United States
Environmental Protection
Agency
Environmental Research
Laboratory
Duluth MN 55804
Research and Development
EPA-600/S3-83-076 Nov. 1983
Project Summary
Detection of Mutagenic/
Carcinogenic Alteration
in Fish
David E. Hinton, James E. Klaunig, Michael M. Lipsky, Rhona M. Jack, and
Benjamin F. Trump
The feasibility of using fish as bioassay
organisms to detect mutagenic/carcino-
genic substances in the aquatic environ-
ment was tested in species not common-
ly employed in chemical carcinogenesis
bioassay. Microsomal fractions from
livers of channel catfish (Ictalurus
punctatus). fathead minnow (Pimephales
promelas). bluegill sunfish (Lepomis
macrochirus), brown bullhead (Ictalurus
nebulosus). and mummichogs (Fundulus
heteroclitus) were used. Data obtained
from these species were compared to
those obtained in rainbow trout (Salmo
gairdneri) - a more commonly employed
fish - and mammalian species. The
presence and relative amount/activities
of microsomal protein, cytochromes P-
450 and bs, NADPH cytochrome C
reductase, aminopyrine demethylase,
and aryl hydrocarbon hydroxylase were
determined. The effects, both morpholog-
ic and biochemical, in exposure to the
known MFOS inducing agents, PCBs,
benzo(a)pyrene, and 3-methylcholanth-
rene were studied. Exposure caused
induction of enzymes and proliferation
of endoplasmic reticulum membranes
of hepatocytes. High pressure liquid
chromatographic analysis of benzo(a)py-
rene (BP) metabolism in catfish and
trout were performed. Both species
produced the following metabolites of
BP: 9,10-diol BP; 4,5-diol BP; 7,8-diol
BP. 9-OH BP; 3-OH BP; and quinones.
Catfish postmitochondrial supernatant
converted BP and 2-acetylaminofluorene
(AAF) into mutagenic intermediates in a
microbial mutagen system. Catfish liver
cells were isolated and maintained with
high viability (98%) for 10 days. When
incubated with H-BP, these cells
showed preferential accumulation of
label over nuclei. Subsequent liquid
scintillation analysis of cell fractions
obtained by cesium chloride centrifuga-
tion revealed radioactivity in DNA
fractions. Foci of hepatocellular altera-
tion, hyperptastic areas and bile ductular
hyperplasia were seen in channel
catfish chronically exposed to the
chemical carcinogen AAF. These data
indicate the suitability of conducting
further studies on this ubiquitous
species designed to determine dose-
response characteristics to various
chemical carcinogens. On the basis of
microsomal metabolism and cellular
response, it appears feasible to use fish
tissue to test for mutagenic/carcinogenic
compounds in the aquatic environment
and to develop bioassay methodology
for testing possible carcinogenic proper-
ties of new chemical formulations prior
to their introduction into the aquatic
environment.
This Project Summary was developed
by EPA's Environmental Research
Laboratory, Duluth, MN. to announce
key findings of the research project that
is fully documented in a separate report
of the same title (see Project Report
ordering information at back).
Introduction
A majority of human cancers are due to
chemical carcinogens in the environ-
ment. Since the oceans ultimately be-
come the reservoir for every pollutant en-
tering the biosphere, the importance of
the aquatic environment in considera-
tions of carcinogenic effects of chemical
pollutants cannot be overly emphasized.
-------
Correlated Biochemical and
Morphologic Studies of Effects
of Xenobiotics on Fish Liver
To characterize the response of certain
microsomal components in channel
catfish, we administered a commercial
PCB mixture (Aroclor 1254*) as seven
daily intraperitoneal injections of 50
mg/kg b.w. Via correlated biochemical
studies on hepatic microsomal mixed
function oxidative system (MFCS)enzymes
and ultrastructural studies of hepatocytes
we analyzed acute and subacute responses.
A similar approach was used with
rainbow trout exposed to 3-MC (50
mg/kg i.p. x 4 days). Our results on
hepatic MFOS components of the channel
catfish and rainbow trout and their
response to Aroclor 1254 and 3-MC
exposure are summarized in the full re-
port.
Acute exposure (7 days) resulted in a
moderate increase in some MFOS com-
ponents. Cytochrome P-450 increased
approximately 33% in channel catfish.
Specific activity of NADPH-cyt-c-reductase
was increased to 1.6 times control
values. The greatest change was observed
in amount of cytochrome b5, which
increased 2.5-fold. Aminopyrine deme-
thylase activity, microsomal protein and
liver to body weight ratios did not change
after acute exposure. In contrast, rat
MFOS components were markedly increased
after an identical concentration and
duration of exposure.
Subacute (21 days) of PCB treatment
resulted in a large increase in catfish
MFOS components with respect to
controls and acutely exposed fish.
Amounts of cytochromes P-450 and bs
increased three and two times over
controls respectively. NADPH-cyt-c-
reductase was elevated to a level similar
to that seen after the acute exposure.
Aminopyrine demethylase, unchanged
by acute treatment, was increased three-
fold over controls after subacute treatment.
Again, microsomal protein and liver to
body weight ratios were unchanged.
Thus, the response of the catfish MFOS to
acute PCB treatment is a moderate
induction, much less than is seen in the
rat. Subacute exposure resulted in a
greater degree of induction.
When compared to control morphology,
changes were also seen in hepatocyte
ultrastructure of channel catfish after
acute PCB exposure. Dilated cisternae
and meandering tracts of endoplasmic
reticulum (ER) were seen. Minimal
•Mention of trade names or commercial products
does not constitute endorsement or recommendation
for use.
increases in agranular ER profiles were
noted. These appeared as focal, discrete
aggregations. Increased lipid droplets
also characterized hepatocytes of acutely
exposed channel catfish.
Subacute exposure of catfish resulted
in alterations in agranular ER not seen in
acutely exposed fish liver. Tubular and
vesicular profiles, nearly absent in
control catfish hepatocytes, were increased
in number and often accumulated in large
aggregates. In addition, parallel stacks of
agranular ER in continuity with granular
ER were observed. Often these patterns
contributed to large whorls of ER membranes
in affected cells.
Biochemical levels of induction follow-
ing acute and subacute Aroclor 1254
exposure in the catfish correlated well
with ER content and morphology. Higher
levels of MFOS induction were accompanied
by apparent increases in agranular ER
content and variations of ER morphology.
Acute 3-MC exposure caused significant
induction of rainbow trout MFOS com-
ponents. Cytochrome P-450 increased to
over three times control amounts.
NADPH-cyt-c-reductase activity was
elevated two times over controls. As in
the catfish, neither microsomal protein
nor liver to body weight ratios of trout
were altered by 3-MC treatment.
Morphologically, acute MFOS induction
correlated with apparent increases in
trout liver endoplasmic reticulum. Ultra-
structural alterations caused by acute
PCB and 3-MC exposure involved granular
ER content and morphology, although
focal increases in agranular membranes
were noted. The major response after 21
days of exposure to Aroclor 1254 was
extensive proliferation of ER.
ARYL Hydrocarbon
(Benzo-a-Pyrene) Hydroxylase
(AHH) in Channel Catfish Liver
Polycyclic aromatic hydrocarbons
(PAH), an important class of environmen-
tal pollutants which cause tumors in
experimental animals including fish and
have been implicated in human carcino-
genesis, are activated by the NADPH-
dependent MFOS. That portion of the
MFOS responsible for this metabolic
activation is the AHH system. AHH from
livers of control fish and from livers of fish
exposed to 3-MC or to Aroclor 1254 was
characterized as to pH, temperature, and
protein concentration optima. Linearity of
reaction over time was established.
With respect to temperature, an
optimum of 30°C was found for control
catfish liver microsomes. This corresponds
to the temperature optima reported for
rainbow trout, Finnish lake trout, and
various marine species. A shift to a higher
temperature range (30°C - 40°C)followed
treatment with 3-MC or Aroclor 1254 in
the catfish. The induced system seems to
have quite a broad temperature range
with no sharp drop at higher temperatures
such as that found in the trout.
The effect of in vitro addition of
benzoflavone (BF) on AHH was studied in
control, Aroclor 1254 and 3-MC-pretreated
fish. Inhibition of activity was seen in 3-
MC-pretreated samples at all ranges of
BF tested (Figure 1). Inhibition ranged
from 12% at .001 mM BF to 76% at 1 mM
BF. PCB treatment left AHH less sensitive
to BF inhibition at most concentrations
than did 3-MC treatment. In control
samples, .001 mM BF had no effect on
AHH levels; however, at concentrations
of .01 mM, up to 200% enhancement of
activity was seen. Enhancement decreased
with increasing BF concentrations, until
at 1 mM BF, 22% inhibition was seen. No
enchancement was seen in samples from
either treated group at all concentrations
tested.
Studies thus far have demonstrated
that two forms of AHH can be distinguished,
one in control fish liver microsomes and
the other in microsomes from 3-MC or
PCB-pretreated fish. The enzymes are
distinguished by the differential effect of
BF on AHH. Both 3-MC and Aroclor 1254
treatment in vivo cause the behavior of
the AHH enzyme to change. Characteris-
tically, the induced enzyme is similar to
the control enzyme with respect to pH
optimum but differs in temperature
optimum and BF sensitivity (at the same
concentration).
Benzo-a-pyrene, administered as a
single i.p. injection of 100 mg/kg b.w. in
corn oil, induced its own in vitro metabo-
lism within 48 hr. in livers of three of four
channel catfish (Figure 2-A). Differences
in response of exposed individuals was
apparent with AHH values ranging from
120 to 350% of control values. Taken as a
group, the mean value for exposed fish
was 1.90± 1.41 compared to 0.50 ±0.04
for controls (p<.025).
When BP was administered as six daily
i.p. doses of 25 mg/kg (Figure 2-B),
induction of AHH was seen and individual
variation among fish was reduced. The
mean AHH value in exposed fish was
2.67 ± 0.97. This is, on the average, a
513% increase over the control mean of
0.52 ±0.01. These values were significant-
ly different (p<0.01). When BPwas given
as a single gastric intubation, induction of
AHH occurred in 80% of treated fish
(Figure 2-C). Interindividual variation
was apparent. The mean for exposed fish
-------
O Microsomes from control liver
• Microsomes from 3-MC-treated livers
A Microsomes from PCB-pretreated livers
200 -
/50 -
c
o
CJ
700
.0007
.007 .07 .7
mM 7,8 Benzoflavone
Figure 1.
Effect of 7,8 benzoflavone concentration on in vitro A HH activity. Control equals the
same sample without BF. Data illustrated are from a single representative
experiment. Triplicate assays were performed with variation between replicates
less than 10%.
a 5-°
o
1 40
AHH
WBP/20 m
\> Co
b b
o
nmole 3-
b
0
4 r+f
& n
rf
1
T
1
©
3
f3
rf
rt
•4-
-i-
©
V
5 T
+
•h
*
Figure 2. Effects of BP on hepatic AHH activity in channel catfish. Slashed bars represent
the average of control fish ±S.D. between animals. Numbers over the bars represent
the number of fish in the experiment. Clear bars represent individual treated fish ±
S.D. for triplicate assays on each animal. Treatment: (A) one i.p. injection of 100 mg <
BP/kg b. w.. sacrificed after 48 hours; (B) six daily injections of 25 mg BP/kg b. w..
sacrificed after 24 hrs; (C) one gastric intubation of 100 mg BP/kg b. w., sacrificed
after 14 days.
(1.73 ± 0.95) was 208% of control values
and was significantly different (p <.025).
3-MC Induction of Channel
Catfish AHH
The effects of five daily i.p. injections of
20 mg 3-MC/kg body weight upon
channel catfish liver AHH are shown
(Figure 3). All fish exposed inthis manner
exhibited induction with the individual
response ranging from 10 to 25 times
control values. The experimental mean of
4.20 ±1.73 was statistically different
from the control mean of 0.27 + 0.12 (p
<.01).
When constant dose (25 mg 3-MC/kg
b.w.) was maintained for 4 days and fish
were then killed daily up to one week
after exposure, it was possible to deter-
mine the duration of effect. These data
are presented in the full report.
AHH was increased dramatically by
one day after the cessation of exposure.
At day 4 following cessation of exposure,
the AHH activity was 24 times the control
mean, and by day 7 after treatment, AHH
was 19-fold the control mean. The
deviation between individual fish decreased
markedly as the time of exposure length-
ened. The highest values for NADPH-
cytochrome-c-reductase and cytochrome
P-450 were seen at day 4. At this time,
the mean cytochrome P-450 estimation
was 2.2 times over the control value. By
day 7, cytochrome P-450 values were
only slightly higher than controls.
Microsomal protein concentration in
catfish appeared to increase in the fish
killed 4 and 7 days after treatment ended;
however, no clear cut correlation between
induction and protein concentration was
seen. The mechanism for induction in
fish has not yet been explained. New
enzyme synthesis or enzyme modification
is one possibility; however, changes in
the membrane composition or conforma-
tion should also be considered.
Metabolism of Benzo-a-Pyrene
by Microsomal Fraction of Fish
Liver
Metabolites formed by the reaction of
fish liver microsomal fractions with 80-
100 nmoles of 3H-BP (specific activity
150-200 fiC\/umo\e were analyzed by
high pressure liquid chromatography
(HPLC). Each ml of assay volume included
0.1 M phosphate buffer, pH 7.0-7.2, 3.0
mM MgCI, O.1 mM EDTA, 0.4 mM NADP,
10 mM glucose-6-phosphate, 15-20/uCi
BP, and 1.5 Units of glucose-6-phosphate
dehydrogenase as the NADPH generating
system. BF (0.1 mM) was added in 20/ul of
acetone in some experiments. Protein
-------
6.0
.c
r
?!
•S
o
I
4.0
2.0
Figure 3.
Effect of 3-MC on hepatic AHH activity in catfish. Five fish were injected on five
consecutive days with 20 mg 3-MC/kg b. w.. and sacrificed after 24 hrs. The slashed
bar represents the mean of three control fish ± 1 S.D. between animals. Clear bars
represent individual treated fish ± 1 S.D. for triplicate assays.
concentration was maintained at 0.1 mg
per assay. After incubation in a shaking
water bath at 30°C for 20 minutes, the
reaction was stopped by the addition of 1
ml acetone. The contents of 10 assay
tubes were combined, and metabolites
were extracted three times with ethyl
acetate. Radioactivity remaining in the
aqueous phase was less than 0.1 % of the
total. The ethyl acetate extracts were
pooled, flash evaporated, and the meta-
bolites were resuspended in 0.1 ml of
glass distilled methanol.
Metabolite separation was performed
using a Varian high pressure liquid
chromatograph with a 25 cm Whatman
Partisil column (PXS10/250DS) of inside
diameter, 4.6mm. Column temperature
was ambient. For analysis, 10-20//I of
the methanolic extract was injected into
the HPLC.
A methanol-water gradient was used
to elute metabolites. Two solvents
designated 'a' and 'b' were used. Solvent
'a' was 30:70 methanol: water, and
solvent 'b' was 70:30 methanol water.
Initially, concentration of 'b' in 'a' was
25%. This was increased linearly at
3%/min and at a flow rate of 1.2 ml/min
to 100% 'b'. This reverse phase chroma-
tography, proceeding from a more to less
polar solvent, elutes polar components
first. Approximately 200 fractions were
collected (0.4 min/fraction) in order to
elute all metabolites and unmetabolized
parent BP which, due to its nonpolar
nature, is eluted last.
All samples were co-chromatographed
with 14C-labeled BP metabolites from
rat liver microsomes as an internal
standard or with pure individual metabo-
lites to validate identification of peaks
(absorbance monitored at 254 nm). Since
the rat liver BP metabolite pattern is
well characterized, it was used as a
biological standard. Peaks were also
identified by retention times established
with pure metabolite standards.
Radioactivity of eluted fractions was
determined by liquid scintillation counting.
The 3H counts were computed to obtain
specific activity for each metabolite, and
expressed as pmoles BP metabolized/min/
mg protein. The 14C counts were used
only for identification purposes. The sum
of counts from fractions collected prior to
thefinal BPpeakrepresentedtotal BPme-
tabolized. Individual metabolites were ex-
pressed as a percent of this total. In this
manner, quantitative differences in acti-
vity of various metabolites were com-
pared in control and treated fish to estab-
lish effect of in vivo exposure of xenobi-
otics upon in vitro BP metabolism.
Table 1 provides a summary of results
in terms of percent metabolism of BP into
the various metabolites. Three different
control and 3-MC-pretreated catfish
were used to obtain these data. The 3-MC-
pretreated catfish showed an apparent
increase in activity toward the formation
of all metabolites. Control and 3-MC val-
ues for 9,10-diol BP and 7,8-diol BP were
significantly different by the T test
(p<0.05). Of the total radioactivity added
per assay, 3-MC-pretreated microsomes
metabolized 12-28% (3.75-4.8 fjC\) and
control microsomes metabolized 14-22%
(3.0-5.6 A»Ci). The PCB-pretreated catfish
also show an increase in 9,10-diol BPand
7,8-diol BP, although total metabolism of
BP was only 3% (0.43 /uCi). 3-MC-pre-
treated trout also showed increases in
9,10-diol BP and 7,8-diol BP over control.
Control trout metabolized only 3% of the
labeled BP (0.64 //Ci), while 3-MC-pre-
treated trout metabolized 13% (2.78//CJ).
The in vitro addition of 0.1 nM BF to
microsomes from untreated catfish
caused an increase inall metabolites over
the straight control preparations, plus a
28.8% metabolism of BP (97.9 //Ci)
corresponding to the enhancement of
AHH discussed earlier. The presence of in
vitro BF in 3-MC-pretreated microsomal
4
-------
Table 1. Summary of BP Metabolite Formation as % of Metabolism
Metabolite
Catfish
Trout
Control
+BF
3-MC
+BF PCB Control 3-MC
BP-9,10-diol
BP - 4,5-diol
BP - 7.8-diol
9-OH - BP
3- OH - BP
quinones
total BP
metabolized, %
AHH
nmoles/20 min/mg
protein
1.97±15
3.77 ±.93
7.47±1.27
14.5±2.3
13.17±3.17
12.5 ±1.32
14-22
0.6
3.9
14.0
20.0
33.0
22.0
28.8
1.0
3.77±1.07
6.03±4.03
1.25±1.09
19.2 ±2.85
13.6 ±4.07
16.4 ±3.38
12-28
6.08
4.0
1.9
5.4
5.7
12.0
10
0.7
9.5
2.6
12.6
19.6
5.5
13.8
3
2.71
1.7
1.8
3.3
13.0
8.5
45.0
3
0.54
5.9
1.0
14.0
10.6
12.6
22.0
13
7.37
Data from three individual fish are given for control and 3-MC-pretreated catfish (50 mg/kg b. w. x
4 days). The concentration of BF when added was 0.1 nM. Data for trout represent "pooled"
microsomal fractions from 4 control and 3-MC treated fish (50 mg/kg b. w.).
preparations markedly inhibited formation
of most metabolites and AHH activity.
Total metabolism was only 10% (2.76
yuCi). A great deal of variation was seen in
BP metabolism among fish; however,
treated fish generally metabolized more
BP. Some 9,10-diol BP and 7,8-diol BP
formation may be at the expense of 4,5-
diol BP, which decreased in 3-MC-
pretreated trout, PCB-pretreated catfish,
and one 3-MC-pretreated catfish. This
study has demonstrated that in vivo
exposure of catfish and trout to xenobiotics
affects the in vitro metabolism of BP.
BF reduced metabolism of BP significant-
ly in samples from 3-MC-pretreated fish
and enhanced metabolism in control
samples. This correlates well with
characterization in which AHH was
enhanced in control samples by addition
of 0.1 mM BF. AHH, however, was
inhibited in microsomal fractions from 3-
MC-pretreated fish after the in vitro
addition of 0.1 mM BF. Since the
formation of every metabolite is affected,
the inhibition must take place at the
oxidase level.
Microbial Mutagen Assays
These followed standard methods
using Salmonella typhimurium tester
stains TA-1535, TA-1537, TA-98, and
TA-100. In order to incubate NADPH
generating system, S-9 and test compound
directly on petri dishes with bacterial
tester strains, an incubation temperature
of 37°C was used. In previous experiments
with catfish liver AHH, 70% of the activity
at temperature optimum (30°C) remained
when assay temperature was maintained
at 37°C. Following incubation, the
number of revertant colonies was estab-
lished by direct counting. For a biological
control, S-9 from livers of rats previously
exposed to Aroclor 1254 was used in
companion trials with fish S-9.
The results of the microbial mutagen
assay using S-9 from fish with the four
tester strains of Salmonella typhimurium
and AAF, BP and MNNG are given (Table
2). AAF and BP, when incubated with S-9
and cofactors of the MFCS, were positive
in all four tester strains. MNNG showed a
variable effect (i.e., positive in TA-100
and TA-1535 but negative in TA-98 and
minimal in TA-1537).
The effect of fish protein (S-9 concentra-
tion upon the number of revertant col-
onies observed when concentration of
test compound is kept constant (20 fjg/
plate) is shown (Figure 4).
When AAF and BP were used, in-
creases in S-9 concentration were asso-
ciated with increases in numbers of re-
vertant colonies. This response was near-
ly linear between S-9 protein concentra-
tions of 0.17 and 0.35 mg/plate but flat-
tened at higher protein concentrations.
By contrast, changes in fish S-9 protein
did not affect the number of MNNG-in-
duced revertants. This compound does
not require microsomal activation to act
as a mutagen in the Ames assay.
The microbial mutagen assay results
show that postmitochondrial supernatant
(S-9) from channel catfish liver converted
AAF and BP into mutation-causing
metabolites.
Culture of Isolated
Fish Hepatocytes
The liver is the major site of experimen-
tally-induced teleost tumors, and as such,
would provide an ideal subject for in vitro
assay. The liver also possesses an active
and inducible MFOS and has been shown
to be sensitive to a broad spectrum of
procarcinogens. The epithelial nature of
the liver also qualifies it as a model for
epithelial carcinogensis in general. For
these reasons, the culture of isolated liver
cells was attempted.
Optimal methods for isolation of catfish
liver cells were developed. Liver pieces
were trypsinized at 25°C for variable
periods of time. The results of this study
are shown in Table 3. Eight hours
trypsinization gave the greatest yield of
viable liver cells as established via trypan
blue staining and direct counting with a
hemocytometer. The yield of viable cells
was 3.25 x 106 per gram body weight (2.8
x106/g liver). An average viability (trypan
blue exclusion) of 98% was obtained.
Following isolation, liver cells were
plated on to 90 mm2 culture dishes and
allowed to stabilize for 24 hrs. Represen-
tative plates were sampled for viability by
phase contrast microscopy and trypan
blue staining after 5 and 10 days. Cells
were also sampled for electron microscopy.
The ultrastructural characteristics of
the isolated liver cells were indistinguish-
Table 2.
Induction of Mutations Using Salmonella typhimurium Strains and S-9 Fraction from
Catfish Liver
Tester Strain
TA-98
TA-100
TA-1535
TA-1537
Compound*
AAF
BP
MNNG
AAF
BP
MNNG
AAF
BP
MNNG
AAF
BP
MNNG
Revertants/ ug CMPD/mg Protein*,
72.7±21.5
155.1 ±85. 8
No response
110.7±36.1
112.0±68.0
43.2+8.3
86.0±38.0
68.5±22.3
56.6+6.8
41.8±14.8
32.9±13.3
1.3+0.4
*Compounds: AAF (2-acetylaminofluorene); BP (Benzo(aipyrene); MNNG
(N-methyl-N'-nitro-N-nitrosoguanidine).
fFive concentrations of S-9 protein (0.17 - 2.51 mg/plate) used per each of six concentrations
(5-30 ug/'plate) test compound.
Values represent the number of revertants per jjg of compound tested per mg protein ± S. D. sub-
sequent to the subtraction of the number of spontaneous revertants (background level).
-------
AAF = 2-acetylaminofluorene 20 fig/plate;
BP = benzo(a)pyrene 20 fig/plate;
MNNG =
N-methyl-N'-nitro-N-nitrosoguanidine
20 fjg/plate.
Table 3. Effect of Duration of Trypsinization on Catfish Liver Cell Yield
s
5 8
•S> 6
o
I 2
s
tL
Q:
MNNG x-
Figure 4.
0.17 0.34 0.84
Protein Concentration
Effect offish S-9 protein concen-
tration upon the number of
revertant colonies (TA-1535).
able from those of the intact liver. After 5
and 10 days of culture, increases in
glycogen content were noted. Cells
during these periods began to aggregate
andjunctional complexes were frequently
noted.
These cultures were used to determine
uptake and intracellular localization of
3H-BP. When cells were exposed to 1.25
fjg 3H-BP (32 //Ci/ml of medium) for 24
hrs. and autoradiograms prepared, grains
were concentrated over nuclei (Table 4).
Net nuclear grains (nuclear number less
cytoplasm and background) averaged 10
(Table 4). Radioactivity of DNA isolated
from liver cells following 24hrs incubation
with 3H-BP averaged 490 dpm//yg.
One limitation of studies restricted to
subcellular fractions is the occurrence of
artifacts inherent in preparation which
may damage key enzymes and/or disrupt
compartmentation of others. The use of
intact cells provides a system more akin
to that of the whole organ or tissue from
which the cell type of interest has been
taken. To date, the results have been
most encouraging. High yields of viable
cells have been routinely obtained. Initial
experiments employing autoradiographic
localization techniques after incubation
of cultures with 3H-BP showed preferen-
tial localization over liver cell nuclei. This
finding, coupled with recovery of radioac-
tivity in DMA fractions suggests interaction
of BP with cellular components. Additional
studies are needed to determine whether
binding to cellular macromolecules has
occurred and, if so, to elucidate the
Duration
hrs
2
4
8
12
Total Number
of Cells
Isolated
x10e
36.8
98.3
243.5
7.3
%
Viable
96
99
98
75
Total Number
of Cells
Per g bw
x/06
0.43
1.31
3.25
0.08
Total Number
of Viable
Cells Per g bw
x/06
0.47
1.30
3.18
0.06
Temperature of trypsinization was 25°C.
Table 4.
Localization and Quantification of 3H-BP in Subcellular Components of Cultured
Primary Fish Liver Cells
Experiment Number
Autoradiographic Analysis*
(grain counts over equal areas)
Liquid Scintillation f
(dpm/fjg DNA)
Nuclei
Cytoplasm
Intercellular
(space background)
1
2
3
33.4+14.7
30.8±11.8
37.8±12.8
18.4±6.3
19.5±7.1
15.2±5.4
6.2+1.7
6.4+1.2
5.8±1.4
480
380
630
*1xW6 cells exposed to 1.25 3H-BP (32 ud/ml of medium).
Grain counts = mean ± S.D. of 100 nuclei, 100 randomly-selected cytoplasmic and 100
randomly-selected intercellular spaces of equivalent area/experiment.
\5x1Oe cell as above.
Each experiment was performed on a separate cellular isolate from individual channel
catfish.
nature of such binding. Since the
metabolism of BP varies between species
and between organs in the same species
as well as with the type of preparation
used (i.e., whole cells, microsomal
fractions, or nuclear fractions), further
studies on metabolism, mutagenesis and
in vitro carcinogenesis using intact
epithelial cells from fish are needed.
Morphologic Findings in
Chronic Carcinogen
Exposures
Chronic dietary exposure of channel
catfish to AAF or FBPA for up to 14
months produced no grossly observable
tumors. Since major emphasis on the
morphologic alterations following carcin-
ogen exposure was placed on liver,
extensive analysis of this organ was
performed. Control channel catfish liver
of this study closely resembled earlier
published descriptions. Exocrine pancreas
was found in adventitia of portal veins.
Cords of cells existing as a dual-plated
muralium were seen. Some regions of
hepatocyte cytoplasm were opaque while
other areas were nearly transparent in
H&E stained preparations. The latter
corresponded to regions where glycogen
was present. At later time periods (12 and
14 months), regions of control liver
showed alterations in architectural
pattern. In these areas hepatocyte
margins were indistinct and areas were
interpreted as necrotic. In addition,
control liver at 14 months showed
occasional foci of round cell accumulation.
These were interpreted as inflammatory
foci and were comprised primarily of
mononuclear cells. The above were
located in portal and in some instances
midzonal regions of hepatic lobules. High
resolution light microscopic analysis
(HRLM) (toluidine blue-stained sections
of Epon-embedded material) revealed
cytologic properties of control hepatocytes.
In these, dark staining material was
arranged as a perinuclear cuff with
extensions to cell periphery. The hepato-
cytes contained one nucleus and were
cuboidal to pyramidal in shape.
After 9 months of exposure to AAF,
focal sites of necrosis with vascular
congestion were encountered in eight of
nine fish. Fat vacuoles were present in
centrolobularand midzonal regions of the
hepatic lobules. In two-thirds of the
animals, peribiliary fibrosis was seen. A
common finding in all fish chronically
exposed to carcinogens was the biliary
epithelial response. This included cyto-
plasmic vacuolization and pyknosis of
nuclei in bile ductular and ductal epithe-
lium. In addition, diffuse inflammation
throughout the hepatic lobule was noted
in one of nine fish after 9 months of
exposure.
After 12 months of exposure to AAF,
one of 12 livers showed a hyperplastic
focus in which hepatocytes contained.
-------
increased eosinophilia and basophilia,
and existed as trabeculae some 8-10cells
in thickness. Mitotic figures were seen in
some hepatocytes of the foci. Evidence of
a chronic necrotizing process was seen in
7 of the 12 animals studied. Necrotic cells
were seen throughout entire regions of
the liver. In 3 of the 12 animals, necrotic
areas were associated with localized
vascular congestion. Two-thirds of the
animals at this time showed evidence of
peribiliary fibrosis and 7 of 12 animals
showed diffuse inflammatory foci through-
out liver lobes. Fatty change was encoun-
tered in a single animal at this time.
Nonspecific tubular epithelial changes
were seen in some kidneys. All other
organs were nonremarkable. Morphologic
alteration occurred in livers of all 27 fish
surviving 14 months exposure to AAF.
Microscopic findings in other organs
were nonremarkable. In general, hepatic
architecture was maintained in all fish.
Foci and areas of cellular alterations were
encountered in 14 of 27 fish. These
lesions were typically comprised of cells
which showed increased acidophilia and
basophilia. They were larger than those
seen at 12 months. In addition, these
clumps of cells showed numerous mitotic
figures. No tumors were encountered in
any of the fish studied at this time.
Hepatocyte nuclei in foci of necrotic
hepatocytes was observed. Two of the
five fish showed small foci of inflammatory
cells in liver parenchyma. Cytologic
features of individual hepatocytes were
identical to that described in control fish.
Four fish were killed after 2 months of
exposure to FBPA and were processed for
HRLM. In these individuals, normal liver
architectural pattern was seen. In two of
the four animals, small areas were
observed in which cells showed a
disorganized pattern of dark staining
material and it was difficult to distinguish
between individual cells in these areas.
These foci were interpreted as necrotic.
No fat was observed. Bile ductular
proliferation was not seen. Peribiliary
fibrosis occurred in two of the four
animals studied. All other features were
identical to controls. After 3 months of
exposure to the compound, the only
alterations observed were small sites of
necrosis similar to areas described above.
Cytologic features of individual hepatocytes
were identical to controls. After 6 months
of exposure to FBPA, a normal liver
architectural pattern was seen. In one of
three treated fish, dark staining regions
within cytoplasm were irregularlyarranged;
however, the response was focal. No
necrosis, fat, bile duct proliferation,
•eribiliary fibrosis or inflammation was
observed. Cytology of individual hepato-
cytes revealed dispersal of dark staining
material peripherally with central portion
of cells containing light staining material.
This pattern of cytoplasmic rearrangement
is similar to that seen in hepatocytes in
which smooth endoplasmic reticulum
has proliferated. Livers from six treated
and six control fish were studied by
routine light microscopy after 9 months of
exposure to FBPA. In general, liver
architecture was well-maintained. How-
ever, in two of the six fish, areas were
seen in which sinusoids were not
apparent. Cytoplasm of cells in these
regions revealed a hyalinized appearance.
Necrosis was not found at this time. Fat
was seen as clear vacuoles within
hepatocytes in one of the six animals. In
addition, the same liver showed diffuse
small foci of inflammatory cells. After 12
months of exposure to FBPA, five of six
livers observed showed normal architec-
tural pattern. In the other, the outer
margins of the liver revealed numerous
bulges and alternating constrictions
resulting in a "scalloped" appearance.
Necrosis was seen in two of the six livers
examined at this time. Sites of necrosis
and pyknosis were common. In three of
the six livers, areas were seen in which
cytoplasm of hepatocytes revealed in-
creased opacity. In addition, these areas
were sites of vascular congestion. Fat
was observed in two of the six animals as
clear vacuoles within hepatocytes of
centrolobular and midzonal regions. Bile
duct proliferation, not seen in animals
exposed for shorter periods of time to
FBPA, now appeared in four of the six
animals. Bile stasis was indicated by
expanded, perfectly rounded lumina of
ductules and ducts and thin lining
epithelial cells suggested increase intra-
luminal pressure. Five of the six animals
studied showed inflammation particularly
pericholangitis. Numerous mitotic figures
were seen in bile ductular epithelium. In
livers of three fish, bile ductal epithelial
hyperplasia was apparent. One of these
had progressed to papillarly projections
within the lumen. This configuration with
numerous mitotic figures is consistent
with a diagnosis of cholangioma.
When compared to controls maintained
for identical periods of time, livers of
channel catfish exposed to FBPA for 14
months revealed changes. Both control
and treated livers showed necrotic areas
although these were by far greater in
treated fish. Areas of necrosis in both
groups were associated with fibrosis.
Granulomas were seen in the liver of one
control fish but not in treatedfish. Hepatic
architecture was similar in both groups.
However, cytoplasm of hepatocytes from
treated fish contained more stamable
area—often completely filling the cell.
Controls apparently contained abundant
glycogen which, by H&E, did not stain,
making these cells nearly transparent.
Cytologic features of hepatocytes from
the 14-month FBPA group included
abundant acidophilic inclusions which
were frequently as large as nuclei. In
basophilic regions of cytoplasm, a fine
vacuolization was seen. Nuclei, generally
one per cell, were lucent, rounded and
had a single prominent nucleolus.
Treated livers revealed oval cell prolifera-
tion in 15 of 32 fish surviving the 14-
month exposure. Peribiliary fibrosis,
percholangitis and melanomacrophage
centers were common. In 8 of 32 livers,
areas were seen in which cytoplasm
appeared hyalinized and gave a general
basophilic hue with H&E. Nuclei in these
regions were more opaque and generally
uniform. However, some nuclei were
enlarged, oval in shape and indented.
Cells in these regions existed as con-
tinuous sheets and it was difficult to
visualize sinusoids. In one fish, the above
described region was seen as a nodule
which compressed adjacent hepatocytes.
This led to a diagnosis of minimal
deviation hepatocellular carcinoma. Mito-
tic figures were seen in the above.
In the interpretation of liver lesions,
previous reports in various teleost
species were reviewed. The response of
the channel catfish liver to AAF and FBPA
was encouraging. However, the latency
period was long. In light of the amount of
water required to maintain large numbers
of this size fish and the problem of
disposal of large amounts of contaminated
water, use of smaller species such as
small aquarium fishes may be advisable.
Further study of carcinogenesis in the
catfish could be extended to egg, embryo-
larval exposures in which concentrated
dosages of carcinogen are followed by
rinsing and subsequent culture under
routine conditions. These procedures
may constitute a feasible alternative to
the above-noted problems. Egg, embryo-
larval exposures need to be extended to
other species in an effort to determine an
ubiquitous, sensitive, indicator organism
for aquatic carcinogen bioassay.
In light of the projected increasing
national reliance on coal-based energy
production, regional contributions of
environmental PAH from high point
source emission associated with coal
combustion, coking and conversion
processes require surveillance of environ-
mental quality. The response of the
channel catfish to BP and 3-MC coupled
-------
with its ubiquitous occurrence in streams
and lakes gives added importance to
continued studies with this species
including: varying routes of exposure,
bioaccumulation, acute and chronic
morphologic alterations and possible
tumorigenesis. Such would help to
determine the suitability of this species to
serve as a sentinel organism for aquatic-
borne PAH in fresh water habitats.
Conclusions
Based upon the data obtained from this
study, the following conclusions have
been made.
• All fish species studied were found to
contain a hepatic mixed-function
oxidative system (MFOS) qualitatively
similar to that of other teleosts and
rodents.
• Enzyme activity of the fish MFOS was
increased by exposure to polychlorin-
ated biphenyls and polycyclic aromatic
hydrocarbons, benzo(a)pyrene (BP)
and 3-methylcholanthrene (3-MC).
• Increased enzyme activity correlated
with proliferation of endoplasmic
reticulum of hepatocytes.
• Channel catfish and rainbow trout
liver microsomal fractions metabolized
BP into diols, hydroxy derivatives, and
quinones.
• Pretreatment of catfish and trout with
3-MC increased formation of 7,8- and
9,10-diols of BP. Since diol formation
proceeds via epoxide formation,
evidence indicates fish MFOS activated
BP to a reactive carcinogen.
• Catfish MFOS formed mutagenic
intermediates of BP and AAF in a
microbial mutagen system.
• Isolated primary hepatocytes of chan-
nel catfish can be maintained with
high viability for 10 days and permit
detailed analysis of cellular events in
chemical carcinogenesis as weft" as
direct interspecies comparison.
The presence of foci and areas of
hepatocellular alteration, nodular
lesions and bile ductular hyperplasia
suggest neoplastic responses to
chronic carcinogen exposure in chan-
nel catfish liver.
It is feasible to use fish to test for the
presence of mutagenic/carcinogenic
substances in the aquatic environment
and to determine whether new chem-
ical formulations, proposed for wide-
spread usage, would have mutagenic/
carcinogenic potential in aquatic
species.
The use of fish species with relatively
large body size coupled with the
chronic nature of laboratory exposures
designed to demonstrate carcinogeni-
city of compounds results in sizeable
quantities of contaminated water
which require decontamination prior
to discharge. The volume of contamin-
ated water can be diminished by use
of closed system exposures but
remains a problem to be considered in
projects of this nature.
The establishment of regional centers
of excellence to provide a safe
environment for testing of potentially
carcinogenic substances under con-
trolled conditions is strongly recom-
mended. Such would provide opportun-
ity for collaboration between govern-
ment, industrial and university person-
nel to establish necessary prerequisites
for aquatic carcinogenesis bioassay.
Defined nutritional requirements of
these species used in long term assay
are needed.
D. E, Hinton is with West Virginia University. Morgantown, WV 26506; J. E.
Klaunig is with the Medical College of Ohio. Toledo, OH 43699; M. M. Lipsky. R.
M. Jack, and B. F. Trump are with the University of Maryland School of
Medicine. Baltimore, MD 21201.
Gary E. Glass is the EPA Project Officer (see below).
The complete report, entitled "Detection of Mutagenic/Carcinogenic Alteration
in Fish." (Order No. PB 83-253 559; Cost: $ 13.00, subject to change) will be
available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Environmental Research Laboratory
U.S. Environmental Protection Agency
6201 Congdon Blvd.
Duluth, MN. 55804
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Official Business
Penalty for Private Use $300
------- |