effects of perylene (P) as a carcinogen or cocarcinogen on A.
tigrinum. under the direction of Dr. F. Rose and 2) the metabolic activation
and/or detoxification of P, and a related environmental carcinogen
benzo(a)pyrene (BaP), by tissue enzymes of £. tigrinum. under the direction of
Dr. R. Anderson. The progress of this effort through the first two years 1s
summarized in this report.
Methodological developments. A radioisotopic method used to measure BaP
metabolism in mammals was adapted for use with A. tigrinum to quantify BaP and
P metabolism. Basically, the parent compounds are converted to metabolites and
conjugates which can be extracted based on their solubility in water and/or
NaOH. This technique is useful to obtain an indication of the total rate of
metabolism of these substrates. Once the in vitro conditions of. reaction had
been optimized this reaction procedure was used to obtain metabolite extracts
in ethyl acetate which were used for HPLC separation of metabolites on a
Perkin-Elmer HC ODS/SilX reverse-phase column. Metabolites of H-P or
14C-BaP were eluted in a 60-80% MeOH-H20 gradient, the eluted samples
were collected for radioactivity determinations and UV-absorbance was monitored
continuously. A complete set of BaP authentic standards were used to identify
by coelution all of the BaP metabolites; unfortunately a set of P metabolite
references is not currently available.
Hepatic metabolism of 14C-BaP and 3H-P in normal and induced A.
tigrinum. Liver homogenates from tiger salamanders mediate the in vitro
metabolism of both BaP and P. We studied the effect of two known aryT
hydrocarbon hydroxylase (AHH) inducers: aroclor 1254 (PCB) and
methylcholanthrene (MC), and P on the metabolism of BaP and P. Both the
metabolites in the aqueous phase (conjugates and macromolecule-bound species)
and the metabolites in the NaOH phase (certain dihydrodiols and the phenolic
derivatives) were quantified; their sum allowed us to estimate the total
nmoles/ml liver protein/30 min produced under the various conditions.
29
-------
Induction procedures used were those found effective for standard laboratory
rodents: 25 mg/kg MC ip, 48 hr; 500 mg/kg PCB ip, 96 hr; 60 mg/kg P ip, 48 hr
(this dosage was based on that commonly used for its isomer BaP); olive oil was
the vehicle in all cases.
Table 1 summarizes the data on ^C-BaP metabolism. Uninduced animals
produced about o.2 total 14C-8aP metabolites/mg protein/30 min; the
alkali-soluble metabolites were produced in quantities more than twice that of
aqueous phase metabolites. MC was the only chemical tried that produced
induction, this induction would have been highly statistically significant if
the variation in the induced data had been somewhat reduced by having a larger
sample size. Interestingly in the MC-induced animals aqueous phase > NaOH
phase metabolites, an effect also seen in the PCB-treated salamanders, although
PCB injection at this level did not produce an over all AHH induction.
Perylene injection had no significant effect on metabolite partitioning and was
not an effective AHH inducer. 14C-BaP metabolism by Mutazyme™ (a
commercial preparation of aroclor induced rat hepatic microsomes designed for
the Ames bacterial mutagenesis assay) was measured for reasons of comparison.
Its activity per mg protein was 100-fold greater than the uninduced salamander
with aqueous phase = NaOH phase metabolites.
Table 1.
HEPATIC 14C-BaP METABOLISM*
Ambystoma
Inducer
NaOH
Metabolites
Aqueous phase
Total
Rat
None
Aroclor
MC
Perylene
Aroclor
0.131 +_ 0.148
0.055 + 0.052
0.228 +_ 0.230
0.118 + 0.117
10.104 + 1.098
0.059 + 0.045
0.089 + 0.038
0.318 + 0.339
0.056 + 0.016
8.044 + 1.925
0.190 + 0.130 (6)
0.144 i 0.072 (6)
0.546 + 0.562 (4)
(P<0.2)
0.174 + 0.116 (3)
18.148 i 3.021 (3)
*Nmoles/mg protein/30 min: X _+ SO (n).
30
-------
Table 2.
Rat
Aroclor
HEPATIC 3H-PERYLENE METABOLISM*
Ambystoma
Inducer
None
Aroclor
MC
Perylene
Metabolites
NaOH
0.
0.
0.
0.
005
192
144
000
± °-
± °-
± °-
+ 0.
010
289
184
000
Aqueous phase
1.
1.
1.
2.
302
305
365
477
+ 0.528
i 0.873
+ 0.527
+ 0.288
1
1
1
2
Total
.307
.498
.509
.477
1 °-
± °-
t °-
+ 0.
529 (6)
733 (5)
641 (6)
288 (3)
16.425 + 1.280
(p<0.01)
56.169 i 19.014 72.593 + 18.483 (3)
Nmoles/mg protein/30 min; X jf SO (n).
When assays using the same protocol as those for ^C-BaP metabolism
were run using 3H-P as substrate an estimate of perylene metabolism was
obtained (Table 2). In both induced and uninduced salamanders the majority of
P metabolites were extracted in the aqueous phase. Perylene was more
extensively metabolized than BaP, -9-fold more for uninduced A. tigrinum and
* 4-fold more for PCB-induced rat. Although neither PCB nor MC treatment caused
significant changes in overall 3H-P metabolism, both treatments produced a
significant increase in the production of NaOH-soluble metabolites. Perylene,
while not a BAE-OHase inducer (Table 1), did significantly increase the rate of
metabolims of H-P in hepatic homogenates. This inductive event was
accomplished by a marked increase in the production of aqueous-phase
metabolites. As was the case for BaP-OHase, the specific activity of _A.
tigrinum P-metabolizing enzymes was much less (-70-fold) than those of the
rat.
Effects of inducer on 14C-BaP metabolite profiles by HPLC. In Table 3
the specific BaP metabolites produced by uninduced and induced salamanders are
listed. Metabolites marked with an "X" were produced during the 30-min j_n
vitro incubation, the number of "Xs" for a given metabolite indicates the
numbers of animals in a given group that produced that metabolite. Metabolites
that were not only produced by the majority of animals within a group, but were
also quantitatively the major metabolite(s) of their class (diols, quinones,
phenols) for that group of animls, are marked with an arrow. Basal animals
produced many readily identified BaP dihydrodiols, quinones, and phenolic
derivatives. The major diol is the 7,8-diol, which can serve as a precursor of
the ultimate carcinogenic BaP metabolite, the 9,10-epoxide of the
7,8-dihydrodiol. In uninduced A. tigrinum the major monohydroxylated
metabolite appears to be 7-OH-BaP, in contrast to mammals which produce mainly
3-OH and 9-OH BaP. In PCB- and MC-induced salamanders 7,8-diol is also a major
31
-------
14C-BaP METABOLITES PRODUCED BY AMBYSTOMA LIVER: RESPONSE TO AHM INDUCERS
BASAL (4)
AROCLOR 1254 (3)
MC (3)
PERYLENE
60 mg/kg (2)
PERYLENE
20 mg/kg (1)
X
X
X
X
X
X
X
x
X
X
X
X
X
X
X
X
X
X
X
X
X
t
X
X
X
X
x
X
X
X
X X
X X
X X
X X
X
X
X X
X X
X
x
x
X
V
X
X
X X
XX X XX
X
XX X
X
XXX X
X
X XX XX
X X
X
X
x
X
X
x
X
X
X
X
X
X
x
X
X
x
9,10 4,5 7,8 4,5 1,6 3,6 6,12 (11 or 12) (4 or 5) 6 9 (8 or 10) 2 7 1 3
Dihydrodiols Quinones Phenolic derivatives
BaP METABOLITES IN ORDER OF ELUTION
-------
diol, as is 9,10-diol in the case of MC induction. The pattern of 3aP
metabolism is shifted toward 3-OH-BaP production by PCS and MC, and 1-OH-BaP in
MC-induced animals. Attempts to induce with 20 mg P/kg produced no significant
alteration from the normal metabolite profile, but treatment with 60 mg P/kg
caused an apparent shift away from dihydrodiol production.
Table 4. EFFECTS OF AHH INDUCERS ON AMBYSTOMA 14C-BaP METABOLITE PROFILES (HPLC)
Basal
(4)
Dihydrodiols
Qui nones
HO-BaPs
54
14
30
.8
.8
.4
i 13.5
+ 14.2
+ 10.9
Aroclor 1254
(3)
52
11
36
.3
.3
.4
± 6-
± 6-
+ 4.
0
4
7
40
11
48
MC
(3)
.3 •»•
(p<0.
.7 +
.0 +
Perylene
60 mg/kg (2) 20 mg/k<
4.0 0
2)
5.0 20.5 +_ 10.6
3.6 79.5 + 10.6
31.0
12.0
57.0
(p<0.025)
X% total C-BaP metabolites in each category _+ SD (n), peak identity confirmed by
coelution with authentic metabolites standards.
Another way to look at the effect of putative AHH inducers on C
metabolite profiles is presented- in Table 4. In this table the actual amount
of metabolites produced from each major class is presented as the % of total
metabolites in each category. For example, in basal animals approximately 55%
of the total BaP metabolites produced are dihydrodiol s, 15% are qui nones, and
30% are phenolic derivatives. Of the three inducers tested, only MC produced
significant overall BP-Phase induction (Table 1); however, both MC and P cause
alterations in BaP metabolite profiles (Table 4). Aroclor 1254 had no effect
on relative composition of the metabolite categories. MC-treatment tended to
cause a shift from diol production to phenol production. This effect was much
more pronounced in the case of 60 mg P/kg, where virtually no dihydrodiols were
generated and -80% of the metabolites were phenols. The distribution of
metabolites started to revert to normalcy when the dose was reduced to 20 mg
P/kg. It would appear that P treatment might have a protective effect in the
case of subsequent BaP exposure because it shifts £. tigrinum metabolism away
from the production of potentially carcinogenic epoxides and diol -epoxides
toward generation of comparatively inactive phenol derivatives. AHH induction
in higher animals also is frequently reported to be protective.
Mutagenicity of BaP, P, and aromatic amines in the Ames bacterial system.
We have been able to identify and quantify numerous BaP metabolites produced by
A. tigrinum enzymes, and quantify P metabolism in the same species (although
The exact identity of the P metabolites is unknown). However, the biological
activity of the metabolites produced by salamanders has not been determined
using conventional testing systems. Therefore, we tested the ability of A_.
tigrinum hepatic enzymes to activate BaP, P, and some carcinogenic aromatic
33
-------
amines using the Ames Salmonella typhimurium tester strains. The results are
given in Table 5.
Microsomal enzyme preparations from the livers of Aroclor-induced
salamanders mediated the production of mutagenic aromatic amine metabolites.
These preparations were generally less active, on the basis of protein
concentration, than comparable rat S9 preparations, with the exception of
2-aminofluorene and 4-aminostilbene. Therefore, higher substrate
concentrations were usually required to get significant mutagenesis with
Ambystoma S9; in no case were the concentrations used highly toxic or directly
mutagenic to the bacterial tester strains. All data presented in Table 5 have
been corrected for this background, as well as for spontaneous revertants.
After the appropriate corrections, it was seen that salamander mixed-function
oxygenase (MFO) activated 2-aminoanthracene, 2-aminofluorene,
2-acetylaminofluorene, 4-ami no-trans-sti1bene, and mono- and diacetylbenzidine;
however, benzidine and 4-aminobiphenyl were poorly mutagenic in this system.
As was expected, the frameshift mutagen detector strains TA 1538 and TA 98 (TA
1538/pK M101) were most sensitive to the activated aromatic amines. Strain TA
100 (TA 1535/pK M101) was also useful because it detects frameshift, as well as
base-pair substitution mutagens.
The generation of mutagenic aromatic amine metabolites by Ambystoma
microsomes was totally inhibited by prior heating of the S9 to 56° C for 30
min. The reaction did not proceed in the absence of the NADPH-generating
system usually included in the S9 mix.
Rat microsomal enzyme induction with phenobarbital produced a marked
increase in the mutagenesis by benzidine, N-acetylbenzidine, and
N,N-d1acetylbenzidine. However, phenobarbital was not a more effective inducer
than Aroclor in the activation of these substrates, or other aromatic amines,
by Ambystoma enzymes.
In this study there was no evidence that either BaP or perylene were
mutagenic after reaction with Ambystoma liver microsomes. Strains TA 98 and TA
100 were mutated by BaP with Mutazyme^, as had already been reported.
Another frameshift detector, TA 1537, was most useful in perylene activation
studies using rat S9. Although data from only 1 concentration of BaP or
perylene + Ambystoma S9 are given in Table 1, concentrations ranging from 5-250
ug/plate were tried, without any sign of mutagenesis. BaP was neither toxic
nor directly mutagenic for TA 98 or TA 1537 over this range; it was slightly
toxic over 100 ug/plate for TA 100. Ambystoma enzymes produce no BaP
activation as measured by TA 98, TA 100 or TA 1537. Likewise perylene
concentrations of 5-250 ug/plate were tested against TA 1537, the only tester
strain to give a good response to rat S9-activated perylene. No perylene
concentration tested was directly toxic or mutagenic; reaction with Ambystoma
microsomes had no effect.
In summary, a number of carcinogenic aromatic amines when activated by
liver microsomes from a salamander, Ambystoma tigrinum, are mutagenic for
Salmonella tester strains sensitive to frameshift mutagens. However, 2
polyeyelie aromatic hydrocarbons (PAH) (BaP and perylene) that are rendered
mutagenic by mammalian microsomes are not activated by Ambystoma mixed-function
34
-------
TABLE 5
COMPARATIVE ACTIVATION OF CARCINOGENS
Compounds*
Aromatic Amines
Mg
S9b
his' revertants/platec
TA 1538
TA 98
Polycyclic aromatic
hydrocarbons
Mg
S9b
TA 1537
TA98
TA100
2-Aminoanthracene (PC)
2-Aminofluorene (PC)
2-Acetylaminofluorene (PC)
4-Aminobiphenyl (PC)
4-Amino-rron5-stilbene (PC)
Benzidine (PC)
A/-Acetylbenzidine (NT)
,
NJV'-Diac«tylb«nzidine (NT)
25
5
10
10
100
5
500
100
50
50
500
500
500
500
200
10
10
200
20 1000 revertants/plate).
35
-------
xidases. These results tend to support the observation that amphibians are
uite resistant to PAH carcinogenesis and suggest that aromatic amines may be
:ore appropriate model carcinogens; possibly because amphibian mixed-function
xidases carry out N-oxidation more efficiently than C-oxidation.
From the point of view of environmental carcinogenesis in relation to
jiibystoma, it is of interest that the recognized carcinogen benzidine can be
ojjndln water supplies. In addition to benzidine and its salts,
.-aminobiphenyl and 2-acetylaminofluorene are included in a list of
:arcinogenic substances for which regulatory standards were promulgated by the
iccupational Safety and Health Administration.
listopathological Response of Ambystoma to Perylene:
Determination of maximum tolerated dose. Three groups of 10 neotenes each
/ere injected ip with 10 mg, 50 mg, or 100 mg P/0.5 olive oil. No lethality or
pther harmful effects noted after 60 days. Autopsy showed much residual P in
leritoneal cavity, especially in higher dose groups; P frequently triggered
jranulomatous reactions.
Injection of perylene. A control group of 19 neotenes each received 2 ml
)live oil ip; 19 experimental were injected ip with 100 mg P/2 ml olive oil.
hnimals in both groups were maintained for about 3 months. Only 2 of the
:ontrols died during this period; one died almost immediately (probably of
:auses unrelated to the study), the other died at about 2 months. None of the
:ontrol animals had tumors of any kind. Thirteen of the experimental group
iied during the course of the study. Of 11 perylene-treated animals sent to
)r. John Harshbarger for study, two had epidermal papillomas and one had an
jnusual hepatocellular carcinoma (differential diagnosis: cholangio
:arc1noma). This is the first unequivocal liver cell neoplasm reported in
salamanders. A commonly observed reactive lesion characteristic of the
experimental group was diagnosed as a lipid granuloma arising in response to
the olive oil vehicle, although they were uncommon in control animals which
Deceived olive oil alone. These lesions were composed of mixed populations of
:ells in which foamy histiocytes and collagen-producing fibrocytes
predominated. These granulomas were often found on the surface of the lungs,
liver, and kidneys.
Another major study of the effects of P, and P and BaP in combination, is
:urrently underway; results are not yet available because of the length of time
required in the carcinogen-testing protocols. Attempts to transplant cutaneous
lelanomas are also in progress.
36
-------
Title: Carcinogens and Neoplasia in Indigenous Populations of Aquatic
Organisms
Principal Investigator: Michael C. Mix
Cooperative Agreement Number: CR808000-01-0
Project Officer: John A. Couch
Major research activities during the past year were directed toward: (1)
measuring baseline levels of arsenic (As), cadmium (Cd) and nickel (Ni) in
indigenous populations of bivalve molluscs; (2) developing methods for
measuring trace levels of polynuclear aromatic hydrocarbons (PNAH) in seawater;
(3) developing a crustacean (barnacle) bioassay system that could be utilized
for testing environmental levels of carcinogens and/or mutagens; (4)
determining whether or not viruses are associated with the neoplastic disorders
of Mytilus edulis from Yaquina Bay; and (5) evaluating data, gathered during
the past five years, on the proliferative disorders in M. edulis. Results from
studies in each of these areas is summarized below.
Baseline Levels _of Inorganic Carcinogens (As, Cd, NI) ijn Shellfish
Freshwater mussels (Margaritifera margaritifera) and bay mussels (Mytilus
edulis) were examined monthly or bimonthly during a one-year period and tissue
concentrations of As, Cd, and Ni were determined by either flame atomic
absorption or neutron activation analysis. For M_. margaritifera. the
concentration ranges of metal (yg/g, dry weight) were: Cd, 0.9-6.2; and Ni,
1.6-19.4. These levels are similar to those reported in organisms from
non-industrial streams and less than, or in the lower range of, those from
industrialized rivers. For M_. edulis, the concentrations of As ranged from
9-15 ug/g (dry weight), Ni, 1.0-6.8 yg/g and Cd, 7.0-12.5 ug/g. Both species
were judged to be excellent monitors for evaluating levels of these important
metals in freshwater (M_. marqaritifera) and marine (M_. edulis) environments.
Measurement _of PNAH in Seawater
The U.S. EPA's Method 610 (U.S. EPA 1979) Polynuclear Aromatic
Hydrocarbons - Method 610. Federal Register, 44(223), (69514-69517) was
modified for detecting and quantifying phenantFrene, fluoranthene, pyrene,
benzo(c)phenanthrene, tnphenylene, benzo(a)anthracene, chrysene,
benzo(b)fluoranthene, benzo(k)fluoranthene, dibenz(a,c)anthracene,
benzo(a)pyrene, dibenz(a,h)anthracene, benzo(g,h,i)perylene,
indeno(l,2,3-c,d)pyrene and coronene in seawater. This method is applicable to
the determination of PNAH in water and was designed to be used to meet the
monitoring requirements of the National Pollutant Discharge Elimination
System.
Procedures described in EPA Method 610 and an alternative extraction
process using C\Q mini-columns (Sep-Paks; Waters Assoc. Inc.) were
investigated for application. Further evaluations of various analytical
components are currently being completed. These studies were undertaken to
evaluate the use of Sep-Paks for extracting PNAH from water since they seemed
to offer certain procedural advantages. Experiments were designed to determine
percent recoveries of individual PNAH, reproducibility and an optimum flow
rate. A system to pump water through the Sep-Paks was also developed, tests
and evaluated for adsorptive loss of PNAH.
37
-------
Development of _a Crustacean Bioassay System
In this study, an in vitro system for culturing the eggs and larvae of the
gooseneck barnacle, PolTTcipes polymerus. was developed and tested in two
bioassay experiments using the phthalate ester plasticizer dibutyl phthalate.
The culture system employed aereated culture chambers composed of chemically
inert substances and designed to be inexpensive, convenient to use, and to
minimize external contamination of the cultures.
Pollicipes eggs were grown in synthetic seawater at 14° +_ 1° C under
lighting conditions of alternating darkness and very low light. Water was
changed every 24 hours. Antibiotics were not necessary if prescribed methods
were employed. An overall mean hatching success index of 75.5 was achieved
with egg masses not exposed to dibutyl phthalate (DBF). Experiments indicated
that allowing 86-96 hours after the first hatch before ending the experiment
resulted in sufficient stage II larvae to be able to draw conclusions regarding
molting success.
One of the DBP experiments showed there was a significant decrease in
molting success in larvae from eggs exposed to 1000 ppb DBP from a week prior
to first hatching to the end of the experiment. Several phthalate esters were
found to be persistent contaminants in synthetic sea salts.
The results of these studies indicate that the £_. polymerus assay system
has great potential for testing environmental carcinogens and mutagens.
Viral Studies
Because of a recent report (Oprandy, et jil_. 1981. J_. Invertebr. Pathol..
38:45-51) which indicated that henric proliferative disorders in Mya arenaria
may be caused by a B-type retrovirus, studies were undertaken to determine
whether or not such a virus was associated with the neoplastic disorders in M_.
edulis. Extensive investigation, employing a variety of techniques and methods
did not reveal the presence of any RNA tumor virus in either affected or
control M_. edulis.
Statistical Evaluations of Proliferative Disorders _UT, M. edulis
The occurrence, prevalence, seasonality and histopathological progression
of a cellular disorder, thought to be a hemic neoplasm, were studied in
subpopulations of Mytilus edulis inhabiting different sites in Yaquina Bay
Oregon, from 1976-1981.There were significant differences in the occurrence
of the disorder that were related to geographical location. In the
subpopulation with the highest levels of the disease, the prevalences ranged
from 0.0% to 20% with a five-year mean of 9.8%. There was a statistically
significant relationship between prevalence and season. During the five-year
study period, there was a consistent pattern characterized by highest
prevalences during January through March followed by a period of decline to
lower levels during the summer and early fall, after which there was an
increase. Data analyses revealed that there was no seasonal histopathological
progression of the disorder. Numbers of stage 1 (early), 2, 3 and 4 (advanced)
cases were not related to season but, rather, occurred in a random manner
throughout the entire year.
38
-------
XII. Publications, Reports and Abstracts
from Program - Cumulative list
(1978 - 1981)
*
Anderson, Robert S. 1978. Benzo(a)pyrene metabolism in the American Oyster,
Crassostrea virginica. Ecol. Res. Series, EPA-600/3-78-009. pp. 18.
Anderson, R.S., Doos, J.E., Rose, S.L. 1982. Differential ability of Ambystoma
tiqrinum hepatic microsomes to produce mutagenic metabolites from
Polycyclic Aromatic Hydrocarbons and Aromatic Amines.
Bunting, O.B. 1979. An evaluation of benzo(a)pyrene metabolism in an Oyster
(Ostrea edu1is)-Bacteria System. M.S. (Thesis) Oregon State.
Coker, S. 1980. Benzo(a)pyrene metabolism in the Sea Catfish (Arius fell us).
Ala. Acad. Sci. Meeting, March 21, 1980.
Couch, J.A. 1979. Vertebral dysplasia in young fish exposed to the herbicide
Trifluralin. Journal of Fish Diseases. 2: 35-42.
Couch, J.A. 1979. Pollution ecology of Penaeid Shrimps. In: Pollution Ecology
of Estuarine Invertebrates, pp. 236-258. Hart/Fuller (eds.) Academic
Press, New York.
Couch, J.A. 1979. Carcinogens in the Aquatic Environment. Interagency
Collaborative Group on Environment Carcinogenesis, NIH, Bethesda,
Maryland January 10, 1979. (Minutes of Meeting).
Couch,, J.A., Lee Courtney, James T. Winstead and Steven Foss. 1979. The
American Oyster (Crassostrea virginica) as an indicator of carcinogens in
the Aquatic Environment. In: Animal Models and Wildlife as Monitors, pp.
65-84. National Academy of Sciences, Washington, D.C.
Couch, J.S. and W.P. Schoor. 1979. First EPA/NCI Annual Report. Effects of
Carcinogens. Mutagens. and Teratogens on Non-Human Species (Aquatic
Animals). February, 1980, NCI Publication.
Couch, JA. and W.P. Schoor. 1980. Second Annual Report to NCI/EPA. Effects
of Carcinogens. Mutaqens and Teratogens on Non-Human Species (Aquatic
Animals). April, 1981. NCI Publication.
Couch, J.A. and James T. Winstead. 1979. Concurrent Neoplastic and Protistan
Disorders in the American Oyster (Crassostrea virginica). Haliotis, 8,
1977- pp. 249-253.
Couch, J.A., Frank G. Lowman and Ford A. Cross. 1980. Biomonitoring of Coastal
Waters, - An Overview. In: Biological Monitoring for Environmental
Effects, Douglas L. Wolf (ed.), Lexington Books, Mass. pp. 93-95.
Couch, J.A., Lee A. Courtney and Steven S. Foss. 1981. Laboratory of Symposium
of Marine Fishes as Carcinogen Assay Subjects. Proceedings of Symposium
of Princess Takamatsu Cancer Research Fund, Phyletic Approaches to Cancer,
C.J. Dawe et al. (eds), Japan Sci. Soc. Press, Tokyo, (In Press).
Couch, J.A. 1982. Aquatic Animals as Indicators of Environmental Exposures,
Journal Envion. Sci. Health, A17(4) 473-476.
Couch, J.A., Clyde Dawe. 1982. Mouse vs Minnow: The Future of Fish in
Carcinogenicity Testing. A Symposium on the use of Small Fish Species in
Carcinogenicity Testing. National Cancer Institute Monograph. In press.
Courtney, L.A., J.A. Couch. 1982. Usefulness of Cyprinodon variegatus and
Fundulus grandis in Carcinogenicity Testing"! Advantages and Special
Problems. A Symposium on the use of Small Fish Species in Carcinogenicity
Testing. National Cancer Institute Monograph. In Press.
39
-------
Dunn, J.E. 1982. Development of a Marine Bioassay System for Priority
Pollutants Using Larvae of the Gooseneck Barnacle, Pollicipes polymerus:
A Feasibility Study. M.S. Thesis.
El am, D., M.V. Kilgore, B. Tan and W.P. Schoor. 1979. Mixed Function Oxidase
Inducibility in the Mullet and Killifish. 4th Intl. Symp. on PAH,
Columbus, Ohio, October 4, 1979.
Elam, D., M.V. Kilgore and W.P. Schoor. 1979. Induction of Mixed Function
Oxidase and Metabolism of Polyaromatic Hydrocarbons in Marine Organisms.
llth Intl. Congress of Biochemistry, Toronto, Canada, July 11, 1979.
Elnenaey, Elsayed, and W. Peter Schoor. A Simple High Performance Liquid
Chromatography Method for the separation of benzo(a)pyrene metabolites.
Presented at American Association for Clinical Chemistry 32nd Annual
Meeting, Kansas City, Missouri, July 18-23, 1981.
Gregory, P.E., P.M. Howard-Peebles, R.D. El lender and B.J. Martin. 1980.
C-Banding of chromosomes from three established marine fish cell lines.
Copeia, 3: 545-547.
Gregory, P.E.% P.N. Howard-Peebles, R.D. Ellender and B.J. Martin. 1980.
Analysis of a marine fish cell line from sheepshead: chromosomal
alterations in Archosargus probatocephalus. J. Heredity 71: 209-211.
Grizzle, J.M., T.E. Schwedler, A.L. Scott. 1981. Papillomas of Black Bullhead
(Ictalurus me!as Rafinesque) Living in a Chlorinated Sewage Pond. J. Fish
Diseases.
Harshbarger, John D., Elliot R. Jacobson, Charles E. Smith and John A. Couch.
1980. Hematopoietic Neoplasms in Invertebrates and Cold Blooded
Vertebrates. In: Advances in Comparative Leukemia Research. 1979. David
S. Yohn, Boris A. Lapin and James R. Blakeslee (eds.) Elsevier/North
Holland. New York/Amsterdam/Oxford, pp. 223-225.
Hemingway, S.J. 1979. (1) Storage Sites of Benzo(a)pyrene in contaminated Bay
Mussels (Mytilus edulis) inhabiting different sites in Yaquina Bay,
Oregon. (2) Uptake and depuration of Benzo(a)pyrene in contaminated Bay
Musses! (Mytilus edulis) inhabiting different sites on Yaquina Bay. M.S.
Thesis, Oregon State University.
Hendricks, J.D. 1980. Chemical carcinogenesis in fish. In: Aquatic
Toxicology. L.Weber (ed.). Raven Press, New York. (In press).
Hendricks, J.D., J.H. Wales, R.O. Sinnhuber, J.E. Nixon, P.M. Loveland and R.A.
Scanlan. 1980. Rainbow Trout (Salmo gairdneri) embryos: A sensitive
animal model for experimental carcinogenesis. Fed. Proc. (In press).
Hendricks, J.D., R.O. Sinnhuber, P.M. Loveland, N.E. Pawlowski and J.E. Nixon.
1980. Hepatocarcinogenicity of gland!ess cottonseeds and refined
cottonseed oil to rainbow trout (Salmo gairdneri). Science (In press).
Hendricks, J.D., and T.R. Meyers. Ganglioneuroblastomas in cono salmon reared
in chlorinated-dechlorinated river water. J. Natl. Cancer Inst. (In
review).
Hendricks, J.D., Meyers, T.R., Shelton, D.W., and Sinnhuber, R.O. (1982).
Liver neoplasia and induction of hepatic mixed function oxidase enzymes in
the rainbow trout following dietary exposure to benzo(a)pyrene. Proc.
Amer. Assoc. Cancer Res. 23:58.
Hendricks, J.D., Meyers, T.R., and Shelton, D.W. Histologic progression of
hepatic neoplasms in rainbow trout (Salmo qairdneri). Nat!. Cancer Inst.
Monogr. (In press).
40
-------
Hendricks, J.D., Meyers, T.R., Casteel, J.L., Nixon, J.E., Loveland, P.M., and
Bailey, G.S. Rainbow trout embryos: Advantages and limitations for
carcinogenesis research Natl. Cancer Inst. Monogr. (In press).
Hendricks, J.D., Meyers, T.R., Shelton, D.W., and Casteel, J.L. The
hepatocarcinogenicity of benzo(a)pyrene to rainbow trout by dietary
exposure and intraperitoneal injection. (Final stages of preparation).
Hillebert, S.A., B.J. Martin and R.D. Ellender. 1980. Chronic exposure of a
teleost cell line to suspect carcinogens. J. Miss. Acad. Sci. (In press).
Kilgore, M.V., and 0. Elam. 1979. Mixed-Function Oxidase Inducibility in
Marine Organisms. Ala. Acad. of Sciences Meeting, April 14, Florence,
Ala.
Kilgore, M.V., and D. Elam. 1979. A comparatiave study of induction of mixed
function oxidase activity in the rat, mullet and killifish. Ala. Acad.
Sci. Meeting, March 30, Florence, Ala.
Koenig, Christopher C., M. Chasar 1982. Usefulness of Hermaphroditic Marine
Fish Rivulus marmoratus as a Test Animal for carcinogenicity testing. A
symposium on the use of small fish species in carcinogenicity testing.
National Cancer Institute Monograph. In Press.
Koening, Christopher C., C. Abel, C.W. Klingensmithe, M.B. Maddock, 1982.
Usefulness of the self-fertilizing Cyprinodontid fish, Rivulus marmoratus
as an experimental animal in studies involving carcinogenesis,
teratogenesis and mutagenesis. EPA Research Assistance Grant (R805469) 129
pp.
LaTouche, Y.D. 1981. Metal levels in a population of Mytilus edulis from
Yaquina Bay, Oregon. PH.D. Thesis, Oregon State universiTyT
LaTouche, Y.D., and M.C. Mix. 1981. Seasonal variations in trace metal levels
in a Mytilus edulis population in Yaquina Bay (Newport, OR). The 21st
Hanford Life Sciences Symposium.
LaTouche, Y.D., C.W. Bennett and M.C. Mix. 1981. Determination of Vanadium in
a marine mollusk using a chelating ion exchange resin and neutron
activation. Bull. Environ. Contam. Toxicol., 26: 224-227.
LaTouche, Y. D. and M. C. Mix. 1982. The effects of depuration, size and sex
on trace metal levels in bay mussels. Marine Pollution Bulletin, 13:27-29
LaTouche, Y. D. and M. C. Mix. In Press. Seasonal variations of arsenic and
other trace elements in bay mussels (Mytilus edulis). Bulletin of
Environmental Contamination and Toxicology. 1982.
Long, R.L., and B.J. Martin.1980.Morphology of peripheral blood cells of
Cyprinodon variegatus. ABSTR. J. Miss. Acad. Sci., Vol. XXV, Suppl:
122.
Martin, B.J. 1980. Effects of petroleum compounds on estuarine fishes. Ecol.
Res. Series. EPA-600/3/80-019. January, 1980.
Martin, B.J., and W.W. Greenwich. 1980. Exposure of two teleost species to
polycyclic aromatic hydrocarbons. ABSTR. J. Miss. Acad. Sci., Vol. XXV,
Suppl: 120.
Martin, B.J., and W.W. Greenwich. 1981. Benzidine Toxicity. J. Miss. Acad. Sci.
26 (Suppl.): 127.
Martin, B.J., and W.W. Greenwich. The Induction of Proliferative Lesions in
the Sheepshead Minnow, Cyprinodon variegatus, with Benzidine
Dihydrochloride. (In preparation).
41
-------
Martin, B.J., R.D., Ellender, S.A. Hillebert, and M.M. Guess. Primary Cell
Cultures from the Teleost, Cyprinodon variegatus: Culture Establishment
and Application in Carcinogen Exposure Studies. National Cancer Institute
Symposium on the Use of Small Fish Species in Carcinogenicity Testing,
Bethesda, MD. December 8-10, 1981.
Meador, C.B., B.L. Middlebrooks, and B.J. Martin. Serologic Alterations in
Carcinogen-Exposed Teleosts: Procedures for Preparation and Analysis of
Samples From Small Fish. National Cancer Institute Symposium on the Use
of Small Fish Species in Carcinogenicity Testing, Bethesda, MD. December
8-10, 1981.
Melius, P., B. Tan, M. Kilgore, D. Elam, W.P. Schoor. 1979. Product pattern
and kinetic analysis of benzo(a)pyrene metabolism in Marine Organism by
HPLC. ACS 31st Southeast Regional Meeting, Florence, Ala. April 14,
1979.
Melius, P., D. Elam, M.V. Kilgore, B. Tan and W.P. Schoor. 1979.
Mixed-Function Oxidase Inducibility and Polyaromatic Hydrocarbon
Metabolism in Mullet, Sea Catfish and Gulf Killifish. In: Polynuclear
Aromatic Hydrocarbons: Chemistry and Biological Effects, Bjorseth and
Dennis (eds.)i PP« 1059-1075. Batelle Press, Columbus, Ohio.
Melius, P., D. Elam, M.V. Kilgore and W.P. Schoor. 1979. Induction of
Mixed-Function Oxidase and Metabolism of Polyaromatic Hydrocarbons in
Marine Organisms. Eleventh International Congress of Biochemistry,
Toronto, Canada. July 7-11.
Melius, P., B. Tan, M.V. Kilgore, and D. Elam. 1979. Metabolites of B(a)P in
Aroclor-induced fish. Fourth ASTM Symposium on Aquatic Toxicology,
Chicago, Illinois. October 16-17.
Meyers, T.R. and J.D. Hendricks. Case reports of spontaneous neoplasms in
feral and laboratory-reared-salmonids. J. Fish Dis. (In review).
Meyers, T.R. and Hendricks, J.D. Histopathological methods. In Principles of
Aquatic Toxicology- (Rand, G.M. and Petrocelli, S.R., ed!7). Hemisphere
Publishing Co., New York (In press).
Meyers, T.R. and Hendricks, J.D. A summary of tissue lesions in aquatic
animals induced by controlled exposures to environmental contaminants,
chemotherapeutic agents and potential carcinogens. Marine Fish. Review
(In press).
Meyers, T.R. and Hendricks, J.D. Histopathology of four spontaneous neoplasms
in three species of salmonid fishes. J. Fish Dis. (In press).
Meyers, T.R. and Hendricks, J.D. A limited epizootic of multi-differentiating
neuroblastoma in coho salmon reared in chlorinated-dechlorinated water.
Submitted to J. Natl. Cancer Inst.
Mix, M.C., R.T. Riley, K.I. King, S.R. Trenholm and R. L. Schaffer. 1977.
Chemical carcinogens in the marine environment. Benzo(a)pyrene in
economically important bivalve mollusks from Oregon estuaries. In: Fate
and Effects of Petroleum Hydrocarbons in Marine Organisms and Ecosystems,
D. A. Wolfe (ed.), pp. 421-431. Pergamon Press, New York.
Mix, M.C., J.W. Hawkes and A.K. Sparks. 1978. The ultrastructure of cells
associated with proliferative disorders in mussels (Mytilus edulis) from
Yaquina Bay, Oregon. Proceedings of a Colloquium on Invertebrate
Pathology: Neoplasms in Invertebrates.
42
-------
Mix, M.C. and R.L. Schaffer. 19797. Benzo(a)pyrene concentrations in mussels
(Mytilus edulis) from Yaquina Bay, Oregon during June, 1976 - June, 1978.
Bull. Environ. Contain. Toxicol., 23: 677 - 684.
Mix, M.C., J.w. Hawkes, and A.K. Sparks. 1979. Observations on the
ultrastructure of large cells associated with putative neoplastic disorder
of mussels, Mytilus edulis, from Yaquina Bay, Oregon. J. Invert. Path.,
34: 41-56.
Mix, M.C. 19797. Chemical Carcinogens in Bivalve Mollusks from Oregon
Estuaries EPA Ecological Research Series, EPA600/3-79-034. pp. 33.
Mix, M.C. 1979. Seasonal variation in benzo(a)pyrene in bivalve mollusks from
Oregon estuaries. Symposium on Carcinogenic Polynuclear Aromatic
Hydrocarbons in the Marine Environment. U.S. EPA., Gulf Breeze, Florida.
Mix, M.C. 1979. Leukemia-like disorders in bay mussels (Mytilus edulis) from
Yaquina Bay, Oregon, U.S.A. Proceedings of a Symposium on Comparative
Leukemia and Related Diseases.
Mix, M.C., D.L. Bunting and D.T. Abbott. 1979. Preliminary studies to
evaluate the potential of using embryo and larval stages of the goose
barnacle, Pollicipes polymerus, for marine bioassays. Proceedings of the
Second Biennial Crustacean Health Workshop, Texas A M Univ.,
TAMU-SG-79-114:361-381. College Station, Texas.
Mix, M.C., S.R. Trenholm and K.I. King. 1979. Benzo(a)pyrene body burdens and
the prevalence of cellular pro!iferative disorders in mussels, Mytilus
edulis, from Yaquina Bay, Oregon. In: Animals as Monitors of
Environmental Pollutants. 52-62. National Academy of sciences,
Washington, D.C.
Mix, M.C., R.L. Schaffer and Y.D. LaTouche. 1980. Environmental contaminants
and the occurrence of certain pathological conditions to bivalve mollusks.
Proceedings of the Society for Invertebrate Pathology.
Mix, M.C., and W.P- Breese. 1980. A cellular proliferative disorder in Ostrea
chilensis from Chilor, Chile, South America. J. Invert. Pathol.
36:123-124.
Mix, M.C. 1980. Utilization of bivalve mollusks. for monitoring carcinogenic
polynulclear aromatic hydrocarbons in estuarie environments. The Eleventh
International Symposium of the Princess Takamatsu Cancer Research Fund.
(In press).
Mix, M.C. Polynuclear Aromatic Hydrocarbons in Bivalve Molluscs from Oregon
Estuaries. EPA Ecological Research Series. (In press).
Mix, M.C., R.L. Schaffer and S.J. Hemingway. Polynuclear aromatic hydrocarbons
in bay mussels (Mytilus edulis) from Oregon. In: Proceedings of the
Eleventh International Symposium of the Princess Takamatsu Cancer Research
Fund. (In press).
Mix, M.C. Cellular prol iferatiave disorders in bivalve mollusks from
contaminated marine environments. To be submitted to Journal of
Environmental Pathology and Toxicology.
Mix, M. C., S. J. Hemingway and R. L. Schaffer. 1982. Benzo(a)pyrene
concentrations in somatic and gonad tissues of bay mussels, Mytilus
edulis. Bulletin of Environmental Contamination and Toxicology,
28:46-51.
43
-------
Mix, M. C. and R. L. Schaffer. In Press. Concentrations of unsubstituted
polynuclear aromatic hydrocarbons in bay mussels, Mytilus edulis, from
Oregon. Marine Environmental Research.
Mix, M. C. and R. L. Schaffer.In Press.Concentrations of unsubstituted
polynuclear aromatic hydrocarbons in softshell clams (Mya arenaris) from
Coos Bay, Oregon. Marine Pollution Bulletin.
Mix, M. C. In Press. The neoplastic disease of bay mussels, Mytilus edulis,
from Oregon: occurrence, prevalence, seasonality and histopathological
progression. Journal of Fish Diseases.
Moreno, M., and B.J. Martin. 1979. Embryologic development of sheepshead
minnow, (Cyprinodon variegatus). ABSTR. J. Miss. Acad. Sci. Vol. XXIV,
Suppl : 116.
Porter, R.D., and B.J. Martin. 1980. The histology of the postpharyngeal
digestive tract of the sheepshead minnow, Cyprinodon variegatus. ABSTR. J.
Miss. Acad. Sci., Vol. XXV. Suppl: 123.
Riley, R.T. 1978. The effects of chemical perturbation by naphthalene in
glucose metabolism in the European flat oyster, Ostrea edulis: An in vivo
kinetic analysis. Ph.D. Thesis Oregon State University.
Riley, R.T., and M.C. Mix. 1980. Thin layer separation of citric acid cycle
intermediates, lactic acid, and the amino acid taurine. Journal of
Chromatography, 189: 286-288.
Riley, R.T., and M.C. Mix. 1981. An ion -exchange technique for the
concentrating of ammonia in small volumes of seawater. Marine Chemistry.
10: 159-160.
Riley, R.T., M.C. Mix, R.L. Schaffer and D.L. Bunting. Uptake and accumulation
of naphthalene by the oyster, Ostrea edulis, in a flow-through system.
Marine Biology. (In press).
Riley, R.T., and M.C. Mix. The effects of naphthalene on glucose metabolism
in the European flat oyster, Ostrea edulis. Marine Biology, (In press).
Riley, R.T. Stimulatory effect of naphthalene on glucose transport in the
oyster. Submitted to Nature.
Riley, R.T., and M.C. Mix. The pathways of glucose metabolism in the oyster,
Ostrea edulis. To be submitted to Comparative Biochemistry and
Physiology.
Schaffer, R.L., and M.C. Mix. A method for determining environmental levels of
polycyclic aromatic hydrocarbons in bivalve mollusks by reverse phase
liquid Chromatography. To be submitted to Analytical Chemistry.
Schneider, S.R., J.D. Hendricks, G.H. Constantine and R.E. Larson. 1980.
Tobramycin nephrotoxicity in coho salmon at subtherapeutic doses.
Toxicol. Appl. Pharmacol. (In press).
Schoor, W.P. 1979. Fluorimetric confirmation of metabolites of benzo(a)pyrene
from a marine fish. Fourth International Symposium on Polynuclear
Aromatic Hydrocarbons, Columbus, Ohio, October 2-4, Battelle Institute.
Schoor, W.P., and J.A. Couch. 1979. Correlation of mixed-function oxidase
activity with ultrastructural changes in the liver of a marine fish.
Cancer Biochemistry and Biophysics, 4: 95-103. _
Schoor, W.P., Elsayed Elnenaey and Barrie Tan. 1981. Benzo(a)pyrene Metabolism
in 3-Methylcholanthrene-treated sea catfish. Submitted to Carcinogenesis.
Schoor, W.P. Exposure of Fishes to Benzo(a)pyrene and Source Aspects of
Analysis of Metabolites, J. Natl. Cane. Inst. in press.
44
-------
Sigismondi, L.A. 1982. The Accumulation of Cadmium, Copper and Nickel in
Freshwater Mussels (Marqaritifera marqaritifera), Sediment and Algae
(Cladophora sp.) from Big Elk River, Oregon. fT.S. Thesis.
Smith,~A.C., and M.C. Mix. 1978. The effects of sodium chloride concentration
on electrophoretic patterns of adductor muscle proteins from bivalve
mollusks. Comparative Biochemistry and Physiology (Part B), 61: 169-171.
Strength, R., H.H. Daron, J.L. Aull, J.F. Wilson and W.P. Schoor. 1980. The
induction of glucuronide and sulfate transferases by phenobarbital and
polycyclic aromatic hydrocarbons. Fed. Proc. 39: 1694.
Strength, D.R., K.V. Sarambadal, S.L. Wang, H.H. Darron, and W.P. Schoor,
Oxidation and Conjugation of BaP in Mullet. Fed. Proceed.,
41,, 1147(1982).
Strength, D.R., H.H. Darron, and W.P. Schoor. Comparison of Polycyclic
Aromatic Hydrocarbon Metabolism in Rodent and Fish. J. Natl. Cane. Inst.,
in press.
Tan, B. 1979. Product Pattern and Kinetic analysis of benzo(a)pyrene
metabolism in marine organisms by HPLC. S.E. Regional A.C.S. meeting,
Roanoke, Va. October 25, 1979.
Tan, B. 1980. Indirect atomic absorption assay of epoxide hydrase. Ala. Acad.
Sci., March 21.
Tan, B. 1980. Indirect atomic absorption assay of epoxide hydrase activity and
determination of 1,2-diols via digested lead periodate. English Society
of Analytical Chemists, 5th Intl. Symp., Lancaster, England, July 20-26.
Tan, B. 1980. Benzo(a)pyrene metabolism in PAH and Biphenyl treated Tilapia.
Presented October 28-30, 5th Intl. Symp. on PAH's, Columbus, Ohio.
Tan, B., and J.M. Grizzle. 1980. The use of benzo(a)pyrene metabolism as a
biochemical indicator in control and tumored bullhead catfish. October
28-30, 5th Intl. Symp. on Polynuclear Atomic Hydrocarbons, Columbus,
Ohio.
Tan, B., P. Melius, M. Kilgore, 0. Elam and W.P. Schoor. 1979. Metabolites of
benzo(a)pyrene in Aroclor-induced fish. XI International Congress of
Biochemistry, Toronto. Canada, July 8-13.
Tan, B., and P. Melius. 1980. Responses of Hepatic Enzymes of Tilapia to PCS
and PAH modifiers. American Assoc. Biol. Chemists Meeting, New Orleans.
June 2-5. Abstract published in Fed. Proc.
Tan, B., P. Melius and M.V. Kilgore. 1980. Determination of 1,2-diols by
direct atomic absorption with digested lead periodate. Anal. Chem.
52:602-604.
Tan, B., D. Elam, E.V. Kilgore and W.P. Schoor. 1979. Metabolites of
Benzo(a)pyrene in Aroclor 1254 treated mullet. 4th ASTM Symp. on Aquatic
Toxicology. October 16-17, 1979. Chicago, Illinois. (In press).
Tan, B. Responses of hepatic enzymes of Tilapia to biphenyls and polyaromatic
hydrocarbons. Submitted to Biochem. Pharmacology.
Tan, B. Comparative metabolism of polyaromatic hydrocarbons in marine
organisms. Invited review article being prepared for Journal of
Comparative Biochemistry and Physiology.
Tan, B., and P. Melius. Kinetic product patterns and mechanistic pathways of
benzo(a)pyrene metabolism in Aroclor-treated mullet. Submitted to
Biochem. Biophys. Acta.
45
-------
Tan, B., D. Elam, M.V. Kilgore, and W.P. Schoor. Mixed function oxidase
inducibility and polyaromatic hydrocarbon metabolism in mullet, sea
catfish and gulf killifish. 4th Intl. Symp. on Polynuclear Aromatic
Hydrocarbons Chemistry and Biological Effects. Battelle Press. (In
press).
Tan, B., M.V. Kilgore, D. Elam and W.P. Schoor. Metabolites of benzo(a)pyrene
in Aroclor 1254 treated Mullet. 4th Symp. on Aquatic Toxicology, ASTM (In
press).
Tan, B. 1981. Indirect Atomic Absorption Spectrometric Assay for Epoxide
Hydrolase. Analytical Letters, 14(85), 311-322.
Tan, B., P. Melius, and J. Grizzle. 1981. Hepatic Enzymes and Tumor
Histopathology of Black Bullheads. Polynuclear Aromatic Hydrocarbons
Chemical Analysis and Biological Fate 5th International Symposium.
Battelle Press.
Tan, B. and P. Melius. 1981. Responses of the Hepatic Enzymes of a Teleost
Fish to trans-Stilbene Oxide Treatment. Bull. Environm. Contam. Toxicol.
26, 801-806.
Tan, B., P. Melius, and P. Zieglor. 1982. A Simple Gas Chromatographic Method
for the Study of Organic Solvents: Moisture Analysis, Hygroscopicity, and
Evaporation. Journal of Chromatographic Science, Vol. 20. 213-217.
Trenholm, S. and M.C. Mix. The lethal and sublethal effects of ionizing
radiation of juvenile Pacific oysters (Crassostrea gigas). Radiation
Research. (In press).
Trenholm, S.R. and M.C. Mix. 1978. Regeneration of radiation damaged digestive
tissue in juvenile Pacific oyster (Crassostrea gigas). J. Invert. Pathol.
32: 249-257.
Trenholm, S.R. 1978. Effects of X- and Gamma irradiation on the juvenile
Pacific oyster, Crassostrea -gigas. M.S. Thesis Oregon State University.
46
-------
i ERL.GB 0267
TECHNICAL REPORT
:fi, -U' /•( Ju ,'•. 'r.,<:;::-i>ts on i!'.: .-(.". t-/Tc
1 rIEPO"0" \C
EPA-600/9-83-005
Problem Oriented Report
EFFECTS OF CARCINOGENS, MLTAGENS, AND TERATOGENS ON
«)NHUMAN SPECIES--AQUATIC ANIMALS
6. PERFORMING ORGANIZATION CODE
AUTHOR(S)
.A. Couch, W.P. Schoor, W. Davis, and Lee Courtney
8. PERFORMING ORGANIZATION REPORT \O
9. PERFORMING ORGANIZATION NAME AND ADDRESS
10 PROGRAM ELEMENT NO
•11 CONTRACT'GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
U.S. Environmental Protection Agency
iivironmental Research Laboratory
Dffice of Research and Development
Gulf Breeze, FL 32561 '
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16, ABSTRACT
This report describes the fourth year of a joint U.S. Environmental Protection
Agency (EPA) and National Cancer Institute (NCI) effort to develop laboratory and
field assays, using fishes as indicators of the presence of certain carcinogens
(polycyclic aromatic hydrocarbons). The study has revealed that fish have
metabolic pathways similar to mammals for disposition of certain carcinogens.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b. IDENTIFIERS-OPEN ENDED TERMS
COSATi F-ield/Croup
,'18. DISTRIBUTION STATEMENT
! Release to public
U9. SECURITY C._ ASS . 7~'iu
! Unclassified
; 20 SECL/RIT _ _ __
i Unclassified
EPA rorm 2220-1 (Rev. J-T
PREVIOUS E - i
n 9 s c - E - s
------- |