AN ABSTRACT OF THE THESIS OF
()Ilan
Zhang
for
the degree of
Master of Science
in the
Department of Fisheries and Wildlife presented on Mav 7, 1992
Title: Temperature Modulated Aflatoxin BI Hepatic Disposition,
and Formation and Persistence of DNA Adducts in Rainbow
Trout.
Redacted for Privacy
Abstract approved:_
Lawrence R. Curtis
Previous work showed that incidence of chemically-induced
tumors
in
fish
increased with environmental
temperature
(Hendricks et al., 1984; Kyono-Hamaguchi, 1984).
The present
study assessed genotoxicity as a mechanism to explain such
results.
Rainbow trout (2 g) were acclimated to 10 or 18°C
for one month, then immersed in 0.1 ppm [3H]aflatoxin Bl (AFB1)
solutions
for
30
min
temperatures, or at 14°C.
at
their
respective
acclimation
The total radioactivity in liver
immediately after immersions was 50% higher for 18 than 10°C
acclimated
and exposed
fish.
Conversely,
adduction
of
[3H]AFB1 to hepatic DNA of 10°C acclimated fish was higher
than for 18°C fish after exposure.
A similar DNA adduction
result as above was observed after varying [3H]AFB1 immersion
concentrations
equivalents.
to
achieve
similar
hepatic
[3H]AFB1
After acute shifts in temperature (10 to 14°C,
or 18 to 14°C), no differences were found in hepatic [3H]AFB1
equivalents.
However, adduction of [3H)AFB1 to hepatic DNA
was higher for 10 than 18°C acclimated fish 1 day after
exposures at 14°C.
This was probably explained by effects of
temperature acclimation on the status of the
membrane-bound
cytochrome P-450 system, affecting its competition
with a
detoxifying cytosolic enzyme.
After [3H]AFB1 immersions at
respective acclimation temperatures,
or acute temperature
shifts to 14°C, DNA adducts were less persistent in fish
maintained for 21 days at 18 than 10°C.
Our data demonstrated
temperature-modulated AFB1 genotoxicity occurred via three
mechanisms:
hepatic
disposition,
and
formation
and
persistence of DNA adducts.
Temperature Modulated Aflatoxin B1 Hepatic Disposition,
and Formation and Persistence of DNA Adducts in
Rainbow Trout
by
Quan Zhang
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Completed May 7, 1992
Commencement June, 1992
APPROVED:
Redacted for Privacy
Professor of Fisheries and Wildlife in charge
of major
Redacted for Privacy
Chairman of department of Fisheries and Wildlife
Redacted for Privacy
Dean of Graduate
Date thesis is presented
Typed by Kelly Schmidt for
May 7, 1992
Quan Zhang
ACKNOWLEDGEMENT
My sincere appreciation goes to Dr. Larry Curtis,
my
major professor, for his guidance, patience,
encouragement and
support throughout my whole education and this
research.
I am especially grateful to Dr.
George S Bailey
Mr.
Wayne Seim and Dr. Hillary Carpenter for their intellectual
,
advise on technical aspects of this project.
I am thankful for the friendship, and
assistance of the
graduate students and staff at Oak Creek
Laboratory of
Biology, particularly Katherine Super for
her cooperation,
Curt
Bambush
for
his
knowledge,
maintenance of
the
experimental equipment, and Ms Kelly Schmidt
for manuscript
preparation.
This investigation was supported by
a research grant
(No.ES04766) from the National Institutes
of Environmental
Health Sciences.
TABLE OF CONTENTS
INTRODUCTION
1
MATERIALS AND METHODS
4
Animals
4
Chemicals
4
Hepatic Accumulation of AFB1
5
Adduction of AFB1 to Hepatic DNA
5
Analysis of AFB1 Metabolites
9
Statistics
RESULTS
10
11
Hepatic Accumulation of AFB1
11
Adduction of AFB1 to Hepatic DNA
14
Analysis of Hepatic AFB1 Metabolites
18
DISCUSSION
Hepatic AFB1 Disposition
20
20
AFL Formation Shifts DNA Adduction
21
Temperature Effects on DNA Adduction
23
Persistence of DNA Adducts
24
DNA Adduction and Tumor Incidence
25
LITERATURE CITED
27
APPENDIX
Appendix A : in vitro AFB1 -DNA Binding and
Biotransformation of AFB1 to AFL
36
Figure
LIST OF FIGURES
1. Effect of time and temperature on hepatic
disposition of total [3H]AFB1 equivalents in rainbow
trout after immersion exposures.
2. [3H]AFB1 hepatic disposition and DNA
in rainbow trout exposed to 0.1 ppm AFB1adduction
for 30 min
at their respective acclimation temperatures
(10 or 18°C).
3. [3H]AFB1 hepatic disposition and DNA
adduction
in 10 or 18°C acclimated rainbow
trout
exposed
to
either 0.12 or 0.08 ppm AFB1 respectively
at their
acclimation temperatures for 30 min.
4. [3H]AFB1 hepatic disposition and DNA
binding in 10 or 18°C acclimated rainbowcovalent
trout
exposed at 14°C to 0.1 ppm [3H]AFB1 for 30 min.
page
12
15
16
17
Table
LIST OF TABLES
page
1. Manipulations of temperature used for AFB -DNA
binding bioassay in rainbow trout.
7
2. Effect of temperature on biological half-life
of
total [3H]AFB1 equivalent in rainbow trout
after
immersion exposures.
13
3. Effect of temperature on in vivo
hepatic [3H]AFB1
metabolism in rainbow trout immediately
after
immersion in 0.1 ppm [3H]AFB1 for 30 min.
19
LIST OF FIGURES IN APPENDIX
Figure
Page
1. Effect of temperature on Tris buffer pH.
38
2. Effect of pH on in vitro AFB1 DNA binding.
39
3. Effect of microsome concentration and incubation
time on in vitro AFB1 -DNA binding.
40
4. Arrhenius plots of in vitro AFB1 -DNA binding by
rainbow trout hepatic microsomes.
41
5. Arrhenius plots of in vitro AFB1 -DNA binding by
rainbow trout hepatic microsomes treated with
a-naphthoflavone (ANF).
42
6. Effect of rainbow trout microsomal and
cytosolic
enzymes on in vitro metabolism of aflatoxin
Br
43
7. Effect of temperature on in vitro biotransformation
of AFB1 to AFL catalyzed by cytosolic
enzymes.
44
Temperature Modulated Aflatoxin B1 Hepatic
Disposition, and
Formation and Persistence of DNA Adducts in
Rainbow Trout
Introduction
Temperature influences incidence of
chemically-induced
tumors in fish (Hendricks et al., 1984;
Kyono-Hamaguchi,
1984), with warmer temperatures being
associated with higher
cancer incidence.
The mechanisms for temperature-modulated
chemical carcinogenesis in fish, however,
have not been
described
in
detail.
Liver
is
a
major
site
of
the
tumorigenesis in rainbow trout following
exposure to many
chemical
carcinogens
including
aflatoxin B1
(AFB1)and
benzo[a]pyrene
(Bailey
and
Hendricks,
1988).
The
carcinogenicity of these and many other
agents is linked to
their
metabolism
and
subsequent
binding
of
reactive
metabolites to DNA (Scribner, 1985; Silber
and Loeb, 1985).
More specifically, AFB1 genotoxicity is
associated with AFB18,9- epoxide binding with DNA to form
the principal covalent
product
8,9-dihydro-8-(N7-guany)-9-hydroxyaflatoxin B1 in
rainbow trout (Croy et al., 1980) and
rodents (Croy and Wogan,
1981)
.
To
better
understand
temperature's
role
in
fish
carcinogenesis,
we have employed warm or cold temperature
acclimation of rainbow trout. Over a month-long
temperature
acclimation period, biochemical adaptations
occur which allow
2
physiological processes to continue at similar
rates at the
new temperature; that is, an "ideal temperature
compensation"
occurs (Precht, 1958).
Supporting this hypothesis, hepatic
microsomes from cold and warm acclimated
fish exhibit similar
monooxygenase activities when assayed at their
respective
acclimation temperatures (Blanck et al., 1989;
Koivusaari et
al., 1981).
termed
Adaptive change in membrane lipid composition,
"homeoviscous
adaptation"
(Sinensky,
1974),
helps
explain this result.
In fish acclimated to cold relative to
warm water, a higher proportion of unsaturated and
long-chain
fatty acids are incorporated in membranes
(Hazel, 1979; 1984).
Thus,
enzyme
membrane fluidity is maintained and
activities
are
similar
at
membrane-bound
different
acclimation
temperatures.
When cold acclimated fish experience
acute temperature
increase, a temperature-induced phase transition
may occur
such that membranes become more fluid than
at the acclimation
temperature.
Greater
membrane
fluidity
may
enhance
accessibility to active sites, and
higher activity
of
membrane-bound enzymes may result.
To the contrary, when warm
acclimated fish experience an acute temperature
decrease,
membranes may become more rigid, and lower
microsomal enzyme
activities may result.
Indeed, when assayed in vitro at a
common,
intermediate
temperature,
microsomes
from
cold
acclimated fish exhibit much higher activity
than those from
warm acclimated fish (Egaas and Varanasi,
1982; Carpenter et
3
al., 1990).
Furthermore, acute temperature changes
modulated
in vivo benzo(a)pyrene metabolism in rainbow
trout in a manner
consistent with the above arguments (Curtis
et al., 1990).
All results reviewed
hypothesis that
above are consistent with the
temperature-induced phase
transitions
of
membrane lipid biophysical states can affect
cytochrome P-450
activities in fish. Microsomal
monooxygenases metabolize many
precarcinogens (including AFB1) to ultimate
carcinogens (Croy
et al., 1980; Scribner, 1985; Silber and Loeb,
1985). In the
case of AFB1 a major detoxication pathway
is formation of
aflatoxicol by a cytosolic enzyme
(Schoenhard et al., 1976).
The soluble enzyme may respond to
temperature changes in a
different manner than membrane-bound
cytochrome P-450s.
Temperature acclimation-dependent modification of membranes
in
fish combined with acute temperature
changes could influence
bioactivation and,
thus,
modulate genotoxicity without
xenobiotic pretreatment. The primary objective
of the present
study
is
investigation
of
temperature-modulated
AFB1
genotoxicity as a mechanism for temperature
effects on
hepatocarcinogenesis in rainbow trout. Therefore, fish
are of
a size and age commonly used for
initiation with chemical
carcinogens in rainbow trout tumor
studies (Bailey and
Hendricks, 1988).
4
Materials and Methods
Animals
Shasta strain rainbow trout (Oncorhynchus
mvkiss) were
provided by the Marine and Freshwater
Biomedical Center core
facility at Oregon State University.
These fish weighed
approximately 2 g and were acclimated at either 10
or 18°C for
one month and fed Oregon Test Diet (Sinnhuber
et al., 1977).
Rations were adjusted to maintain similar
growth rates between
acclimation groups (Kemp and Curtis,
1987).
During the
experiments, fish were held in flow-through
tanks and kept
under a 12 hr light-dark cycle.
The fish were not
fed for 48
hr before ( 3H)AFB1 exposure.
Chemicals
Uniformly labeled [4.1)AFB1 (24 mmol/mCi in
methanol) was
purchased from Moravek Biochemicals,
purity
verified
by
TLC
on
Inc.
silica
(Brea,
gel
CA.)
and
G
with
benzene:acetone:ethylacetate (55:15:30) and by radioscanning.
Unlabelled AFB1 (Sigma Chemical, Co., St.
Louis, MO, or
Aldrich Chemical Co., Milwaukee, WI)
stock solutions were
prepared by injection of methylene chloride
into a sealed vial
of crystals and transferring the
resultant solutions
volumetric
flasks.
The
solution
concentrations
to
were
determined by making an approximately 2-4
ppm solution in
ethanol, reading the absorbance at 362 nm, and calculating
with an extinction coefficient
of 21,800.
Purity of
unlabelled AFB1 was verified by TLC
as described above and by
5
scanning spectrophotometry from 220-440
nm (Rodricks et al.,
1970).
Working [3H]AFB1 solutions were prepared
in ethanol
while removing methanol and methylene
chloride by rotary
evaporation.
The ethanol concentration of exposure solutions
was 0.1%.
Hepatic Accumulation of AFB1
The effects of time and temperature on total [3H]AFB1
equivalents in rainbow trout liver
were determined by
immersing fish fry in 0.07 ppm [3H]AFB1 solution
for 30 min at
their acclimation temperatures (10
or 18°C) or an intermediate
temperature
(14°C).
The
fish
were
then
maintained
in
uncontaminated water at their respective
exposure temperatures
for an additional 0.25, 1, 4 and 24 hr.
Six fish were killed
after each interval from each temperature
and two groups of
three livers were pooled.
The total [3H]AFB1 equivalent
residues were quantified
by liquid scintillation counting
(LSC) after base digestion and
decolorization (Carpenter
and
Curtis, 1989).
Half-life of elimination was calculated for
a
one-compartment, first order model as described by O'Flaherty
(1981)
.
Adduction of AFB1 to Hepatic DNA
Immersions were
also
conducted
for
all
temperature
regimens with 0.1 ppm [3H]AFB1 for 30 min.
After these
immersions 9 to 18 fish from each
regimen were
immediately
killed and groups of 3 livers
were pooled (3 to 6 individual
samples per regimen) and homogenized
at 4°C as described
6
below.
The
remaining
animals
were
transferred
to
uncontaminated water for one day in tanks
maintained at their
exposure temperature
and equipped with submersed charcoal
filters.
After one day fish were returned
to their
acclimation temperature for an additional 2 or 20 days (Table
1).
After each interval, 9 to 18 fish from
each group were
killed and groups of 3 livers
were extracted to determine the
concentrations of [3H]AFB1- derived DNA adducts.
In the next
experiment, the [3H]AFB1 concentrations of immersion
solutions
were varied between 0.08
and 0.12 mg/1 so that equivalent
target organ (liver) residual 3H
was observed in 10 and 18°Cacclimated and exposed fish. Here, 10°C fish
were immersed in
0.12 ppm and 18°C fish were immersed in 0.08
ppm [3H]AFB1 for
30 min.
Livers were taken from nine fish and
pooled in three
groups of three immediately after immersions
and target organ
[3H]AFB1 was characterized
as described below. Fish were held
as
described above for 1,
3
or
21 days
and liver
concentrations
of
[3H]AFB1-derived
DNA
adducts
were
determined.
Each DNA sample analyzed consisted of
three pooled livers
with a combined weight of approximately
100 mg.
Each sample
was homogenized in
chloride,
1.5 ml
of DNA buffer
50 mM sodium-4-aminosalicylate,
(0.1 M sodium
pH
7.2).
The
homogenates were vortexed in 1.5 ml lysis
buffer (2% sodium
dodecyl-sulphate,
2%
sodium
chloride,
12%
sodium-4aminosalicylate, 12% 2-butanol) on a rotary
shaker for 1 hr
7
TABLE 1.
Manipulations of temperature used for AFB1 -DNA
binding bioassay in rainbow trout.
TEMPERATURE REGIMEN (°C)
Groups
10-10
10-14
18-14
18-18
Acclimation 30 days
10
10
18
18
Exposure 0.5 hour
10
14
14
18
Rearing 1 day
10
14
14
18
10
10
18
18
Rearing 20 days
8
and then shaken for another hour
with 1.5 ml 4% isoamyl
alcohol/96% chloroform (IAC) and 1.5 ml of phenol, which
was
presaturated with an equal volume of 1 M Tris and
phenol
saturation solution (300 mM sodium chloride,
100 mM Tris, pH
8.0).
After centrifugation at 3000 g for 15 min, the
upper
aqueous layers were transferred to
a new tube and extracted
twice with equal volumes of IAC. DNA was precipitated
with
0.4 ml of 5 M sodium perchlorate and
3
volumes of cold
ethanol.
The precipitates were redissolved in 1.5
ml of DNA
buffer for 24 hr. The nucleic acids
were digested with 100 Al
RNase (RNase A, 50 mg/ml, Sigma R-5000;
RNase 5000 units/ml,
Sigma R-8251) at 37°C for 1 hr, and
then RNA and proteins were
removed by adding 10 Al of proteinase k
incubating for another 2 hr at 37°C.
(
20 mg/ml)
and
The nucleic acid
mixtures were then extracted with Tris
saturated phenol and
IAC.
After centrifugation, the aqueous
upper layers were
transferred to clean centrifuge tubes
and extracted three
times with IAC alone.
Finally, the DNA was isolated by
precipitation with sodium perchlorate
and cold ethanol as
mentioned above. The concentration
of DNA was determined by
the Burton (1956) method.
Briefly, DNA was redissolved in
distilled water and hydrolyzed by
heating for 20 min in 1 M
perchloric acid at 70 °C.
The hydrolyzed DNA samples and
standards were incubated at 30°C in the
dark for 18 hr with
0.5 M perchloric acid and Burton
reagent (diphenylamine,
glacial acetic acid,
concentrated sulfuric acid,
1.6%
9
acetaldehyde:
1.5/100/1.5/0.5, w/v/v/v), then quantified by
measuring the UV absorption at 595 nm.
adducts were determined by LSC, using
solubilizer
and
AquasolR
as
scintillant.
The [3H]AFB1-DNA
ProtosolR
tissue
Results were
expressed as nmol of [3]flAFB1 per g of DNA.
Analysis of AFB1 Metabolites
Livers removed immediately after
immersions at 0.1 ppm
were homogenized in
0.8 ml of distilled water and then
vortexed for 3 min after addition of 2 ml of ethanol.
hepatic [3HJAFB1 equivalents were
determined by
duplicate
0.1
ml
aliquot
of
homogenate.
The
Total
LSC
of
remaining
homogenate then was vortexed for 3 min
after addition of 3 ml
of hexane and centrifuged at 8000
g for 5 min.
The hexane
layer was aspirated, found to contain
less than 1% of total
OHJAFB1 and discarded. The aqueous layer
was adjusted to 10%
methanol and applied to a C18 Sep PakR (Waters
Associates,
Inc., Milford, MA.) column with a syringe.
The columns were
rinsed with 15 ml of 10% methanol in 20 mM potassium
acetate
buffer followed with 10 ml of 95%
methanol in this buffer.
The 95% methanol eluent was evaporated
to 0.3 ml and separated
by reverse phase HPLC on a jBondapak
Cui column with 10 mM
potassium
acetate
(pH
5.0)
acetonitrile-methanoltetrahydrofuran (57:16:22:3)
at 1 ml/min.
An Applied
Biosystems (Ramsey, NJ)
Model 757 u.v. detector monitored
flow off the column at 345 nm.
Thirty-drop fractions were
collected for determination of radioactivity
by LSC.
Known
10
standards for AFB1, aflatoxicol and aflatoxin
M1 served for
metabolite
identification.
Recovery
of
radioactivity
throughout the analysis was 86%.
Statistics
The time-course data for hepatic [41]AFB1
disposition and
[3H]AFB1 -DNA adduct concentrations
for the fish from different
temperature regimens were compared by two-way
analysis of
variance (ANOVA) followed by point
comparison using the Tukey
test.
Comparisons of two means were made by the
t-test.
Results were expressed as means ± SE.
11
Results
Hepatic Accumulation of AFB1
This experiment
employed
four temperature
regimens:
acclimation and exposure at 10°C, acclimation
and exposure at
18°C, acclimation at 10°C with acute
temperature shift to 14°C
for exposure, and acclimation at 18°C with
acute temperatureshift to 14°C for exposure (Table 1).
These groups hence will
be described respectively as
10-10°C, 18-18°C, 10-14°C, and
18-14°C.
Exposure temperature significantly affected
total [31-1]AFB1
equivalents in rainbow trout liver during
the first 4 hr after
30 min immersions in 0.07 mg[311]AFB1/1
(Fig.
1).
For fish
exposed at their respective acclimation
temperatures and to
the
same
(0.07
ppm
[3H]AFB1)
concentration,
hepatic
concentrations were significantly higher
for 18-18°C than 1010°C fish at 0.25, 1 and 4 hr after
immersions (p=0.0003,
0.0009 and 0.0016 respectively).
For acute temperature
shifted fish, 18-14°C fish had higher
hepatic concentrations
of total [3]flAFB
equivalents 0.25 hr after immersion than 1014°C fish (p=0.0025).
1
Total hepatic [3]flAFB1 equivalents declined
to similar
concentrations in all temperature regimens
after 24 hr. The
half-life of elimination for total [3H]AFB1
equivalents was
about twice as long in the liver
from 10°C acclimated and
exposed fish than other groups
(Table 2).
Half-lifes
calculated from mean liver concentrations
at 1, 4 and 24 hr
12
1.8
1.5
1.2
0.9
0.6
0.3
0.0
0
5
10
15
20
25
Time (hours)
0
0 18-18 °C
- 18-14 °C
A
A 10-14 °C
A 10-10 °C
Figure 1.
Effect of time and temperature on hepatic
disposition of total (41]AFB1 equivalents in rainbow trout
after immersion exposures. Both 10 or 18°C acclimated rainbow
trout fry were immersed in 0.07 ppm [3H]AFB1 solution for 30
min at their respective acclimation temperatures
or an
intermediate temperature (14°C).
Fish were transferred to
clean water and then maintained at their respective exposure
temperatures for an additional 0.25, 1, 4 and 24 hr. Results
are means ± SEM for n=6 individual fish.
One asterisk
indicates a significant difference between 10-14°C and 18-14°C
at 0.25 hr after exposure. Two asterisks indicate significant
differences between 10-10°C and 18-18°C at various times.
13
TABLE 2.
Effect of temperature on biological half-life of
total [3H]AFB1 equivalent in rainbow trout after
immersion exposures.
Temp. (°C)
B
R-squared(%)
Halflife (hr)
18-18
18-14
10-14
10-10
0.0357
0.0395
0.0309
0.0151
97.67
99.88
98.42
90.64
18.6
18.1
22.0
Results are for 12 groups of 6 pooled livers.
Equation: Y=exp(a+bX) & log(Y)=a+bX
43.2
14
after immersions were
14°C,
hr;
18
10-10°C, 43 hr; 10-14°C, 22 hr; 18-
:
18-18°C,
19
hr
(respective
correlation
coefficients for regressions which yielded slope estimates
were 0.91, 0.98,
1.00, and 0.98).
Adduction of AFB1 to Hepatic DNA
Hepatic concentrations of total
immediately
after
a
30
min
[3H]AFB1
immersion
in
equivalents
ppm were
0.1
significantly (50%) higher in 18-18°C fish than 10-10°C fish
(Fig.
2,
inset).
Despite this, hepatic concentrations
of
[3H]AFB1 DNA adducts were significantly higher 1 day after
immersions in 0.1 ppm [3H]AFB1 in 10 than 18°C acclimated fish
(p=0.0038, Fig. 2).
In addition, higher concentrations of
hepatic [3H] AFB1-DNA adducts were observed in 10 than 18°C
acclimated fish after varying [3H]AFB1 exposure concentrations
to 0.12 and 0.08 ppm, respectively to achieve equivalent
target organ concentrations immediately after immersions (Fig.
3) .
There
were
no
significant
differences
in
hepatic
concentrations of total [3H]AFB1 equivalents for 10-14°C and
18-14°
fish immediately after immersion
(Fig.
4,
inset).
However, the concentration of hepatic [3H]AFB1 DNA adducts was
2-fold higher 1 day after immersion for 10-14°C than 18-14°C
fish (p=0.022).
Hepatic [3H]AFB1 adducts appeared somewhat
more persistent in 10°C than 18°C fish exposed
at their
respective acclimation temperatures (Fig. 2) or at 14°C (Fig.
4). From 1 to 21 days after exposure, the average concentration
15
40
Time = 0
30
20
0.0
10 -10°C
18 -18°C
10
10-10
18-18
Temperature Regimen
1 day
Figure 2.
KNI
3 days
1..
(°C)
21 days
[3H]AFB1 hepatic disposition and DNA adduction in
rainbow trout exposed to 0.1 ppm AFB1 for 30 min at their
respective acclimation temperatures (10 or 18°C). Total
[3H]AFB1 hepatic disposition was determined immediately after
immersion while DNA adduction was measured after 1, 3, or 21
days.
Results are means ± SEM for three groups and three
pooled livers, except at three days where results are for six
groups of 3 pooled livers. Asterisks indicate significant
differences between temperature regimens at particular times
after exposure.
16
35
CI
Time = 0
30
on
10-10C
15-15C
0
10-10
18-18
Temperature Regimen (°C)
1 day
k\-1
3 days
A
21 days
Figure 3.
[3H]AFB1 hepatic
and DNA adduction in
10 or 18°C acclimated rainbowdisposition
trout exposed to either 0.12 or
0.08 ppm AFB1 respectively at their acclimation
temperatures
for 30 min.
Total [3H]AFB1
disposition was determined
immediately after immersion while DNA adduction
was measured
after 1, 3, 7 or 21 days.
Results are means ± SEM for three
groups of three pooled livers. Asterisks indicate
significant
differences between temperature regimens
at particular times
after exposure.
17
(4-
Z
50
S-o
Q) 1.5
tu)
0
0
Time = 0
ri>
..-1
.--0
40
r
00
'''. 1.0
T
0
5
0
'sr* 0.5
30
1-1
12:1
t10
0
;a4
0.0
*
20
10-14°C
18-14C
r4.5
<4
10
*
P
0
10-14
18-14
Temperature Regimen (°C)
1 day
Figure 4.
IN \
3 days
... A
21 days
[3H]AFB1 hepatic disposition and DNA covalent
binding in 10 or 18°C acclimated rainbow trout
exposed at 14°C
to 0.1 ppm [31.1]AFB1 for 30 min.
Fish were maintained at 14°C
for 24 hr after immersion and then
returned to their
acclimation temperature.
Total [31-1]AFB1 disposition was
determined immediately after immersion while DNA adduction was
measured after 1, 3, or 21 days. Results are means ± SEM
for
five groups of three pooled livers.
Asterisks
indicate
significant differences between temperature
regimens at
particular times after exposure.
18
concentration of adducts decreased 15 ± 4% when
all data for
all 10°C acclimated fish were pooled. The pooled average
for
adduct loss in 18°C-acclimated fish was 30 ± 10%
after 21
days.
Because all
exposure groups were held at their
acclimation temperatures for at least 20 of 21 days after
exposure,
pooling
data
for
this
comparison
was
deemed
appropriate.
Analysis of Hepatic AFB1 Metabolites
HPLC analysis of liver extracts prepared
immediately
after exposure to [3H]AFB1 indicated unreacted
parent [3H]AFB1
constituted about 70% of total
radioactivity at all
temperatures (Table 3).
There were no significant differences
in concentrations of aflatoxicol
or AFM1 extracted from livers
of fish between temperature
regimens.
There were no
significant peaks other than those which
co-eluted with AFB1,
AFM1 and aflatoxicol standards.
No AFM1 or aflatoxicol
occurred in chromatographic profiles of liver
homogenates from
untreated fish to which [3H]AFB1 was added in vitro (data
not
shown).
19
TABLE 3.
Effect of temperature on in vivo hepatic [3H]AFB1
metabolism in rainbow trout immediately
after
immersion in 0.1 ppm [3H]AFB1 for 30 min.
TEMPERATURE REGIMEN (°C)
10-10
AFB
1
AFM
10-14
18-14
18-18
%
71.7±9.4
76.0±8.9
65.6±3.5
66.3±8.8
%
24.1±8.9
18.9±8.6
28.9±5.0
29.9±7.3
4.2±1.9
4.9±2.9
5.4±2.4
3.7±2.3
1
AFL%
Results are means ± SD of the percentage of total recovered
[3H]AFB1 equivalents found in three
HPLC peaks for three
groups of three pooled livers.
Details for the assay were
presented in the text.
Extraction efficiency of [ 5H]AFB1
added to liver homogenates
of untreated trout was 92%.
Recovery of total radioactivity from the "clean-up"
column was
95%.
20
Discussion
Hepatic AFB1 Disposition
The liver burden of total [3H]AFB1 equivalents
up to 4 hr after
immersions increased with increased
exposure temperature
(Figs. 1-3).
This may be partially explained by increased
hepatic blood flow (Kemp and Curtis, 1987)
and ventilation
associated with higher oxygen demand at warmer
temperatures
leading to greater gill uptake and distribution
of [3H]AFB1 to
liver.
Temperature acclimation
per se may have influenced
hepatic AFB1 disposition
as
indicated by higher
hepatic
concentrations of [3H]AFB1 in livers of
18-14°C than 10-14°C
fish 0.25 hr after 0.07 ppm immersions (Fig. 1).
However, we
concluded that acclimation was not a
major factor in
disposition
because
hepatic
concentrations
were
not
significantly different immediately after 0.1
ppm immersions
(Fig. 4, inset).
HPLC analysis (Table 3) of liver extracted
immediately
after
immersion confirmed that total
hepatic
[3H]AFB1
concentrations at this time were reasonable
estimates of
target organ dose in various temperature
regimens.
This
allowed us to design exposures in which
temperature-dependent
differences in [3H]AFB1 DNA adduction
were more readily
explained by intrahepatic events.
Incidence of AFB1-induced
liver cancer and formation of AFB1 DNA adducts
were dosedependent in rainbow trout (Bailey et al.,
1988; Hendricks et
21
al.,
1984; Sinnhuber et al.,
temperature-modulated hepatic
1977).
AFB1 disposition identified
temperature/exposure concentration
similar target organ doses.
Our experiments on
regimens which yielded
At similar target organ doses,
temperature-modulated DNA adduction was more directly related
to rates of competing
bioactivation/detoxication pathways for
AFB1.
While hepatic
equivalents
interpretation
tissue
elimination
appeared
rates
higher
at
for
total
warmer
temperatures,
was complicated by differences
concentrations
(Fig.
1).
Our
[3H]AFB1
in initial
data
did
not
unequivocally demonstrate increased hepatic
elimination of
[3H]AFB1 with temperature,
but the literature suggested
xenobiotic excretion was influenced by temperature
in fish.
Biological half-lifes of pentachlorophenol,
hexachlorobenzene
and mirex in rainbow trout were reduced at
warm relative to
cool temperatures
(Niimi and Palazzo,
1985).
Biliary
excretion of benzo[a]pyrene metabolites was
significantly
higher in 18 than 10°C acclimated and
exposed rainbow trout
(Curtis et al., 1990).
AFL Formation Shifts DNA Adduction
In
fish
exposed
at
their
respective
acclimation
temperatures, temperature modulated the formation
of hepatic
[3H]AFB1 DNA adducts by at least two
mechanisms.
The first of
these was disposition to the target
organ, as described above.
The second was probably related to the formation
and adduction
22
of AFB1's genotoxic
characterization
8,
9-epoxide metabolite.
An in vitro
of rainbow trout AFB1 metabolism (Williams
and Buhler, 1984) showed that cytochrome P-450
isozyme LM-2
almost exclusively catalyzed this
epoxide's formation.
However, immunoquantitation revealed no differences in
hepatic
microsomal cytochrome P-450 LM-2 content for 10 and 18°Cacclimated rainbow trout (Carpenter et al., 1990).
Perhaps
temperature modulation of [3H]AFB1 DNA adduction was regulated
by increased activity of a competing detoxication
reaction.
Conversion of AFB1 to aflatoxicol by a cytosolic
dehydrogenase
was identified as the first step in a major detoxication
pathway in rainbow trout (Schoenhard et al., 1976).
AFL, the
most toxic and carcinogenic of AFB1 metabolites,
is formed by
the cytosolic
enzyme NADPH-dependent
17-hydroxy-steroid
dehydrogenase (Cambell and Hayes, 1976). Since AFL can be
oxidized
to
yield
AFB1
by
NADP-dependent
microsomal
dehydrogenase and further actived to form
a
reactive
metabolite and other metabolites to initiate
cytotoxicity and
carcinogenicity,
it
has
been proposed to provide
an
intracellular reservoir for AFB1 (Patterson, 1974). For these
reasons,
reduction
of
AFB1
to
AFL
is
questionable
detoxification step. However, modulation of AFL formation
may
evidently shift the amount of 8,9-epoxide and DNA adduction.
If equivalent concentrations of the same cytosolic
isozyme
existed in both 10 and 18°C acclimated
fish, increased
activity of this soluble enzyme system with temperature
would
23
be expected. A resent study in this lab
demonstrated that in
vitro biotransformation of AFB1 to AFL catalyzed
by cytosolic
enzymes was
modulated by incubation temperature.
The
transformation of AFB1 to AFL was significantly higher in 18°C
than
10°C.
This
result
may
explain
the
decreased
concentrations of [3H] AFB1 DNA adducts at
warmer temperature.
Lack of concurrent stimulation of membrane-bound
cytochrome P-
450 LM-2 (or other microsomal pathways for that matter)
was
explained by "ideal temperature compensation"
(Precht, 1958),
the
mechanism
for
which
was
"homeoviscous
adaptation"
(Sinensky, 1974).
Temperature Effects on DNA Adduction
Increased [3H]AFB1 DNA adduction after acute temperature
increase (10-14°C) as opposed to acute
temperature decrease
(18-14°C)
(Fig.
4) was due to increased AFB1 bioactivation
(cytochrome P-450 LM-2 mediated).
Both in vivo (Curtis
et
al., 1990) and in vitro (Carpenter et al., 1990)
metabolism of
benzo[a]pyrene was higher for 10 than 18°C-acclimated
rainbow
trout when exposed at 14°C.
Further, covalent binding of
benzol[a]pyrene to calf thymus DNA was much higher in 29°C
incubations of 10,000 g supernatents from 7
than 16°C-
acclimated rainbow trout
(Egaas and Varanasi,
1982).
Our
interpretation of the temperature shift experiments
was that
differences in phase transition temperatures
for microsomal
lipids from 10 and 18°C-acclimated fish
were important in
modulating AFB1 8,9 epoxide formation at 14°C.
The higher
24
degree of liver membrane phospholipid unsaturation
for cold
acclimated rainbow trout was previously established
(Hazel
1979; 1984).
Persistence of DNA Adducts
The half-life of hepatic [3H]AFB1 DNA adducts was 3-4
weeks at 12-14°C in rainbow trout (Bailey et al., 1988; Goeger
et al., 1986).
Our data at 18°C were generally consistent
with this.
There was a reduction in persistence of [3H]AFB1
DNA adducts at 18°C compared to 10°C
occurred
in
acclimation
(14°C).
cases
where
temperatures
exposures
or
an
(Figs.
were
2-4).
at
intermediate
This
respective
temperature
Exposure temperature probably did little to influence
adduct persistence (as opposed to formation) because fish were
maintained at their exposure temperatures for 1 day
and their
acclimation temperatures for at least
20 days of the 21-day
time-course.
Possible mechanisms for [3H]AFB1 DNA adduct loss includes
tritium
exchange,
spontaneous
synthesis and cell turnover.
depurination,
DNA
repair
The literature suggested very
little of the [3H]AFB1 lost from DNA from 1 to 21 days after
exposure
was
attributable
to
tritium
exchange.
Other
researchers showed approximately 20% tritium exchange
from
[3H]AFB1 in the first 12 hr after in vivo
treatment of rainbow
trout, but negligible additional loss
over the subsequent 21
days (Bailey et al., 1988).
Spontaneous depurination,
however, was a likely a non-enzymatic mechanism
for loss of
25
[3H)AFB1 from DNA from 1 to 21 days after exposure.
When
purified [3H]AFB1- adducted DNA was incubated at 14°C for 21
days, 18% loss of radiolabelled substance was attributed
to
this mechanism (Bailey et al.,
1988).
On a simple
thermodynamic basis, spontaneous depurination probably was
higher at 18°C than 10°C in our in vivo experiment.
The rate
of DNA repair synthesis had been shown to increase
more than
two-fold from 15 to 25°C in isolated rainbow trout
liver cells
(Miller et al., 1989).
In our experiments, it was likely that
both increased spontaneous depurination and enzymatic
repair
contributed to reduced persistence of [3H]AFB1
DNA adducts at
18°C relative to 10°C.
However, as previously shown (Bailey
et al.,
1988; Kensler et al.,
adducts
was
of
arguable
hepatocarcinogenesis.
1986), persistence of DNA
significance
in
AFB1-induced
In both rainbow trout (Bailey et al.,
1988) and rats (Kensler et al., 1986) the
concentration of
hepatic AFB1 DNA adducts days after
exposure correlated much
better with tumor response than the concentration
of adducts
which persisted for weeks.
This result indicated cautious
interpretation of our results on adduct persistence.
Finally,
one
might
speculate
increased
hepatocyte
turnover with
temperature but we know of no published evidence for this.
DNA Adduction and Tumor Incidence
Previous work showed chemically-induced tumor
incidence
increased with exposure temperature in fish
(Hendricks et al.,
1984; Kyono-Hamaguchi, 1984), although carcinogen
disposition
26
or DNA adduction was not determined in these studies.
Our
results strongly suggested both target organ dose and the
extent of adduction at measured tissue concentration were
important
mechanistic
considerations
in
understanding
modulation of chemical carcinogenesis by temperature
shift
(Figs. 1 and 4).
While temperature acclimation followed by
acute temperature shift clearly modulated initiation events,
temperature may also act as a modulator of tumor promotion
and/or progression in fish.
Kyono-Hamaguchi (1984) provided
evidence that tumor progression was stimulated
at warmer
temperatures. Medaka exposed to diethylnitrosamine at 22° and
then reared at 22°C exhibited 100% incidence of large
liver
tumors, while those exposed at 22°C and reared at 6°C had
fewer, smaller tumors.
In the case of AFB1, tumor incidence
increased with temperature
in
rainbow trout
acclimated,
exposed and reared at the same temperature (Curtis et al.
unpublished results).
This was not predicted
by
genotoxicity results presented here (Fig.
2 and 3).
the
Taken
together the evidence in the present study clearly indicated
the importance of promotion/progression events in
temperaturemodulated chemical carcinogenesis in fish.
27
Literature Cited
Ahlers, J. (1981). Temperature effects
on kinetic properties
of plasma membrane ATPase from the yeast
saccharomyces
cerevisiae. Biochim Biophys Acta. 649, 550-556.
Bailey,
G.S., Taylor, M.J. and Selivonchik,
D.P.
Aflatoxin B1 metabolism and DNA binding in
hepatocytes from rainbow
Carcinogenesis 3, 511-518.
trout
(Salmo
(1982).
isolated
gairdneri).
Bailey, G.S., M. Taylor, D.
Selivonchick, T.
Hendricks, J. Nixon, N. Pawlowski, and R. Eisele, J.
Sinnhuber
(1982). Mechanisms of dietary modification of aflatoxin
B carcinogenesis, in Fletch,
R.A. and Hollaender, A.E.
(eds.). Genetic Toxicology, An Agricultural Perspective,
Plenum, New York, pp. 149-164.
1
Bailey, G.S., J.D. Hendricks, J.E. Nixon,
and N.E. Pawlowski
(1984). The sensitivity of rainbow
trout
and other fish
to carcinogens.
Drug Metab. Rev. 15, 725-750.
Bailey, G.S., Taylor, M.J., Loveland,
P.M., Wilcox, J.S.,
Sinnhuber, R.O. and Selivonchik, D.P. (1984). Dietary
modification of aflatoxin Bl carcinogenesis: Mechanism
studies with isolated hepatocytes from rainbow
trout.
Natl. Cancer Inst. Monogr. 65, 379-385.
Bailey, G.S., J.D. Hendricks, J.E. Nixon,
and N.E. Pawlowski
(1984). The sensitivity of rainbow trout and
other fish
to carcinogens. Drug Metab. Rev. 15, 725.
Bailey, G.S., D.P. Selivonchick, and J.D.
Hendricks (1987).
Initiation, promotion, and inhibition of
in rainbow trout. Env. Health Perspectivescarcinogenesis
71, 147-153.
Bailey, G.S. and J.C. Hendricks, (1988).
and
dietary modulation of carcinogenesisEnvironmental
in fish. Aqua.
Toxicol. 11, 69-75.
Bailey, G.S., D.E. Williams, J.S. Wilcox,
P.M. Loveland, R.A.
Coulombe and J.O. Hendricks
(1988).
Aflatoxin B1
carcinogenesis and its relation to DNA adduct formation
and adduct persistence in
sensitive and
salmonid fish. Carcinogenesis 9, 1919-1926. resistant
Barron, M.G., B.D. Tarr and W.L. Hayton
(1987). Temperaturedependence of cardiac output and regional
rainbow trout, Salmo gairdneri richardson. blood flow in
J. Fish Biol.
31, 735-744.
28
Black, J.J. (1988). Carcinogenicity tests with
rainbow trout
embryos: a review. Aquatic Toxicology 11, 129-142.
Blank, J., Lindstrom-Seppa, P., Agren, J.J., Hanninen,
O.,
Rein,
H.,
and Ruckpaul,
K.
(1989).
Temperature
compensation of hepatic microsomal cytochrome P-450
activity in rainbow trout. I. Thermodynamic regulation
during water cooling in autumn. Comp. Biochem. Physiol.
93C: 55-60.
Buhler, D.R. and M.E. Rasmusson (1968).
The oxidation of
drugs by fishes. Comp. Biochem. Physiol.
25, 223-239.
Burton, K. (1956). A study of the conditions
and mechanisms
of the diphenylamine reaction for
the colorimetric
estimation of deoxyribonucleic acid.
Biochem. J.
62:
315-322.
Cambell, T.C. and J.R. Hayes (1976). The role of aflatoxin
metabolism in its toxic lesion. Toxicol. Appl.
Pharmacol.
35, 199-222.
Carpenter, H.M., and Curtis, L.R. (1989). A characterization
of chlordecone pretreatment-altered pharmacokinetics
in
mice.
Drug Metab. Dispo.
17: 131-138.
Carpenter, H.M., L.S. Fredrickson, D.E. Williams, D.R. Buhler
and L.R. Curtis (1990). The effect of thermal
on the activity of arylhydrocarbon hydroxylaseacclimation
in rainbow
trout (Oncorhynchus mykiss).
Comp. Biochem. Physiol.
97C, 127-132.
Coulombe R.A. (1983). Aflatoxin Mutagenesis
and metabolism
and their dietary modification in rainbow
trout (salmo
gairdneri).
PhD
Thesis,
Oregon
State University,
Corvallis, OR 97331.
Coulombe R.A., D.W. Shelton, R.O. Sinnhuber,
and J.E. Nixon
(1983). Comparative mutagenicity of aflatoxins
using a
salmonella/trout hepatic enzyme activation
system.
Carcinogenesis 3, 1261-1264.
Croy, R.G., Nixon, J.E., Sinnhuber, R.O., and
Wogan, G.N.
(1980). Investigation of covalent aflatoxin in B1-DNA
adducts formed in in vitro in rainbow trout (Salmo
gairdneri) embryos and liver. Carcinogenesis 1, 903-909.
Croy, R.G. and Wogan, G.N. (1981). Quantitative
comparison of
covalent aflatoxin-DNA adducts formed in rat
and mouse
livers and kidneys. NCI. 66, 761-768.
Curtis,
L.R.,
Kemp,
C.J.
and Svec,
A.L.
(1986).
Biliary
29
excretion of [14C] taurocholate by rainbow trout (Salmo
aairdneri)
is
stimulated
at
warmer
acclimation
temperature. Comp. Biochem. Physiol. 84C, 87-90.
Curtis, L.R., Fredrickson, H.M. Carpenter (1990).
Biliary
excretion appears rate limiting for hepatic elimination
of benzo[a]pyrene by temperature acclimated rainbow
trout, Fundam. Appl. Toxicol.
15, 420-428.
Dalezios, J.I. and G.N. Wogan (1972). Metabolism of aflatoxin
B1 in rhesus monkeys. Cancer Res. 32, 2297-2303.
Dashwood, R.H., D.N. Arbogast, A.T. Fong, C.
Pereira, J.D.
Hendricks,
and
G.S.
Bailey
(1989).
Quantitative
interrelationships between aflatoxin B1 carcinogen dose,
indole-3-carbinol anti-carcinogen dose, target organ DNA
adduction and final tumor response. Carcinogenesis
10,
175-181.
Decad, Gary M., Kathleen K. Dougherty, Dennis
P.H. Hsieh, and
James L. Byard (1979). Metabolism
of aflatoxin B1 in
cultured mouse hepatocytes: coparision with rat and
effects of cyclohexene oxide and diethyl
maleate.
Toxicol. Appl. Pharmacol. 50, 429-436.
Degen G.H. and Neumann H-G. (1981). Differences
in aflatoxin
B1- susceptibility of rat and mouse are correlated with
the capability in vitro to inactivate aflatoxin
epoxide. Carcinogenesis 2, 299-306.
Egaas, E. and U. Vasanasi (1982).
biphenyls and environmental
B1-
Effects of polychlorinated
temperature on
in vitro
formation of benzo[a]pyrene metabolites by liver of
trout
(Salmo aairdneri). Biochem. Pharmacol.
31,
561-566.
Erickson, R.J. and J.M. Mckim (1990). A model
for exchange of
organic chemicals at fish gills: flow and diffusion
limitations. Aquatic Toxicology 18, 175-198
Essigmann J.M., R.G. Croy, W.F. Nadzan, V.N.
Busby Jr.,
V.N.Reinhold, G. Buchi, and G.N. Wogan (1977). Structural
identification of major DNA adduct formed by aflatoxin B1
in vitro. Proc. Natn. Acad. Sci. U.S.A.
74, 1870-1874.
Garner,
R.C., E.C. Miller and J.A. Miller
(1972). Liver
microsomal activation of aflatoxin B1 to a reaction
derivation toxic to Salmonella typhimurium TA 1530.
Cancer Res. 32, 2058-2066.
Goeger, D.E., D.W. Shelton, J.D. Hendricks and G.S.
Bailey
(1986).
Mechanisms of anti-carcinogenesis by indole-3carbinol: effect on the distribution
and metabolism of
30
aflatoxin B1 in rainbow trout.
2031.
Carcinogenesis
7, 2025-
Hazel, J.R. (1979).
Influence of thermal acclimation on
membrane lipid composition of rainbow trout liver. Am.
J. Physiol. 236, R91-R101.
Hazel, J.R. (1984).
Effects of temperature on the structure
membranes in Fish.
Am. J.
246, R460-R470.
and metabolism of cell
Physiol.
Hazel, J.R. and P.A. Sellner. The Regulation of Membrane
lipid
composition
in
thermally-acclimated
poikilotherms.
Department of Zoology, Arizona State University, Tempe,
Arizona 85281, USA
Hendricks, J.D.,T.P. Putnam, D.D. Bills, and R.O. Sinnhuber
(1977). Inhibitory effect of a polychlorinated biphenyl
(Aroclor 1245) of aflatoxin B1 carcinogenesis in rainbow
trout (Salmo gairdneri). J. Natl. Cancer Inst.
59, 15451551.
Hendricks, J.D., W.T. Stott, T.P. Putnam, and R.O. Sinnhuber
(1981). Enhancement of aflatoxin B1 hepatocarcinogenesis
in rainbow trout (Salmo gairdneri) embryos by prior
exposure of gravid females to dietary aroclor 1245. Proc.
4th annual symposium on aquatic toxicology,
Society for Testing and Materials. Philadelphia. American
203-214.
Hendricks, J.D. (1982). Chemical carcinogenesis in
fish. In.
Aquatic Toxicology (L.J. Weber ed.), pp. 149-202 . Raven
Press, New York.
Hendricks, J.D., Meyers, T.R., Casteel, J.L., Nixon, J.E.,
Loveland, P.M., R.O. Sinnhuber, K.E.
Berggren, L.M.
Libbey, J.E. Nixon, and N.E. Pawlowski (1977). Formation
of aflatoxin B1 from aflatoxicol by rainbow trout (salmo
gairdneri) liver in vitro. Res. Commun. Chem. Pathol.
Pharmacol. 16, 167-170.
Hendricks, J.D., T.R. Meyers, J.L. Casteel, J.E. Nixon, P.M.
Loveland, and G.S. Bailey (1984). Rainbow trout embryos:
Advantages and limitations for carcinogenesis research.
Natl Cancer Inst. Monograph 65, 129-137.
Kemp,
C.J.
and Curtis,
L.R.
(1987).
Thermally modulated
biliary excretion of [14C] taurocholate in rainbow trout
(Salmo gairdneri) and the Na, K+-ATPase Can. J. Fish.
Aqua. Sci. 44, 846-851.
Kensler, T.W., Egner, P.A.,
Groopman, J.D.
(1985).
Trush, M.A.,
Bueding,
E.
Modification of aflatoxin
and
B1
31
binding to DNA in vivo in rats fed phenolic
antioxidants,
ethoxyquin and a dithiothione. Carcinogenesis
6, 759763.
Kim, D-H. and F.P. Guengerich (1990). Formation
of DNA adduct
S-[-2-(N7-guanyl)ethyl]glutathione
from
ethylene
dibromide: effects of modulation
of glutathione and
glutathine S-transferase levels and lack of a role for
sulfation. Carcinogenesis 11, 419-424.
Kinoshita, N., B. Shears, and H.V. Gelboin
(1973). K-region
and non-K-region metabolism of
Benzo(a)pyrene
by rat
liver microsomes. Canc. Res. 33, 1937-1944.
Klaunig,
J.E.
(1984).
Establishment of fish hepatocyte
cultures for use in in vitro carcinogenicity studies.
Natl. Cancer Inst. Monogr. 65, 163-173.
Koivusaari, U., Harri, M., and lianninen, 0. (1981).
variation of hepatic biotransformation in female Seasonal
rainbow trout (Salmo gairdneri). Comp. Biochem. and male
Physiol.
70C, 149-157.
Kyono-Hamaguchi, Y. (1984).
Effects of temperature and
partial hepatectomy on the induction of liver
tumors in
Oryzias latipes.
65, 337-344.
Laurin, D.L., P.P. Halarnkar, B.D. Hammock
and D.E. Hinton
(1989).
Microsonal and cytosolic epoxide hydrolase and
glutathione-S-transferase activities in the gill, liver,
and kidney of the rainbow trout.
Salmo Gairdneri.
Biochem. Pharmacol.
38, 881-887.
Lee. D.J., J.H. Wales, and R.O. Sinnhuber
(1971). Promotion of
aflatoxin-induced hepatoma growth in trout by methyl
malvate and sterculate. Cancer Res. 31, 960.
Lee, D.J., R.O. Sinnhuber, J.H. Wales, and G.B.
Putnam (1978).
Effect of dietary
protein on the response of rainbow
trout (Salmon gairgneri) to aflatoxin Bl. J.
Natl. Cancer
Inst. 60, 317-320.
Lin J-K., J.A. Miller and E.C. Miller
(1977). 2,3-Dihydro-2(guan-7-y1)-3-hydroxy-aflatoxin
B1,
a
major
acid
hydrolysis product of aflatoxin B1-DNA
or -ribosomal RNA
adducts formed in hepatic microsome-mediated reactions
and in rat liver in vivo. Cancer Res.
37, 4430-4438.
Loveland, P.M., R.O. Sinnhuber, K.E.
J.E. Nixon, and N.E. Pawlowski Berggren, L.M. Libbey,
(1977).
aflatoxin B1 from aflatoxicol by rainbow Formation of
trout (salmo
gairdneri) liver in vitro. Res. Com.
Chem. Path. and
32
pharmacol. 16, 167-169.
Loveland, P.M., J.E. Nixon, N.E. Pawlowski, T.A. Eisele, L.M.
Libbey, and R.O. Sinnhuber (1979). Aflatoxin B1
and
Aflatoxicol metabolism in rainbow trout (Salmo gairdneri)
and the effects of dietary cyclopropene. J. Env. Path.
Toxicol. 2, 707.
Loveland, P.M., R.A. Coulombe, L.M. Libbey, N.E. Pawlowski,
R.O. Sinnhuber, J.E. Nixon and G.S. Bailey
(1983).
Identification
and
Mutagenicity
of
aflatoxicol-M1
produced by metabolism of aflatoxin B1 and Aflatoxicol
by liver fractions from rainbow trout (salmo gairdneri)
fed 13- naphthoflavone. Food Chem. Toxic. 21, 557-562.
Loveland, P.M. and Bailey, G.S. (1984). Rainbow trout embryos:
Advantages limitations for carcinogenesis research.
Natl. Cancer Inst. Monogr. 65, 129-137.
Loveland,
P.M.,
J.E.
Nixon,
and G.S.
Bailey
(1984).
Glucuronides in bile of rainbow trout (Salmo gairdneri)
injected with [31H] aflatoxin B1 and the effects of
dietary 13- naphthoflavone. Comp. Biochem. Physiol.
78C,
13-19.
Loveland, P.M., J.S. Wilcox, J.D. Hendricks and G.S. Bailey
(1988).
Comparative metabolism and DNA binding of
aflatoxin B1, aflatoxin M1, aflatoxicol and aflatoxicol -M1
in hepatocytes from rainbow trout (Salmo gairdneri).
Carcinogenesis. 9, 441-446.
Lutz,
W.K.
(1979). In vivio covalent binding of organic
chemicals to DNA as a quantitative indicator in the
process of chemical carcinogenesis. Mutat. Res. 65, 289356.
Markwell, M.A.K., S.M.
Haas, L.L. Bieber, and N.E. Tolbert
(1978). A modification of the Lowry procedure to simplify
protein determination in membrane
and lipoprotein
samples. Analytical Biochem. 87, 206-210.
McKim, J.M., S.P. Bradbury, and G.J. Niemi
(1987). Fish acut
toxicity syndromes and their use in the QSAR approach to
hazard assessment. Env. Health Perspectives 71, 171-186.
Miller, M.R., J.B. Blair, and D.E. Hinton. 1989. DNA repairs
synthesis in isolated
rainbow trout liver cells.
Carcinogenesis 10, 995-1001.
Miranda, C.L., J.-L. Wang, M.C. Henderson and D.R. Buhler
(1989).Purification and characterization of
steriod hydroxylases from untreated rainbow hepatic
trout.
33
Archives of Biochemistry and Boiphysics
Neal,
268, 227-238.
G.E. and Colley, P.J. (1978). Some high-performance
liquid-chromatographic studies of the metabolism of
aflatoxin by rat liver microsomal preparations. Biochem.
J. 174, 839-851.
Nichols, J.W., J.M. McKim, M.E. Andersen, M.L. Gargas, H.J.
Clewell III and R.J. Erickson (1990). A physiologically
based toxicokinetic model for uptake and disposition of
waterborne organic chemicals in fish. Toxicology and
Applied Pharmacology 106, 433-447.
Niimi, A. J., and V. Palazzo (1985).
Temperature effect on
the elimination of pentachlorophenol, hexachlorobenzene
and mirex by rainbow trout (Salmo crairdneri). Water
Res.
19, 205-207.
Nixon, Joseph E.,
Pereira,
R.O.
J.D. Hendricks,
Sinnhuber,
and
N.E.
G.S.
Pawlowski,
C.B.
Bailey
(1984).
Inhibition of aflatoxin B1 carcinogenesis in rainbow
trout by flavone and indole compounds. Carcinogenesis 5,
615-619.
O'Flaherty, E.J. (1981).
Acute exposure with nonlinear and
mixed kinetics of disposition. In, Toxicants and Drugs:
Kenetics and Dynamics.
Wiley-Interscience.
New York.
Pages 173-221.
Patterson,
D.S.P.
(1973).
Metabolism as a factor in
action of the aflatoxins in
different animal species. Food Cosmet. Toxicol. 11, 287-
determining the toxic
294.
Precht, H. (1958). Concepts of the temperature
adaptation of
unchanging reaction systems of cold-blooded animals. In.
Physiological Adaptation C. L. Prosses, ed. Am. Physiol.
Soc., Washington, D. C. Pages 50-78.
Prosser, C.L. (1990) Temperature 109-165.
Rodricks, J.V., Stoloff, L., Pons, W.A., Robertson,
J.A., and
Goldblatt, L.A. (1970). Molar absorptivity
values
for
aflatoxins and justification for their use as criteria
purity of analytical standards. J. AOAC 53, 96-101. of
Roebuck, B.D. and Wogan, G.N. (1977). Species comparison
of in
vitro metabolism of aflatoxin B1. Cancer Res. 337, 16491659.
Salhab, A.S. and D.P.H. Hsieh (1975). Aflatoxin H1: a major
metabolite of aflatoxin B1 produced by human and
rhesus
34
monkey livers
in vitro.
Pharmacol. 10, 419-431.
Res.
Commun.
Chem.
Pathol.
Salhab, A.S. and Gordon S. Edwards (1977). Comparative in
vitro metabolism of aflatoxicol by liver preparations
from animals and humans. Cancer Res. 37, 1016-1021.
Schoengard, Grant L., D.J. Lee, S.E. Howell, N. E. Pawlowski,
L.M. Libbey, and R.O. Sinnhuber
(1976). Aflatoxin B1
metabolism to aflatoxicol and derivatives lethal to
Bacillus subtilis GSY 1057 by rainbow trout (Salmo
gairdneri) liver. Cancer Res. 36, 2040-2045.
Scribner,
J.D.
(1985).
Chemical
carcinogenesis.
In
Environmental Pathology (N. K. Mottet, Ed.), pp. 17-55.
Oxford University Press, New York.
Shelton, D.W., R.A. Coulombe, C.B. Pereira, J.L. Casteel,
and
J.D. Hendricks (1983). Inhibitory effect of aroclor 1245
on aflatoxin-initiated carcinogenesis in rainbow trout
and mutagenesis
using a
Salmonella/trout hepatic
activation system. Aquatic Tox. 3, 229-238.
Shelton, D.W., J.D. Hendricks, R.A. Coulombe, and G.S. Bailey
(1984).
Effect
of
dose
on
the
inhibition
of
carcinogenesis/mutagenesis by arocolor 1245 in rainbow
trout fed aflatoxin B1. J. Toxicol. Env. Health 13, 649657.
Shelton, D.W., Goeger, D.E., Hendricks, J.D. and
Bailey, G.S.
(1986).
Mechanisms
of
anti-carcinogenesis:
the
distribution and metabolism of aflatoxin Bl in rainbow
trout fed Aroclor 1254. Carcinogenesis 7, 1065-1071.
Sigler, W.F. and J.W. Sigler (1987). Rainbow
trout Salmo
gairderi. in Fish of the great Basin. Univ. of Nevada
press.
Silber, J.R. and L.A. Loeb (1985).
The molecular basis of
environmental mutagenesis. In, Environmental Pathology,
N. K. Mottet, ed. Pages 3-55.
Sinensky, M. (1974). Homeoviscous adaptation - A homeostatic
process that regulates the viscosity of membrane lipids
in Escherichia coli. Proc. Nat. Acad. Sci. 71, 522-525.
Sinnhuber, R.O., J.D. Hendricks, J.H. Wales, and G.B. Putnam
(1977). Neoplasms in rainbow trout, a sensitive animal
model for environmental carcinogenesis. Ann. N.Y.
Acad.
Sci. 298-389.
Stegeman, J.J., R.L. Binder and A. Orren (1979). Hepatic and
35
extrahepatic microsomal electron transport components and
mixed-function oxygenases in the marine fish stenotomus
versicolor. Biochem. Pharmacol. 28, 3431-3439.
Stegeman, J.J., Woodin, B.R. and Binder, B.L. (1984). Patterns
of benzo[a]pyrene metabolism by varied species, organs
and developmental stages of fish. Natl. Cancer Inst.
Monogr. 65, 371-377.
Thakker,
D.R.,
H.
Yagi,
and D.M. Jerina. Analysis of
polycyclic aromatic hydrocarbons and their metabolites by
High-Pressure Liquid Chromatography.
Valsta, L.M., J.D. Hendericks and G.S. Bailey (1988). The
significance of glutathine conjugation for aflatoxin Bl
Metabolism in rainbow trout and coho salmon. Food Chem.
Toxic. 26, 129-135.
Williams,
D.E. and D.R. Buhler (1983).
Purified form of
cytochrome P-450 from rainbow trout with high activity
toward conversion of aflatoxin Bl to aflatoxin B1..2 3epoxide.
Cancer Res.
43, 4752-4756.
Williams, D.E., R.R. Becker, D.W. Potter, F.P. Guengerich
and
D.R.
Buhler
(1983).
Purification
and
comparative
Properties of NADPH-Cytochrome P-450 Reductase from rat
and rainbow trout: Differences in temperature optima
between reconstituted
and microsomal trout enzymes.
Archives of Biochemistry and Biophysics 255, 55-65.
Williams D.E. and D.R. Buhler (1983).
Purified form of
cytochrome P-450 form rainbow trout with high activity
toward conversion of aflatoxin B1 to aflatoxin B1-2,3epoxide. Cancer Res. 43, 4752-4756.
Williams D.E., R.T. Okita, D.R. Buhler, and B.S.S. Masters
(1984). Regiospecific hydroxylation of lauric acid at the
-1)
position by hepatic and kidney microsomal
cytochromes P-450 from rainbow trout. Archives
of
Biochem. and Biophysics 231, 503-501.
(
William, F.S. and J.W. Sigler (1987).
Salmoniformes. In.
Fishes of the Great Basin, pp. 119-127. Univ. of Nevada
Press.
Wood, M.L., J.R.L. Smith and R.C. Garner (1988).
Structural
characterization of the major adducts obtained after
reaction of an ultimate carcinogen
aflatoxin B/dichloride with calf thymus DNA in vitro. Cancer Res. 48,
5391-5396.
APPENDIX
36
Appendix A
in vitro AFB1 -DNA Binding and Biotransformation of
AFB1 to AFL
Aflatoxin
hepatocarcinogen.
B1
is
a
The critical
metabolically
target
for
activated
initiation of
carcinogenesis by AFB1 and its metabolites is believed to be
DNA. The amount of hepatic DNA binding of AFB1 correlates very
well with the development of hepatic tumors in rainbow
trout
(Dashwood et al., 1989), which suggests that the binding of
chemical carcinogens to DNA in vitro is a short-term predictor
of hepatic carcinogenic response (Bailey et al 1982).
The primary phase
I
metabolite of AFB1
is AFL in
isolated hepatocytes of trout(Bailey et al 1982). AFL is also
the major in vitro metabolite after incubation of AFB1 with
trout liver cytosol (Loveland et al 1977).
AFL is slightly
less toxic and carcinogenic than AFB1 and is converted
back to
AFB1 by NADP-dependent microsomal dehydrogenases. For this
reason,
the reduction of AFB1 to AFL is not considered
complete detoxification,
but also storage of the toxin to
produce chronic toxic effects. Glucuronidation of AFL produces
a detoxication product readily excreted in bile. Studies with
anticarcinogens (BNF and PCB) demonstrated that the mechanism
of action for the anticarcinogenesis involved
increased rate
of formation of the detoxification product AFM1 (P-450 1A1
mediated), decreased formation of AFL
(cytosolic enzyme
mediated), and decreased DNA adduction (Bailey et al 1987).
Temperature modulated AFB1
genotoxicity
(Zhang et al
37
1992) may involve modulation of AFB1 metabolic pathways. The
primary objectives of the present study are to
compare
temperature-modulation of (1) in vitro binding of AFB1 to DNA
catalyzed by hepatic microsomes and (2) biotransformation of
AFB
1
to AFL by hepatic cytosol from 10 and 18 °C acclimated
rainbow trout.
38
7.400
7.300
I-14
7.200
7.100
7.000
6
8
10
12
14
16
18
20
22
Temperature (°C)
Figure
Effect of temperature on Tris buffer pH. Tris
buffers (0.2 M) were adjusted to 7.2 and at 14 °C and the
tested at 8, 14 and 20°C respectively. Results indicates a
1.
decrease in pH value with an increase in temperature.
39
260 -
240
220
200
180
160
140
120
6.75
7.00
7.25
7.50
7.75
PH
Figure 2. Effect of pH on in vitro AFB1 DNA binding. Calfthymus DNA was incubated with [3]H]AF131, NADPH and rainbow trout
microsomal protein at different pHs of Tris buffer at 14 °C
for 30 min with shaking. Results indicates that in vitro AFB1DNA binding increases with the increase of pH.
40
700
A
600
500
0
400
300
INCUBATION TIME
200
A
100
A
1 HOUR
oo 30 MIN
0
00
0.2
0.4
0.6
0.8
1.0
MICROSOME CONCENTRATION ( mg / ml )
Figure 3. Effect of microsome concentration and incubation
time on in vitro AFB1 -DNA binding. Calf-thymus DNA was
incubated at 14 °C and pH 7.2 for 30 or 60 min with [3H)AFB1,
NADPH and microsomal protein ( 0.125, 0.25, 0.5 and 1.0 mg/ml
from 14 °C acclimated rainbow trout.
)
41
22
1000
INCUBATION TEMPERATURE (°C)
20
18
18
14
12
10
8
8
ACCLIMATION TIMPERATURE
o 18 °C
- 10 °C
100
3.41
3.45
3.49
3.53
3.57
1/K x 1000
Figure 4. Arrhenius plots of in vitro AFB1 -DNA binding by
rainbow trout hepatic microsomes. The incubation mixture (2
ml)
for the binding of AFB1 to DNA contained 0.35 mg
microsomal protein/ml from 10 or 18 °C acclimated fish, 45 mM
Tris-C1 (pH 7.2 at 14 °C), 1 mM NADPH, 1 mg calf-thymus
DNA/ml, and 0.01 mM [3H]AFB (21.22 mCi/mmol). The incubation
mixtures were shaken at 6, 8, 10, 12, 14, 16, 18, 20, and 22
°C in the dark. The reactions were terminated after 30 min.
1
42
INCUBATION TEMPERATURE (°C)
22
20
3.41
18
18
3.45
14
3.49
12
10
3.53
8
8
3.57
1/K x 1000
Figure 5. Arrhenius plots of in vitro AFB1 -DNA binding by
rainbow trout hepatic microsomes treated with a-naphthoflavone
(ANF). The incubation mixture (2 ml) for the binding of AFB1
to DNA contained 0.35 mg microsomal protein/ml from 10 or 18
°C acclimated fish, 45 mM Tris-Cl (pH 7.2 at each incubation
temperature), 1 mM NADPH, 1 mg calf-thymus DNA/ml, 0.5 mM ANF
and 0.01 mM [3H]AFB1 (21.22 mCi/mmol). The incubation mixtures
were shaken at 6, 8, 10, 12, 14, 16, 18, 20, and 22 °C in the
dark. The reactions were terminated after 30 min.
43
AFB1
50
--- Microsome
40
Cytosol
V 30
a4
q 20
Anil
10
AFL
I/
/c\
....------N.%..----s--4
10
15
20
25
30
.
I
35
FRACTIONS
Figure 6. Effect of rainbow trout microsomal and cytosolic
enzymes on in vitro metabolism of aflatoxin B1. Incubation
consisted of phosphate buffer (50 mM, pH 7.4), 1 mM NADPH,
MgCl2 (5 mM), KC1 (60 mM), microsomes or cytosol (4 mg
protein/ml) from 14°C acclimated fish and [3H]AFB1 (1 nM,
10.22 mCi/mmol).
The mixtures (2 ml) were incubated at 18°C
with shaking for 60 min in the dark.
44
70
ACCLIMATION TEMPERATURE
0
60
)
10 °C
18 °C
50
40
30
8
10
12
14
16
18
20
INCUBATION TEMPERATURE (°C)
Figure 7. Effect of temperature on in vitro biotransformation
of AFB, to AFL catalyzed by cytosolic enzymes. Incubation
consisted of phosphate buffer (50 mM, pH 7.4), 1 mM NADPH,
MgC12 (5 mM), KC1 (60 mM), cytosol (4 mg protein/ml) from 10°C
or 18
°C
acclimated fish and [31flAFB1
(0.5 nM,
10.22
mCi/mmol).
The mixtures (4 ml) were incubated at 10, 14 and
18°C with shaking for 60 min in the dark.