AN ABSTRACT OF THE THESIS OF
Camille R. EI-Zahr for the degree of Doctor of Philosophy in Fisheries Science
presented
on
March
6,
1996.
Title:
Temperature-Modulated
7.12 -,
Dimethylbenz(a)anthracene Carcinogenicity in Rainbow Trout.
Redacted for Privacy
Abstract approved:
Lawrence R. Curtis
Temperature influences the incidence of chemically induced cancer in fish,
with warmer temperatures being associated with higher cancer incidence. The
mechanisms of temperature-modulated chemical carcinogenesis in fish, however,
have not been described in detail. Therefore, one primary objective of this study
was increased understanding of how temperature-modulated genotoxicity of 7,12­
dimethylbenz(a)anthracene (DMBA) corresponded with tumor response. The
second entails the potential of temperature to modulate cancer promotion.
Rainbow trout (Oncorhyncus mykiss) (2 g) were acclimated at 10, 14 or
18°C for one month and then exposed to 1.0 ppm DMBA in their water for 20 hr.
Exposures were at respective acclimation temperatures or 10 and 18°C
acclimated fish were shifted to 14°C for DMBA exposures. Adduction of
[3H]DMBA to hepatic DNA 21 days after exposure was higher in 10°C than 18°C
fish exposed at their respective acclimation temperatures. However, in fish shifted
to 14°C, the concentration of hepatic [3H]DMBA DNA adducts was similar in 10°C
and 18°C acclimated fish at that time. Temperature effects on tumor incidence
were assessed 9 months after DMBA waterborne exposures. Incidence of
stomach, liver and swim-bladder cancer increased with rearing temperature.
Differences in tumor incidence were less marked in fish reared at the same
temperature (14°C).
Retrospective analyses of livers from a tumor study initiated with aflatoxin
B1 (AFB1) was conducted with antibodies to endogenous proliferating nuclear
antigen (PCNA). Proliferating cells were scored by counting labeled nuclei in 5
random 10x fields using an image analysis program (BIOQUANT SYSTEM IV).
There was no significant increase in numbers of PCNA labeled hepatocytes with
temperature. The influence of acclimation temperature on plasma mitogens that
stimulate cell division was assessed in cultured Chinook salmon embryo cells
(CHSE-214). Plasma from rainbow trout (120-150 g) acclimated to either 10 or
18°C for at least four weeks stimulated in vitro proliferation of CHSE-214 cells to
the same extent.
This study demonstrated that chemically induced tumors in fish were
modulated by temperature not only through genotoxin disposition and formation
but also through persistence of DNA adducts. It also discounted the role of
mitogenesis in temperature-modulated chemical carcinogenesis.
Temperature-Modulated 7,12-Dimethylbenz(a)antheracene
Carcinogenicity in Rainbow Trout
by
Camille R. EI-Zahr
A DISSERTATION
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Completed March 6, 1996
Commencement June 1996
Doctor of Philosophy thesis of Camille R. EI-Zahr presented on March 6. 1996
APPROVED:
Redacted for Privacy
Major Professor, representing Fisheries Science
Redacted for Privacy
Head of Department of Fisheries and Wildlife
Redacted for Privacy
Dean of G
uate School
I understand that my thesis will become part of the permanent collection of
Oregon State University libraries. My signature below authorizes release of my
thesis to any reader upon request.
Redacted for Privacy
Camille R. EI-Zahr, Auth
ACKNOWLEDGMENT
Through the years of study at Oregon State University which has provided
me with a valuable learning experience, I have come to admire and respect my
advisor, Dr. Larry R. Curtis for his guidance and friendship. I would not have
been able to successfully complete my research without his help and support.
I
am also especially grateful to Dr. Richard A. Tubb for his support and
encouragement to pursue my goals. I would like also to thank the members of my
doctoral committee, Drs. Phillip N. McFadden, Lavern J. Weber, and Daniel R.
Selivonchick for their advice and encouragement. My thanks goes also to my
colleagues at Oak Creek Laboratory, Deke Gundersen, Quan Zhang, Gene foster
and Regina Donahoe, who were always available to help and have fun.
Finally I would like to express my appreciation to my beautiful wife and
best friend, Kimberly K EI-Zahr and to my two wonderful children, Candace and
Curtis for their support and patience throughout my academic career. I am also
thankful for the support and spiritual guidance of my parents Reda and Souad ElZahr.
CONTRIBUTION OF AUTHORS
Dr. Lawrence R. Curtis was project coordinator and provided advice concerning
all aspects of the project. Cell proliferation assessments were performed in the
laboratories of Drs.
Daniel
Dietrich and David Barnes using PCNA
immunohistochemistry techniques and mitogenesis in cultured cells assays
respectively. C. Samuel Bradford assisted in cell culture assays, while Quan
Zhang assisted in DMBA accumulation, adduction and excretion studies. Dr.
Jerry Hendricks confirmed the presence of tumors in fish histologically.
TABLE OF CONTENTS
Page
INTRODUCTION
1
CHAPTER 1: Temperature Modulated Genotoxicity and Carcinogenicity
of 7,12-Dimethylbenz(a)anthracene Carcinogenicity in
10
Rainbow Trout
ABSTRACT
11
INTRODUCTION
13
MATERIALS AND METHODS
16
RESULTS
21
DISCUSSION
35
REFERENCES
42
CHAPTER 2: Cell Proliferation and Temperature Modulated Chemical
Carcinogenesis in Rainbow Trout
47
ABSTRACT
48
INTRODUCTION
49
MATERIALS AND METHODS
52
RESULTS
55
DISCUSSION
65
REFERENCES
71
SUMMARY
75
BIBLIOGRAPHY
79
APPENDICES
88
LIST OF FIGURES
Page
Figure
1.1
Disposition of total and hexane extractable [3H]DMBA in liver and
gallbladder of rainbow trout exposed to 1.0 ppm DMBA for 20 hr at
22
their respective acclimation temperatures (10 or 18°C)
1.2
Disposition of total and hexane extractable [3H]DMBA in liver and
gallbladder of rainbow trout acclimated at 10, 14, or 18°C and
exposed to 1.0 ppm DMBA for 20 hr at 14°C
1.3
24
Disposition of total [3H]DMBA and DNA adducts in liver of rainbow
trout exposed to 1.0 ppm DMBA for 20 hr at their respective
acclimation temperature (10 or 18°C)
27
1.4
Disposition of total [3HJDMBA and DNA adducts in liver of rainbow
trout acclimated at 10, 14, or 18°C and exposed to 1.0 ppm DMBA
30
for 20 hr at 14°C
1.5
Percentage of rainbow trout bearing swim bladder, liver or
stomach tumors 9 months after 20 hr waterborne DMBA exposure
at 1.0 ppm
32
2.1
Number of cells per well in fish cell culture (CHSE-214) to which
56
trout plasma from 10 or 18°C acclimated fish was added
2.2
Number of labeled cells in normal and tumor tissue in the livers of
rainbow trout exposed to AFB1 at concentrations that yield
equivalent organ dose
2.3
58
Number of labeled cells in the livers of rainbow trout acclimated at
11, 14.5 or 18°C then exposed to 0.1 ppm AFB1 and reared at
their respective acclimation temperature
2.4
61
Number of labeled cells in the livers of rainbow trout acclimated at
11, 14.5 or 18°C then exposed to 0.1 ppm AFB1 and reared at
2.5
14.5°C
63
Liver tumor incidence plotted against percent AFB1 DNA adduct
persistence
67
LIST OF APPENDICES
Appendix
Page
Appendix A: Cell Proliferation Assessment by Immunohistochemical
Methods
Appendix B: Temperature-Modulated Incidence of Aflatoxin B1-Initiated
Liver Cancer in Rainbow Trout
89
99
Appendix C: In vitro and In vivo Temperature Modulation of Hepatic
Metabolism and DNA Adduction of Aflatoxin B1 in Rainbow
134
Trout
LIST OF APPENDICES FIGURES
Page
Figure
A.1
Liver tissue of rainbow trout acclimated, exposed to 0.1 ppm AFB1
and reared at 10°C. This section was fixed with Bouin's solution,
embedded in paraffin, and stained with PCNA for assessment of
91
cell proliferation
A.2
Liver tumor of rainbow trout acclimated, exposed to 0.1 ppm AFB1
and reared at 18°C. This section was fixed with Bouin's solution,
embedded in paraffin, and stained with PCNA for assessment of
91
cell proliferation
A.3
Liver tissue of rainbow trout reared at 18°C for at least four weeks
and then injected intra peritoneally with BrdU (30 mg/kg). This
section was fixed with 70% ethanol, embedded with paraffin and
stained with BrdU for assessment of cell proliferation
A.4
94
Liver tissue of rainbow trout reared at 10°C for at least four weeks
and then injected intra peritoneally with BrdU (30 mg/kg). This
section was fixed with 70% ethanol, embedded with paraffin and
stained with BrdU for assessment of cell proliferation
B.1
Schematic for
temperature
acclimation/ AFB1
exposure
110
regimens
B.2
94
Percent of rainbow trout liver tumors 9 months after 30 min
waterborne AFB1 exposures at the concentrations specified on the
118
ordinant
B.3
Liver tumor multiplicity in rainbow trout 9 months after 30 min
waterborne AFB1 exposures at the concentrations specified on the
120
ordinant
C.1
Upper panel. Arrhenius plots for in vitro [3H]AFB1 -DNA binding to
calf thymus DNA catalyzed by hepatic microsomes from rainbow
trout acclimated to 10 or 18°C. Lower panel. Arrhenius plots of
a-naphthoflavone modulated in vitro [3H]AFB1 -DNA binding
C.2
. . .
145
The effects of temperature acclimation on the in vitro production
of AFL from [3H]AFB1 by the cytosol from rainbow trout acclimated
to 10 or 18°C
147
LIST OF APPENDICES FIGURES (Continued)
Figure
C.3
The effect of temperature acclimation on in vivo [3H]AFB1-DNA
binding versus total hepatic [3H]AFB1 concentration
C.4
In vivo [3H]AFB1-DNA binding versus total hepatic
concentration in temperature shifted fish
C.5
150
[3H]AFB1
152
Representative HPLC chromatogram of the biliary [3H]AFB1
metabolite profile from rainbow trout acclimated and exposed at
14°C
C.6
Pie
154
The percentage of [3H]AFB1 biliary metabolites produced by rainbow
156
trout acclimated to 10 or 18°C
LIST OF APPENDICES TABLES
Ea=
Table
B.1
Absence of an effect of temperature acclimation of general hepatic
112
microsomal parameters in rainbow trout
B.2
Absence of an effect of temperature acclimation on hepatic
microsomal content of CYP-450 isozymes in rainbow trout
B.3
Quantitative extraction data for liver lipids from temperature
acclimated rainbow trout
B.4
113
114
Fatty acid composition of phosphatidylcholine from livers of
temperature acclimated rainbow trout
115
B.5
Fatty acid composition of phosphatidylethanolamine from livers of
117
temperature acclimated rainbow trout
B.6
Percent of tumor bearing fish possessing particular tumor types
9 months after AFB1 exposures
C.1
122
Fatty acid composition of lipid extracts from whole livers of
temperature acclimated trout
158
C.2
Fatty acid composition of lipid extracts from hepatic microsomes
159
of temperature acclimated trout
C.3
The effect of temperature and [31-1]AFB1 on the activities of plasma
161
GOT and GPT
Temperature-Modulated 7,12-Dimethylbenz(a)antheracece
Carcinogenecity in Rainbow Trout
INTRODUCTION
In the aquatic environment, fish encounter a variety of chemical
contaminants. Polycyclic aromatic hydrocarbons (PAHs) are a predominant class
of such environmental contaminants (Eisler, 1987). The presence of PAHs in the
aquatic environment is of concern because of their mutagenic/ carcinogenic
properties (Protic-Sabljic and Kurelec, 1983; Hawkins et al., 1990). Field studies
by Malins et aL, (1984, 1985) have established an association between hepatic
tumor incidence among fish populations and sedimentary concentrations of
PAHs. PAHs themselves are not carcinogenic; metabolic activation to reactive
electrophilic metabolites is required (De Pierre and Ernster, 1978). PAHs are
metabolized by the cytochrome P-450 enzyme (CYP) system to form reactive
intermediates which can interact with nucleophilic sites on tissue macromolecules
such as the amino or hydroxyl groups present in DNA, RNA or proteins (Gelboin,
1980). This covalent binding of reactive metabolites of PAH to DNA appears to
be an essential first step in PAH induced neoplasia (Dipple et al., 1984; Harvey
and Nicholas, 1988).
The PAH 7,12-dimethylbenz(a)anthtracene (DMBA) was chosen as the
constituent of interest in these studies because it is a potent procarcinogen,
causing hepatic and extrahepatic tumors in fish (Schultz and Schultz, 1982;
2
Hawkins et al., 1989, 1990). DMBA is metabolized in rodents via a sequence of
reactions catalyzed by CYP and epoxide hydrolase, to give a variety of
metabolites, among them the bay-region diol epoxide DMBA-3,4-dio1-1,2-oxide,
which is considered to be the ultimate carcinogen (Slaga et al., 1979; Sawicki et
aL, 1983; Vigny et al., 1985). However, there appear to be other possibilities for
DMBA metabolic activation. Flesher and Sydnor (1971) have proposed that
hydroxylation of the 7-methyl group of DMBA subsequently formed a reactive
benzylic ester. Several reports, in support of this concept, have indicated that the
sulfation of 7-hydroxymethyl-12-methylbenz(a)anthracene by rat liver cytosolic
sulfotransferase resulted in the formation of a benzylic sulfate ester and the
generation of an electrophilic carbonium ion capable of covalently binding to
cellular DNA (Watabe et al., 1982). Rainbow trout bile had 3,4-dihydrodiol DMBA
and 3-hydroxy DMBA at much higher concentrations than hydroxymethyl
derivatives (Schnitz et al., 1993). Thus providing strong evidence that the major
pathway of DMBA metabolism in rainbow trout was oxidation of the aromatic ring.
(Slaga et al., 1979; Sawicki et al., 1983; Vigny et at., 1985). The majority of
DMBA-initiated hepatic tumors in trout were found to carry Ki-ras alleles with
activating point mutations on codons 12 or 61 (Fong et at., 1993).
Environmental variables play an important role in an organism's ability to
respond to toxic chemicals in the aquatic environment. Studies with fish have
implicated water temperature as modulating factor in carcinogenesis, with warmer
temperature being associated with higher cancer incidence (Hendricks et al.,
1984; Kyono-Hamaguchi, 1984). Temperature significantly altered the effects of
3
exposure to diethylnitosamine (DENA) in the medaka (Oryzias latipedes);
treatment at 22-25°C led to tumor development in 95% of fish but no tumors
occurred when fish were exposed to DENA at 5-8°C (Kyono-Hamaguchi, 1984).
Although the mechanisms for altered carcinogenesis at different temperatures
have not yet been well explained, one can conveniently manipulate water
temperature before, during or after carcinogen exposure in fish to determine the
effects of temperature on cancer initiation and development. This is possible
because chemical induced-carcinogenesis is a multistage process (reviewed by
Williams and Weisburger, 1991). The first step in the traditional scheme is
"initiation" in which the normal cell is damaged by binding of activated
carcinogens to the nucleic acids. If such lesions are not repaired prior to mitosis
they can become fixed as mutations. Promotion involves stimulated growth of
mutated cells while progression entails sequential genetic change leading to an
obvious neoplasm.
Thermal modulation of chemical carcinogenesis may potentially act at
many levels including carcinogen uptake, metabolism, elimination and adduct
formation. Uptake of toxic chemicals has been reported to be temperature
dependent in many species of fish, with accumulation enhanced at higher
temperature (Cember et al., 1978; Powell and Fielder, 1982; Jimenez et al.,
1987). Results from our laboratory have shown that the uptake of the carcinogen
AFB1 from water by rainbow trout was higher acclimation at 18°C compared to
10°C (Zhang et al., 1992).
4
Metabolism of many organic toxic chemicals in fish is accomplished by the
mixed function oxidase (MFO) system, a multi enzyme complex that metabolizes
both foreign and endogenous compounds through a series of oxidative reactions
(Goksoyr and For lin, 1992). These enzymes are also responsible for the
metabolic activation of some xenobiotic compounds to highly reactive
intermediates which are thought to be ultimate carcinogens (Buhler and Williams,
1988). Interactions between temperature and xenobiotic metabolism are complex
and are affected by many factors including acclimation temperature, exposure
temperature and time fish are allowed to acclimate before exposure.
Fish adaptations to long term changes in environmental temperature
usually results in "ideal temperature compensation". This is characterized by
biochemical adaptations which allow physiological processes to continue at
similar rates across a range of acclimation temperatures (Precht, 1958). The
outcome of such adaptation does not result in homeostasis (preservation of the
same state) but enantiostatis (conserved function), a concept formalized by
Mangum and Towle (1977). Supporting this hypothesis, in vitro metabolic activity
of the CYP system was similar when hepatic microsomes from cold and warm
acclimated fish were assayed at their respective acclimation temperatures (Blank
et al., 1989; Koivusaari et al., 1981).
The CYP system is membrane bound enzyme and it's catalytic function is
influenced by membrane fluidity (Hochachka and Somero, 1984). Biochemical
adaptations to conserve the membrane's lipid physical state, termed
"homeoviscous adaptation" (Sinensky, 1974) helps explain the similar metabolic
5
rate of the enzyme system at different acclimation temperatures. Membrane
fluidity is conserved in cold acclimated fish by incorporating relatively high
percentages of unsaturated lipids (Hazel, 1979, 1984). Therefore, one might
predict similar rates of bioactivation from fish acclimated to cold or warm
temperature that are exposed to a precarcinogen at their respective acclimation
temperature. Blank et a/. (1989) and Koivusaari et al. (1981) reported similar
hepatic microsomal MFO activities in cold- and warm-acclimated rainbow trout
when incubation temperatures were the same as acclimation temperatures. On
the other hand different rates are expected from fish acclimated to a particular
temperature and subjected to an acute temperature shift during exposure (Curtis
et at., 1990).
Under these conditions there will not be sufficient time for
biochemical adaptations to maintain similar enzyme activities. Therefore when
cold acclimated fish experience acute temperature increase, membranes become
more fluid than at the acclimation temperature which will result in higher enzyme
activity probably due to enhanced accessibility to active sites. To the contrary,
lower enzyme activity is expected from warm-acclimated fish that experience an
acute temperature decrease, due to increased membrane rigidity. In fact that was
the case in a recent study, fish acclimated to 10°C and exposed to 0.1 ppm
AFB1 at 14°C had significantly higher DNA adducts than fish acclimated to 18°C
and exposed at 14°C (Zhang et al., 1992).
Environmental temperature also effects elimination of xenobiotics from
tissues of poikilotherms (Curtis et al., 1986 and 1990). Toxicants are eliminated
from the body by several routes. An important route for DMBA elimination in rats
6
is biliary excretion, particularly of glucuronide conjugates (Levine, 1974;
Khanduja et a1.,1981; Vater et al., 1991). Rainbow trout eliminate DMBA as
glucuronide or sulfate conjugates depending on the dose administered; at high
doses (1032 pg/kg), glucuronides predominated whereas at lower doses (258
pg/kg), the major conjugates were sulfates (Schnitz et al., 1993). Previous work
in fish suggested that increased temperature stimulated biliary excretion
(Varanasi et al., 1981). In support of this concept, several studies have reported
that biliary excretion of exogenously provided organic anions was stimulated at
warmer environmental temperatures in rainbow trout (Curtis , 1983; Curtis et al.,
1986). In another study using temperature-acclimated rainbow trout, Curtis et al.,
(1990) reported that biliary excretion of benzo(a)pyrene was stimulated with
increased temperature.
Cell proliferation appears to play an important role in the multistage
process of chemical carcinogenesis (Cayama et aL, 1978). Several of the non­
genotoxic carcinogens seem to increase the levels of tumor formation by inducing
a mitogenic stimulus in normal and neoplastic cells of a target organ (Schutle-
Hermann et aL, 1983). As we have stated earlier; temperature influences the
incidence of chemically-induced tumors in fish with warmer temperature being
associated with higher incidence (Hendricks et al., 1984; Kyono-Hamaguchi,
1984). In a recent investigation rainbow trout acclimated, exposed to AFB1 for 30
minutes and reared for nine months at 11.0, 14.5, or 18.0°C had liver tumor
incidences of 4, 35, and 61% respectively (Curtis et al., 1995). Temperaturedependent differences in tumor incidence were not explained by initial rates of
7
adduct formation, since hepatic [3H]AFB1 DNA adduction was slightly higher at
lower temperature (Zhang et al., 1992). These results suggested that warmer
temperature perhaps promoted tumor formation.
Cell proliferation enhances initiation by increasing conversion of damaged
bases in DNA (e.g., adducts) to mutations. This process occurs through
increasing the number of cells in the S phase which increase the vulnerability of
cells to the genotoxic agent and limit the time available for DNA repair (Bertman
and Heidelberger, 1974; Schulte-Hermann et a/.,1983; Rabes et al., 1985). An
alternative mechanism for increasing tumor incidence as result of cell replication
during the initiation process is spontaneous mutations (Ames and Gold, 1990).
That is, spontaneous errors in DNA replication may occur during mitosis and
become fixed as mutations. The probability of such errors will increase with cell
division rate. Spontaneous mutations leading to cancer were well documented in
rodents (Reynolds et al., 1987; You et al., 1989).
Tumor promotion involves the clonal expansion of mutated cells. The
importance of cell proliferation in tumor promotion were documented by several
studies (Solt and Faber, 1976; Schulte-Hermann et al., 1981). However
comprehensive understanding of the mechanism is not well developed (Popp and
Marsman, 1991). The role that cell proliferation most likely plays
during
progression is increasing the probability of additional mutation in initiated cells
thus pushing the cells further away from normalcy towards malignancy (Pitot,
1989).
8
Traditionally, cell proliferation have been studied by the incorporation of
tritiated thymidine during the S-phase of the cell cycle, followed by
autoradiography. Recently researchers employed immunohistochemical methods
to avoid the use of radioactive materials. Investigators have used monoclonal
antibodies to exogenously provided 5-bromo-2-deoxyuridine (BrdU) and
endogenously synthesized nuclear protein (proliferating cell nuclear antigen,
PCNA) (Weghorst et al,. 1991, Kamel et al., 1991). BrdU is incorporated into
DNA only during the S-phase of the cell cycle, while PCNA is expressed at peak
concentrations during the same phase, therefore immunohistochemical
techniques allows for the rapid detection of BrdU and PCNA laden cells. The
commercial availability of antibody that recognizes the naturally occurring cell
replication marker (PCNA) provides a means of retrospectively identifying
replicating cells. PCNA is a 36,000 molecular weight auxiliary protein of DNA
polymerase delta, an enzyme vital for DNA replication (Bravo et al., 1987; Celis
et al., 1987). Studies have indicated PCNA to be highly stable protein found in
most eukaryotes including fish (reviewed by Dietrich, 1993).
As reviewed earlier, temperature may influence DMBA uptake, metabolism
and elimination and subsequently it's carcinogenicity in poikilotherms. Thus the
primary objective of this study was increased understanding of how genotoxicity
of DMBA could be modulated by temperature. The second entailed examining the
potential of temperature to modulate promotion/progression. Therefore a model
was presented in this study by which environmental temperature of rainbow trout
(Oncorhynchiss mykiss) was conveniently manipulated before, during or after
9
exposure to a carcinogen.
This model allowed us to selectively modulate
different steps of the multistage process of chemical carcinogenesis. The
physiological and biochemical adaptations that occur in the tissue of temperature
acclimated poikilotherms provided the mechanistic basis for this model. This
model also enabled us to investigate the role of temperature on cell proliferation
as a potential mechanism for tumor promotion.
10
CHAPTER 1
Temperature Modulated Genotoxicity and Carcinogenicity
of 7,12-Dimethylbenz(a)anthracene in Rainbow Trout
Camille EI-Zahr, Quan Zhang, Jerry D. Hendricks, and Lawrence R. Curtis
To be submitted for publication in Fundamental and Applied Toxicology
11
ABSTRACT
Temperature modulated hepatic disposition, DNA adduct formation and
persistence, and cancer incidence in rainbow trout (Oncorhyncus mykiss) after
a single exposure to 7,12-dimethylbenz(a)anthracene (DMBA). Fish (2 g) were
acclimated at 10, 14 or 18°C for one month and then exposed to 1 ppm DMBA in
their water for 20 hr. Exposures were at respective acclimation temperatures or
10 and 18°C acclimated fish were shifted to 14°C for DMBA exposures. After 4
but not 20 hr of exposure hepatic [3H]DMBA concentration increased with
temperature for fish exposed at their respective acclimation temperatures (10 or
18°C). Adduction of [3H]DMBA to hepatic DNA was similar after 3 days in fish
acclimated and exposed at their respective acclimation temperatures. However,
in fish exposed at 14°C, the concentration of hepatic [3H]DMBA DNA adducts was
higher in 10°C than 18°C acclimated fish after 3 days. Hepatic [3H]DMBA DNA
adduct concentrations were lower in 18°C than 10°C acclimated, exposed and
reared fish after 21 days. There were no differences between temperature shifted
groups at that time. Temperature effects on tumor incidence were assessed 9
months after DMBA waterborne exposures in fish which were reared at: (1) their
respective acclimation and exposure temperatures, (2) 14°C after exposure at
their respective acclimation temperature, and (3) 14°C after 14°C exposures.
Incidence of stomach, liver and swim bladder cancer increased dramatically with
rearing temperature. Differences in tumor incidence were less marked in fish
12
reared at the same temperature (14°C).
Temperature modulated DMBA
carcinogenicity was partially explained by altered genotoxin disposition, but
probably also involved formation and persistence of DNA adducts.
13
INTRODUCTION
Fish like many other aquatic animals are exposed to toxic chemicals in
their environment. Polycyclic aromatic hydrocarbons (PAHs) are a predominant
lass of such environmental contaminants (Malin et al., 1984). PAH themselves
are not carcinogenic; metabolic activation to reactive electrophilic metabolites is
required (De Pierre and Ernster, 1978). T h e
PAH
7
,
12­
dimethylbenz(a)anthracene (DMBA) is a potent synthetic procarcinogen, which
causes tumors in fish (Schultz and Schultz, 1982; Hawkins et a/., 1989; Hawkins
et a/., 1990). DMBA is metabolized via a sequence of reactions catalyzed by
cytochromes P-450 (CYP)
and epoxide hydrolase, to give a variety of
metabolites, among them the bay-region diol epoxide DMBA-3,4-dio1-1,2-oxide,
which is thought to be the ultimate carcinogen (Slaga et aL, 1979; Sawicki et al.,
1983; Vigny et al., 1985). Fong et a/. (1993) studied carcinogenicity and
metabolism of DMBA in rainbow trout embryos and reported that among 11
hepatic tumors examined, nine carried Ki-ras alleles with activating point
mutations on codons 12 or 61.
Ideal temperature compensation is one aspect of fish adaptation to long
term changes in environmental temperature (Precht, 1958). This is characterized
by membrane constitutive changes which allow physiological processes to
continue at similar rates across a range of acclimation temperatures. The
outcome of such adaptation does not result in homeostasis (preservation of the
14
same state) but enantiostatis (conserved function), a concept formalized by
Mangum and Towle (1977). Supporting this hypothesis, in vitro metabolic activity
of the CYP system was similar when hepatic microsomes from cold and warm
acclimated fish were assayed at their respective acclimation temperatures (Blank
et al., 1989; Koivusaari et al., 1981).
The CYP system is membrane bound enzyme and it's catalytic function is
influenced by membrane fluidity (Hochachka and Somero, 1984). Biochemical
adaptations to conserve the membrane's lipid physical state,
termed
"homeoviscous adaptation" (Sinensky, 1974) helps explain the similar metabolic
rate of the enzyme system at different acclimation temperatures. Membrane
fluidity is conserved in cold acclimated fish by incorporating relatively high
percentages of unsaturated lipids (Carpenter et a/., 1995; Hazel; 1979, 1984).
Therefore, one might predict similar rates of bioactivation from fish acclimated to
cold or warm temperature that are exposed to a precarcinogen at their respective
acclimation temperature. Blank et al (1989) reported similar hepatic microsomal
mixed function oxidase activities in cold- and warm-acclimated rainbow trout
when incubation temperatures were the same as acclimation temperatures. On
the other hand, different rates are expected from fish acclimated to a particular
temperature and subjected to an acute temperature shift during exposure
(Carpenter et al., 1990; Curtis et al., 1990). Under these conditions there will not
be sufficient time for biochemical adaptations to maintain similar enzyme
activities. In a recent study, fish acclimated to 10°C and exposed to 0.1 ppm
aflatoxin B1 (AFB1) at 14°C had significantly higher DNA adducts, as a result of
15
more active metabolites formed, than fish acclimated to 18°C and exposed at
14°C (Zhang et al., 1992).
Toxicants are eliminated from the body by several routes. An important
route for DMBA elimination in rats is biliary excretion, particularly of glucuronide
conjugates (Levine,
1974;
Khanduja et a/.,1981; Vater et al., 1991).
Environmental temperature effects elimination of xenobiotics from tissues of
poikilotherms. Biliary excretion of exogenously provided organic anions was
stimulated at warmer environmental temperatures in rainbow trout (Curtis , 1983;
Curtis et at, 1986). In another study using temperature-acclimated rainbow trout,
Curtis et al. (1990) reported that biliary excretion of benzo(a)pyrene was
stimulated with increased temperature.
Among the factors that affect tumor development in fish, warmer
temperature is associated with higher cancer incidence (Hendricks et al., 1984;
Kyono-Hamaguchi, 1984; Curtis et at, 1995). Temperature may influence DMBA
metabolism and elimination and subsequently it's carcinogenicity in poikilotherms.
Therefore, the primary objective of this study was increased understanding of
how temperature-modulated genotoxicity of DMBA corresponded with tumor
response.
16
MATERIALS AND METHODS
Animals
Shasta strain rainbow trout (Oncorhyncus mykiss) were provided by the
Marine Freshwater Biomedical Center core facility at Oregon State University.
Fish (2 g) 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 lightdark cycle. The fish were not fed for 48 hr before [3FIJDMBA exposure.
Chemicals
Uniformly labeled [3H]DMBA was obtained from Amersham (Arlington, IL),
and was more than 94% pure as assessed by high-pressure liquid
chromatography. [3H]DMBA was diluted with unlabeled DMBA (Aldrich Chemical
Co., Milwaukee, WI) to form a stock solution with a final concentration of 200
ppm and specific activity of 12.8 mCi/mmol. Stock solutions were prepared by
injection of DMSO into a sealed vial of crystals and transferring the resultant
solutions to volumetric flasks.
17
Hepatic accumulation DNA adduction and biliary excretion of DMBA
The effects of time and temperature on total [3H]DMBA equivalents in
rainbow trout liver were determined after waterborne exposures at 1.0 ppm for 20
hr. Fish were exposed at their acclimation temperatures (10, 14 or 18°C) or acute
temperature shift (10 to 14°C and 18 to 14°C). The fish were then maintained
in uncontaminated water at their respective exposure temperatures until sampled.
Eight fish from each temperature regimen were sacrificed at 4 and 20 hr after
starting the exposure, livers and gallbladder plus bile were removed and weighed.
Two livers were pooled and homogenized 4:1 with distilled water. Duplicate 100
pl aliquots were placed in liquid scintillation counting (LSC) vials. Soluene 350
tissue solubilizer (0.5 ml) was added to each vial which was kept at room
temperature for 24 hr. After this incubation, 10 ml of counting fluid was added to
each vial which was then analyzed by liquid scintillation counting (LSC) (Packard
Tri Carb). The total [3H]DMBA equivalent residues in gallbladder plus bile were
quantified by LSC after base digestion and decolorization as described earlier.
The remaining liver homogenates were further homogenized with 2.5 volume of
hexane by 10 up and down strokes with a potter-Elvehjem homogenizer and then
vortexed for 5 min. Samples were then centrifuged (2000g for 10 min), the
hexane layers aspirated, their volumes determined, and subsamples evaporated
prior to LSC.
Eighteen fish from each temperature regimen were sampled 3 and 21
days after [3H]DMBA exposure for analysis of hepatic DNA adducts. Groups of
18
three livers were pooled and homogenized at 4°C. Duplicate 100 pl aliquots
were sampled from the homogenized livers for determination of total [3H]DMBA
equivalents. DNA was extracted from the remaining homogenized livers and the
amounts of [3H]DMBA adducted to DNA were determined by LSC (Zhang et al.,
1992). Fish stomachs were subjected to the same procedure as livers to
determine DNA binding, but sufficient counts were not detected.
Influence of temperature on tumor Incidence
In an effort to selectively modulate different stages in DMBA-induced
carcinogenesis, fish were subjected to three temperature regimens. Components
of these regimens were categorized as follows: (1) acclimation period which
lasted for at least four weeks prior to exposure to allow for biochemical
adaptations for temperature, (2) exposure to 1.0 ppm DMBA for 20 hr and (3) a
rearing period of nine months. Duplicate groups of 100 fish each were tested in
three series of temperature regimens. In the first series, fish were exposed to
DMBA and reared at their respective acclimation temperatures (10, 14 or 18°C).
Under these conditions similar in vivo metabolism of the procarcinogen was
expected due to "ideal temperature compensation". In the second series fish were
exposed at their respective acclimation temperatures (10 or 18°C) and reared at
an intermediate temperature (14°C) to examine the promotional effects of
temperature. The third series of temperature treatment involved 4°C temperature
shift for the exposure period ( 10 to 14°C and 18 to 14°C). Fish were then
19
maintained at the same rearing temperature (14°C). Temperature shift during
DMBA exposure was viewed as a manipulation which preferentially modulated
genotoxicity. Duplicate groups of 100 fish were vehicle (DMSO) exposed and
subjected to the above temperature regimens to assess the influence of
temperature on spontaneous tumor incidence in rainbow trout (control group). At
the end of the rearing period fish were killed by overdose with the anesthetic MS­
222 (90 mg/I) for excision of tissue, gross pathological examination and fixation
for histopathology.
Statistics
The time-course data for hepatic [3H]DMBA disposition and [3H]DMBA­
DNA adduct concentrations for the fish from different temperature regimens were
compared by two-way analysis of variance followed by point comparison using
the Tukey test. Comparisons of two means were made by the t-tests. Tumor
incidence data were subjected to arc-sine transformation (Sokal and Rohlf, 1981)
and analyzed by one-way analysis of variance. Two replicate tanks which
contained fish that were acclimated, exposed and reared at 18°C were subjected
to sudden increase in temperature towards the end of the experiment due to
malfunction in the thermostat. This resulted in high fish mortality which prompted
us to combine the remaining 21 fish in one tank. Another tank which held fish that
were acclimated at 10°C and exposed and reared at 14°C was lost due to
equipment failure (interruption of water flow). The results from these groups
20
were analyzed by assuming that the variability within the two treatments without
replication would be the same as the variability within the treatments which were
replicated. Thus the error terms used in the F-ratios were calculated using only
the treatments where replication occurred. Results were expressed as means ±
standard error (SE). Significance was set at p s 0.05.
21
RESULTS
Hepatic accumulation and biliary excretion of DMBA
Temperature significantly affected total [3H]DMBA equivalents in rainbow
trout liver during the first 4 hr of a 20 hr waterborne exposure to 1.0 ppm
[3H]DMBA There was a two-fold increase in [3H]DMBA hepatic disposition in fish
acclimated and exposed at 18°C when compared to fish acclimated and exposed
at 10°C (Fig. 1.1, upper panel). After 20 hr of exposure livers from 10 and 18°C
exposed fish contained approximately the same equivalents of [3H]DMBA. Two-
way analysis of variance followed by Tukey test for multiple comparisons of
means indicated that hexane extractable [3H]DMBA in liver of rainbow trout
decreased with time and higher temperature (Fig. 1.1, middle panel). HPLC
demonstrated [3H]DMBA equivalents were the primary material extracted by
hexane (86.0%). Biliary excretion was also affected by temperature (Fig. 1.1,
lower panel). Fish acclimated and exposed at 18°C had significantly higher
[3H]DMBA equivalents in their gallbladders than fish acclimated and exposed at
10°C, after 4 hr exposure.
Gallbladder [3H]DMBA equivalents 20 hr after
exposure were too variable to detect statistical differences.
Fish acclimated at 10°C and exposed at 14°C had a significantly higher
hepatic concentration of total [3H]DMBA equivalents than those acclimated and
exposed at 14°C (Fig. 1.2, upper panel) despite similar [3H]DMBA gallbladder
disposition (Fig. 1.2, lower panel). On the other hand, [3H]DMBA hepatic and
22
Figure 1.1
Disposition of total (upper panel) and hexane extractable (middle
panel) [3H]DMBA in liver and gallbladder (lower panel) of rainbow
trout exposed to 1.0 ppm DMBA for 20 hr at their respective
acclimation temperature (10 or 18°C). Samples were taken after 4
or 20 hr of starting waterborne exposures. Results are means t SE
of four individual preparations of two pooled livers and for 6-8
gallbladders. The first number represents acclimation temperature
and the second exposure temperature for each regimen. Asterisks
indicates significant differences between temperature regimens at
particular times during exposure.
O
H
0
N.)
0-P,
0co
µg [3H]DMBA/Gallbladder
0co
I
03
1
oo
0
0
-_
O
*
=II
b.
0
*
0
N)
O
W
% Hepatic [3H]l:IMBA in Hexane
O
0
1
O
-.%
*
0
N)
0
(...1
Hepatic [3H]l)MBA (ng/mg liver)
24
Figure 1.2
Disposition of total (upper panel) and hexane extractable (middle
panel) [3H]DMBA in liver and gallbladder (lower panel) of rainbow
trout acclimated at 10, 14, or 18°C and exposed to 1.0 ppm DMBA
for 20 hr at 14°C. Samples were taken after 4 or 20 hr of starting
waterborne exposures. Results are means ± SE of four individual
preparations of two pooled livers and for 6-8 gallbladders. The first
number represents acclimation temperature and the second
exposure temperature for each regimen. Asterisks indicates
significant differences between temperature regimens.
25
=
*
0
4 hours
20 hours
1
10-14
14-14
18-14
12
0
*
/4
10-14
1
14-14
18-14
200
14-14
0
18-14
Temperature Regimen (°C)
1
26
gallbladder disposition of fish acclimated at 18°C and exposed at 14°C was not
different from fish acclimated and exposed at 14°C while the amount of hepatic
hexane extractable [3H]DMBA was significantly higher. The significantly higher
hexane extractable [3H]DMBA in liver of fish acclimated at 18°C and exposed at
14°C (Fig. 1.2, middle panel) could be explained by lower mixed function
oxygenase (mfo) activity expected at this temperature regimen (Curtis et
al.,1990).
Adduction of DMBA to hepatic DNA
There were no significant differences in hepatic concentrations of total
[3H]DMBA equivalents for fish acclimated at either 10 or 18°C and exposed at
their respective acclimation temperature after 3 and 21 days of exposure (Fig.
1.3, upper panel).
Similarly, the concentrations of hepatic [3H]DMBA DNA
adducts 3 days after exposure were not significantly different (Fig. 1.3, lower
panel). However, the concentration of hepatic [3H]DMBA DNA adducts 21 days
after immersion was two-fold higher for fish acclimated and exposed at 10°C than
those acclimated and exposed at 18°C. Thus, hepatic [3H]DMBA DNA adducts
appeared somewhat more persistent at 10°C than at 18°C in fish exposed and
reared at their respective acclimation temperatures.
Temperature shift during exposure to the genotoxin modulated hepatic
disposition and formation of [3H]DMBA DNA adducts. Two-way analysis of
variance indicated that the hepatic concentration of total [3H]DMBA equivalents
27
Figure 1.3 Disposition of total [3H]DMBA (upper panel) and DNA adducts in
liver (lower panel) of rainbow trout exposed to 1.0 ppm DMBA for
20 hr at their respective acclimation temperature (10 or 18°C).
Samples were taken 3 or 21 days after exposures. Results are
means ± SE of 5-6 individual preparations of three pooled livers.
The first number represents acclimation temperature and the
second exposure temperature for each regimen. Asterisk indicates
significant differences between temperature regimens at particular
times during exposure.
28
10 ­
days
1772 21 days
3
10
10
18 18
40
days
21 days
3
I
30
I
20
*
10
10 -10
18
18
Temperature Regimen
(°C)
29
and the concentration of hepatic [3H]DMBA DNA adducts 3 and 21 days after
exposure were significantly higher in fish acclimated at 10°C and exposed at
14°C than either fish acclimated and exposed at 14°C or acclimated at 18°C and
exposed at 14°C (Fig. 1.4).
Influence of temperature on tumor incidence
There were no acclimation temperature-dependent differences in fish body
weight. The mean body weight for fish acclimated at 10, 14, or 18°C were 5.40
± 0.69, 5.69 ± 0.74, and 5.79 ± 0.68 g respectively. Rearing temperatures had no
effect on fish body weights either (overall average, 87.85 ± 4.0 g).
DMBA induced tumors in swim bladder, liver and stomach of fish at all
temperature regimens tested. Highest tumor incidence was observed in the
stomach and lowest in the swim bladder. Fish exposed to DMSO developed no
spontaneous tumors.
Tumor incidence increased with temperature in fish exposed and reared
at their respective acclimation temperatures (Fig. 1.5, upper panel). However,
rearing the fish at an intermediate temperature (14°C) eliminated acclimation and
exposure temperature differences (Fig. 1.5, middle panel). Temperature shift
upwards from 10°C acclimation to 14°C for DMBA exposure and rearing
increased tumor incidences (Fig. 1.5, lower panel).
Tumor incidence was
significantly higher when rearing temperature was increased 4°C in fish
30
Figure 1.4 Disposition of total [3H]DMBA (upper panel) and DNA adducts in
liver (lower panel) of rainbow trout acclimated at 10, 14, or 18°C
and exposed to 1.0 ppm DMBA for 20 hr at 14°C. Samples were
taken 3 or 21 days after exposures. Results are means ± SE of 5-6
individual preparations of three pooled livers. The first number
represents acclimation temperature and the second exposure
temperature for each regimen. Asterisks indicates significant
differences between temperature regimens.
31
10
3
days
21 days
10-14
14-14
80
3
60
days
EZi 21 days
40
20
10-14
14-14
18-14
Temperature Regimen (°C)
32
Figure 1.5
Percentage of rainbow trout bearing swim bladder, liver or stomach
tumors 9 months after 20 hr waterborne DMBA exposure at 1.0
ppm. Fish were exposed to DMBA and reared at their respective
acclimation temperature (upper panel) or exposed at their
respective acclimation temperature and reared at intermediate
temperature of 14°C (middle panel) or exposed and reared at 14°C
and acclimated at 10, 14, or 18°C (lower panel). The first number
represents acclimation temperature, the second exposure
temperature and the third rearing temperature for each regimen.
Asterisks indicates significant differences between temperature
regimens within a panel, while plus signs indicate significant
differences in temperature regimens between upper and middle
panels.
33
100
80
Swim Bladder
Liver
EZZ2i Stomach
60
40
*
20
1 0 10 10
100
14 -14 -14
I1
18 - 18-18
­
80
60
0
40
20
10 -10 - 14
14 -14 -14
18 - 18 -14
14 -14 -14
18 -14 -14
100
80
60
40
20
0
*
10 14 -14
Temperature Regimen (°C)
34
acclimated and exposed at 10°C (Fig. 1.5, upper and middle panels). On the
other hand lowering rearing temperature 4°C decreased tumor incidence,
particularly for liver and swim bladder in fish acclimated and exposed at 18°C
(Fig. 1.5, upper and middle panels).
35
DISCUSSION
Temperature affected [3H]DMBA hepatic disposition in rainbow trout.
Elevated temperature increased the liver burden of total [3H]DMBA equivalents
after 4 hr of waterborne exposure in both fish exposed at their acclimation
temperature and those subjected to acute temperature shift (Figs. 1.1, 1.2). This
may be partially explained by increased hepatic blood flow (Kemp and Curtis,
1987) and gill ventilation associated with higher oxygen demand at warmer
temperatures, leading to greater gill uptake and distribution of [3H]DMBA to livers.
The movement of DMBA between fish tissue and water is dynamic and involves
DMBA accumulation in tissue due to its lipophilicity and its passive release from
tissue reflecting an equilibrium distribution between the aqueous phase and
lipophilic phase. Accordingly, higher DMBA liver disposition at 18°C up to 4 hr
of exposure in fish exposed at their respective acclimation temperature may have
resulted from more rapid uptake to a steady state concentration.
Equilibrium
concentrations of PAHs were often reached in fish tissues in 24 h or less (Lee et
aL, 1972). Another contributing factor to hepatic disposition was the nonspecific
capacity of intra- and extracellular proteins to bind to [3H]DMBA, thus reducing it's
availability for biotransformation and clearance. Temperature effects on protein
binding capacity have not been studied extensively.
Total hepatic [3H]DMBA equivalents increased to similar concentrations in
fish acclimated and exposed at their respective acclimation temperature after 20
36
hr. However, fish subjected to an acute temperature shift showed differences
in liver burden of total
[3H]DMBA equivalents at this time. Differences
in [3H]DMBA disposition between fish acclimated and exposed at the same
temperature and fish subjected to acute temperature shift during exposure can
be explained by modulation of genotoxin metabolism and excretion. When fish
were acclimated and exposed to the genotoxin at the same temperature similar
in vivo metabolism at 10 and 18°C was expected due to ideal temperature
compensation (Precht, 1958). In fact, hepatic microsomes from cold and warm
acclimated fish exhibit similar monoxygenase activities when assayed at their
respective acclimation temperatures (Blank et al., 1989; Koivusaari, 1983;
Koivusaari et al., 1981). However hexane extractable [3H]DMBA in liver of fish
decreased with higher temperature (Fig. 1.1) indicating higher metabolism at
warmer temperature. These findings were better explained by higher biliary
excretion at 18°C rather than elevated metabolism at higher temperature. Our
data clearly demonstrated increased hepatic elimination of [3H]DMBA with
temperature (Fig. 1.1). Similarly earlier work with benzo(a)pyrene (Curtis et al.,
1990), phenolphthalein (Curtis, 1983) and taurocholate (Curtis et a/., 1986; Kemp
and Curtis,1987) showed that biliary excretion of organic anions increased with
temperature. No differences in [3H]DMBA hepatic disposition occurred after 3 or
21 days of exposure between 10 and 18°C for fish exposed at their respective
acclimation temperature (Fig. 1.3).
Fish acclimated at 10°C and subjected to an acute temperature shift to
14°C during exposure had higher liver burden of total [3H]DMBA equivalents than
37
fish acclimated and exposed at 14°C after 20 hr (Fig.
1.2).
Under these
conditions there was not sufficient time for biochemical adaptations to adjust
enzyme activities. In support of the above argument, total hexane extractable
[3H]DMBA in livers of fish acclimated to 18°C and subjected to an acute shift to
lower temperature (14°C) was significantly higher, indicating lower mfo activity.
Acute temperature shift data indicated no significant differences in biliary
excretion.
From the above discussion it was concluded that temperature
modulated hepatic DMBA disposition in rainbow trout through it's effects on
metabolism and elimination of the genotoxin. Similarly, exposure temperature
selectively modulated benzo(a)pyrene metabolism and biliary excretion in
temperature-acclimated rainbow trout (Curtis et al., 1990).
Formation of [3H]DMBA -DNA adducts correlated with and was dependent
on the target organ concentration of [3H]DMBA, which in turn was influenced by
temperature. Livers of fish exposed at their respective acclimation temperature
contained approximately the same equivalents of [3H]DMBA and similar
concentrations of [3H]DMBA-DNA adducts 3 days after exposure (Fig. 1.3).
However, higher hepatic concentrations of total [3H]DMBA equivalents in fish
acclimated and exposed at 10°C and exposed at 14°C, probably contributed to
higher concentration of hepatic [3H]DMBA -DNA adducts than either fish
acclimated and exposed at 14°C or fish acclimated at 18°C and exposed at 14°
after 3 days of DMBA immersion (Fig. 1.4). Adduct formation was probably also
influenced by temperature through it's effects on DMBA bioactivation rates.
Hepatic microsomes from cold and warm acclimated rainbow trout had similar
38
CYP-mediated activities when assayed at respective acclimation temperatures
(Blank et al., 1989; Koivusaari et al., 1981). On the other hand, arylhydrocarbon
hydroxylase at 14°C was higher for hepatic microsomes from 10 than 18°C
acclimated rainbow trout ( Carpenter et al., 1990; Curtis et al., 1990).
[3H]DMBA-DNA adducts were less persistent at higher temperature (Fig.
1.3). We partially attributed this to higher rate of DNA repair synthesis at warmer
temperature. Miller et al, (1989) demonstrated more than two-fold increase in the
rate of DNA repair synthesis as the temperature increased from 15 to 25°C in
isolated rainbow trout liver cells. Similarly Zhang et al., (1992) reported reduced
persistence of [3H]aflatoxin-DNA adducts in rainbow trout at warmer acclimation
temperature. Increased spontaneous depurination and enzymatic repair were
identified as potential contributors to reduced persistence at higher temperature.
Tumor incidence in stomach, liver and swim bladder increased with
temperature in rainbow trout (Fig. 1.5), in spite of higher concentration of
[3H]DMBA -DNA adducts in livers of fish acclimated and exposed at 10°C
compared to 18°C. Similarly, previous work showed that chemically induced
tumor incidence in fish increased with exposure temperature (Hendricks et al.,
1984; Kyono-Hamaguchi, 1984; Curtis et al., 1995). Initially, increased cell
turnover with temperature was thought to underlie these results, since bulky DNA
adducts may be lost at higher rate at 18°C than at 10°C. In support of the above
argument, the difference in tumor incidence was abolished when fish were reared
at the same intermediate temperature (14°C) after their exposure at their
respective acclimation temperature (Fig. 1. 5, middle panel). Furthermore, tumor
39
incidence in stomach increased more than three-fold when rearing temperature
was increased 4°C. Lowering rearing temperature on the other hand decreased
stomach, liver and swim bladder tumor incidence (Fig. 1.5, upper and middle
panels). A retrospective analyses of fish livers from a tumor study (Curtis et al.,
1995) was conducted using antibodies to endogenous proliferating nuclear
antigen (PCNA) evaluated cell proliferation at different temperature regimens (ElZahr et al., 1994). Immunohistochemistry for PCNA demonstrated no significance
increase in number of labeled hepatocytes with temperature (EI-Zahr et al.,
1994). This discounted the role of mitogenesis in temperature-modulated
chemical carcinogenesis in rainbow trout. An alternative mechanism involved
DNA error prone repair. Equivalent [31-IpMBA-DNA adducts were formed at 10°C
and 18°C but adducts persisted longer at the lower temperature (Fig. 1.3). Miller
et a/. (1989) demonstrated that in vitro DNA synthesis repair of rainbow trout
hepatocytes increased with temperature. In fact a negative correlation between
adduct persistence and tumor incidence was reported (EI-Zahr et al., 1994) which
supported the argument that loss of DNA adducts reflected error prone repair.
Although DNA repair mechanisms have been studied in many different
systems ranging from bacteria to mammalian cells, little is known about DNA
repair in fish. It is generally believed that DNA damage can be repaired by the
following mechanisms: direct repair, excision repair, postreplication repair and
mismatch repair (reviewed by Myles and Sancar, 1989; Sancar, 1994). Among
the DNA repair mechanisms investigated in fish, direct repair appeared to be the
most prominent followed by excision repair (Regan et al., 1982; Mano et al., 1980
40
and 1982). Direct repair mechanisms reverse the covalent modification in DNA
without removal nor replacement of bases or nucleotides (Myles and Sancar,
1989), while excision repair removes the damaged DNA section and replaces it
with a normal base using the complementary strand as a template (Sancar,
1994). The excision repair mechanism is considered as the main repair system
responsible for removing bulky DNA adducts. Ishikawa et al. (1984) studied DNA
repair processes in five species of fish and concluded that liver and intestinal
cells were deficient in the excision repair mechanism. Other studies also
suggested that excision repair was defective in fish cells (Shima et a/., 1981;
Regan et al., 1983; Walton et al., 1983). Defects in excision repair in mammalian
cells could lead to high incidence of cancer (Sankar, 1994; Tanaka and Wood,
1994). Accordingly, it is possible for the higher tumor incidence recorded at 18°C
to result from error prone repair since more DNA adducts persisted at 10°C.
Other factors beside DNA adduct
persistence could also contribute to
differences in tumor incidence between fish acclimated, exposed and reared at
10°C and 18°C. These include hormonal influence, immune status and other
post-initiative factors which could differ between fish subjected to different
temperature regimens.
Tumor incidences were also affected by both target organ dose and the
extent of adduction at measured tissue. Fish subjected to 4°C upwards shift
during exposure had higher hepatic concentration of total [3H]DMBA equivalents
and [31-IJDMBA-DNA adducts (Fig. 1.4) and consequently resulted in higher tumor
incidence even when fish were reared at the same temperature (14°C)(Fig. 1.5).
41
From the above discussion we concluded that chemically induced tumors in fish
were modulated by temperature not only through genotoxin disposition and
formation but also through the persistence of DNA adducts.
42
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Kyono-Hamaguchi, Y. 1984. Effects of temperature and partial hepatectomy on
the induction of liver tumors in Oryzias latipes. Natl. Cancer Inst. Monogr.
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Lee, R.F., R. Sauerheber, and G.H. Dobbs. 1972. Uptake, metabolism and
discharge of polycyclic aromatic hydrocarbons by marine fish. Mar. Biol.
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Levine, W. G. 1974. Hepatic Uptake, metabolism, and biliary excretion of 7,12­
dimethylbenz(a)anthracene in the rat. Drug Metab. Dispos. 2: 169-177.
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P.G. Prohaska, A.J. Friedman, L.D. Rhodes, D.G. Burrows, W.D.
Gomlund, and H.O. Hodgins. 1984. Chemical pollutants in sediments and
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Sci. Technol. 18: 705-713.
Mangum, C.P. and D.W. Towle. 1977. Physiological adaptation to unstable
environments. Ameri. Sci. 65: 67-75.
Mano, Y., H. Mitani, H. Etoh, and N. Egami. 1980. Survival and photoreactivability
of ultraviolet-irradiated cultured fish cells (CAF-MM1). Radiat. Res. 84:
514-522.
Mano, Y., K. Kator, and N. Egami. 1982. Photoreactivation and excision of
thymine dimers in ultraviolet-irradiated cultured fish cells. Radiat. Res. 90:
501-508.
Miller, M.R., J.B. Blair, and D.E. Hinton. 1989. DNA repair synthesis in isolated
rainbow trout liver cells. Carcinogenesis 10: 995-1001.
Myles, G.M., and A. Sancar. 1989. DNA repair. Chem. Res. Toxicol. 2(4): 197­
226.
Precht, H. 1958. Concepts of the temperature adaptation of unchanging reaction
systems of cold-blooded animals. In Physiological Adaptation, ed. C.L.
Prosser, 50-78. Washington, D.C. : Amer. Physiol. Soc.
Regan, J.D., W.L. Carrier, C. Samet, and B.L. 011a. 1982. Photoreactivation in
two closely related marine fishes having different longevities. Mech. Aging
Dev. 18: 59-66.
45
Regan, J.D., R.D. Snyder, A.A. Francis, and B.L. 011a. 1983. Excision repair of
ultraviolet and chemically induced damage in the DNA of fibroblasts
derived from two closely related species of marine fishes. Aquat. Tox. 4:
181-188.
Sancar, A 1994. Mechanisms of DNA excision repair. Science 266: 1954-1956.
Sawicki, J.T., R.C. Moschel, and A. Dipple. 1983. Involvement of both syn and
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of
7,12­
in
the
anti-dihydrodiol-epoxides
dimethylbenz(a)anthrancene to DNA in mouse embryo cell culture. Cancer
Res. 43: 4212-4218.
Schultz, M.E., and R.J. Schultz. 1982. Induction of hepatic tumors with 7,12­
dimethylbenz(a)anthracene in two species of viviparous fishes (Genus
Poeciliopsis). Environ. Res. 27: 337-351.
Shima, A., M. Ikenaga, 0. Nikaido, H. Takebe,
and N. Egami. 1981.
Photoreactivation of ultraviolet light-induced damage in cultured fish cells
as revealed by increased colony forming ability and decreased content of
pyrimidine dimers. Photochem. Photobiol. 33: 313-316.
Sinensky, M. 1974. Homeoviscous adaptation-A homeostatic process that
regulates the viscosity of membrane lipids in Escherichia coli. Proc. Natl.
Acad. Sci. 71: 522-525.
Sinnhuber, R. 0., 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: 389-408.
Slaga, T.J., G.L. Gleason, J. Di Giovanni, K.B. Sukumaran, and R.G. Harvey.
1979. Potent tumor-initiating activity of the 3,4-dihydrodiol of 7,12­
dimethylbenz(a)anthracene in mouse skin. Cancer Res. 39: 1934-1936.
Sokal, R.R., and F.J. Rohlf. 1981. Biometry, 2nd ed., pp. 859. New York:
Freeman.
Tanaka, K., and R.D. Wood. 1994. Xeroderma pigmentosum and nucleotide
excision repair of DNA. Trends Biochem. Sci. 19: 83-86.
Vater, S.T., D.M. Boldwin, and D. Warshawsky. 1991. Hepatic metabolism of
7,12-dimethylbenz(a)anthracene in Male, Female and Ovariectomized
Sprague-Dawley Rats. Cancer Res. 51: 492-498.
46
Vigny, P., A. Brunisson, D.H. Phillips, C.S. Cooper, A. Hewer, P.L. Grover, and
P. Sims. 1985. Metabolic activation of 7,12-dimethylbenz(a)anthracene
and its hydroxy-methyl derivatives. Cancer Res. 40: 3661-3664.
Walton, D.G., AB. Acton, and H.F. Stich. 1983. DNA repair synthesis in cultured
mammalian and fish cells following exposure to chemical mutagens.
Mutation Res. 124: 153-161.
Zhang, Q., K Suorsa-Super, and L.R. Curtis. 1992. Temperature-Modulated
Aflatoxin B1 Hepatic Disposition and Formation and Persistence if DNA
adducts in Rainbow Trout. Toxicol. Appl. Pharmacol. 113: 253-259.
47
CHAPTER 2
Cell Proliferation and Temperature Modulated Chemical
Carcinogenesis in Rainbow Trout
Camille EI-Zahr, Daniel R. Dietrich, C. Samuel Bradford,
David W. Barnes, and Lawrence R. Curtis
To be submitted for publication in Aquatic Toxicology
48
ABSTRACT
Incidence of aflatoxin B1 (AFB1) induced liver cancer increased with
temperature in rainbow trout (Curtis et aL, 1995), a result not explained by initial
rates of adduct formation (Zhang et al., 1992). These results suggested warmer
temperature promoted tumor formation. Cell proliferation played an important role
in the multistage process of chemical carcinogenesis in a number of experimental
models. Retrospective analyses of livers from aflatoxin B1 tumor study was
conducted with antibodies to endogenous proliferating nuclear antigen (PCNA).
PCNA is a highly stable protein expressed at maximum concentrations during the
S-phase of the cell cycle. Proliferating cells were scored by counting labeled
nuclei in 5 random 10x fields using an image analysis program (BIOQUANT
SYSTEM IV). There was no significant increase in numbers of PCNA labelled
hepatocytes with temperature. As a further means of cell proliferation
assessment, the influence of acclimation temperature on plasma mitogens that
stimulated cell division was evaluated in cultured chinook salmon embryo cells
(CHSE-214). Plasma from rainbow trout (120-150 g) acclimated to either 10.0 or
18.0°C for at least four weeks stimulated in vitro proliferation of CHSE-214 cells
to the same extent. This further discounted the role of mitogenesis in
temperature-modulated chemical carcinogenesis in rainbow trout.
49
INTRODUCTION
Several lines of investigation indicated cell proliferation played an
important role in the multistage development of cancer. Replication of irradiated
nonproliferating cultured cells was required for their transformation to the
neoplastic state (Borek and Saches, 1968). Studies on the mode of action of
nongenotoxic chemical carcinogens provided further evidence.
While,
mechanisms were not fully understood, many nongenotoxic carcinogens induced
cell proliferation at the target organ (Ames and Gold, 1990; Cohen and Ellwein,
1990).
Cell proliferation enhances initiation by increasing conversion of damaged
bases in DNA (e.g., adducts) to mutations. This process involves increasing the
number of cells in the S phase which increases the vulnerability of cells to a
genotoxic agent and limits the time available for DNA repair (Bertman and
Heidelberger, 1974; Schulte-Hermann et a/.,1983; Rabes et aL, 1985). Increase
in spontaneous mutation rate is an alternative mechanism for cell replication
increasing initiation and tumor incidence (Ames and Gold, 1990). That is,
spontaneous errors in DNA replication may occur during mitosis and become
fixed as mutations. The probability of such errors will increase with cell division
rate. Spontaneous mutations leading to cancer are well documented in rodents
(Reynolds et at., 1987; You et at., 1989).
50
Tumor promotion involves the clonal expansion of mutated cells. The
importance of cell proliferation in tumor promotion is documented (Solt and
Farber,
1976; Schulte-Hermann et al., 1981). However, comprehensive
understanding of the mechanism is not well developed (Popp and Marsman,
1991). The role that cell proliferation most likely plays during progression is
increasing the probability of additional mutations in initiated cells thus pushing
the cells further away from normalcy towards malignancy (Pitot, 1989).
Temperature influences the incidence of chemically-induced tumors in fish
(Hendricks et al., 1984; Kyono-Hamaguchi, 1984; Curtis et al., 1995) with warmer
temperatures being associated with higher incidence. Aflatoxin B1 (AFB1) is an
extremely potent fish hepatocarcinogen for which carcinogenicity is modulated by
temperature (Hendricks et al., 1984; Curtis et al., 1995). In rainbow trout, AFB1
is metabolized to a reactive intermediate, AFB1-8,9-epoxide, by liver cytochrome
P-450 2K1 (Williams and Buhler, 1983; Buhler et al., 1994). AFB1 genotoxicity
is associated with AFB1-8,9-epoxide binding with DNA to form principal covalent
product 8, 9- dihydro- 8- (N7- guanyl) -9- hydroxyaflatoxin B1 in rainbow trout (Croy
et al., 1980) and rodents (Croy and Wogan, 1981).
In a recent investigation rainbow trout acclimated, exposed to AFB1 for 30
minutes and reared for nine months at 11.0, 14.5, or 18.0°C had liver tumor
incidences of 4, 35, and 61% respectively (Curtis et al., 1995). Temperaturedependent differences in tumor incidence were not explained by initial rates of
adduct formation, since hepatic rHJAFB1 DNA adduction was slightly higher at
lower temperature (Zhang et al., 1992). These results suggested that warmer
51
temperature perhaps promoted tumor formation. This study assessed mitogenicity
of warm temperature in two ways: (1) Comparison of mitogenic potency of plasma
from 10 and 18°C acclimated rainbow trout in cultured CHSE-214 cells. (2)
Retrospective immunohistochemistry of proliferating cell nuclear antigen (PCNA)
in liver from an earlier tumor study (Curtis et al., 1995)
52
MATERIALS AND METHODS
Mitogenesis in cultured cells
Shasta strain rainbow trout (Oncorhynchus mykiss) (body weight 120 -150
g) were acclimated for at least four weeks at either 10 or 18°C. Blood was drawn
from the caudal vein into heparinized syringes. After removing the cells by
centrifugation, the plasma was sterilized by filtration (0.2um filter), and was
incubated at 56°C for 20 min before use in cell culture.
The mitogenicity of trout plasma was assessed in a growth assay
employing CHSE-214 cells (an established cell line derived from chinook salmon
embryos). CHSE-214 cells were maintained in LDF medium (Collodi et a/., 1990
and 1992) under air atmosphere at 18°C in 6-well tissue culture plates (Falcon).
Each well contained 2 ml LDF medium supplemented with trout plasma at a
concentration of either 0.3% or 1% (doses chosen based on a preliminary
concentration-response experiment). After 5 days the culture medium was
replaced with fresh medium; on day 10 the total number of cells in each well was
determined by harvesting well contents using trypsin/EDTA and counting a
suspension of the trypsinized cells in phosphate-buffered saline using a Coulter
particle counter.
53
Retrospective analyses of tumor study
The experimental design and results of the tumor study were previously
described (Curtis et al., 1995). Briefly, Shasta strain rainbow trout (Oncorhynchus
mykiss) (4 to 5 g) were acclimated to 11.0, 14.5, or 18.0°C. Fish were then
exposed to 0.075 to 0.12 ppm waterborne AFB1 for 30 minutes. Following
exposure, fish were returned to holding tanks and reared at 11.0, 14.5, or 18.0°C.
After nine months then were killed and examined for tumors.
Retrospective analyses of liver by PCNA immunohistochemistry was
conducted on 21 livers fixed in Bouin's solution and embedded in paraffin blocks,
selected from seven treatments from the above tumor study. Three livers were
examined for each treatment. The first three treatments were from fish acclimated,
exposed to 0.1 ppm AFB1 and reared at 11.0, 14.5, or 18.0°C. The forth and fifth
treatments were from fish acclimated, exposed and reared at 11.0 or 18.0°C for
which concentrations were 0.12 and 0.075 ppm AFB1 respectively, which
achieved equivalent target organ dose (Zhang et al., 1992). The last two
treatments were from fish acclimated to either 11.0, 14.5 or 18.0°C then exposed
to 0.1 ppm AFB1 at 14.5°C and reared at 14.5°C. Equivalent target organ dose
was also achieved in this protocol (Zhang et al., 1992).
PCNA immunohistochemistry
Two-four micron thick tissue sections were deparaffinized and then
rehydrated in phosphate-buffered-saline (PBS). Unspecific protein binding was
54
blocked via incubation of the tissue sections with a 1:20 dilution of normal mouse
serum (BioGenex) for 20 min. Tissue sections were then incubated with a 1:400
dilution of a mouse monoclonal antibody to PCNA (PC10, Dakopatts) overnight
at 4 °C. Following this incubation the sections were rinsed with PBS thoroughly
and reincubated for 30 min at room temperature with a 1:10 dilution of
biotinylated anti-immunoglobulins specific for mouse (LinkR, BioGenex, San
Ramon CA). After this the sections were washed in PBS and incubated for a
further 20 min at room temperature with a 1:10 dilution of streptavidin-conjugated
alkaline phosphate (LabeiR, BioGenex, San Ramon CA). Following a 2x5 min
wash with PBS, unspecific alkaline phosphate was blocked via a 10 min
incubation of the sections in a 50mM solution of LevamisolR (Sigma, St. Louis
MS). Fast RedR (BioGenex, San Ramon CA) was then applied as the chromogen
which turned positive nuclei red. Following a brief wash with tap water the
sections were then counterstained blue with Mayers-Hematoxylin. Proliferating
hepatocytes were scored by counting labeled nuclei in 5 random x10 fields with
an image analysis program (BIOQUANT SYSTEM IV). Data were represented
as number of labeled cells per mm2.
Statistics
All data were analyzed by analysis of variance and the Tukey test for
multiple comparisons of means. Significance was set at p s 0.05. Results were
expressed as means ± SEM.
55
RESULTS
Rainbow trout plasma stimulated proliferation of cultured CHSE-214 cells.
The mean number of cells per well increased about 4.4-fold in cell cultures that
contained 0.3% plasma from both 10 and 18°C acclimated rainbow trout (Fig.
2.1). The mean number of cells per well in culture with 1.0% plasma increased
to a similar extent (Fig. 2.1). Two-way analysis of variance showed that neither
acclimation temperature nor percent plasma added to LDF medium significantly
modulated plasma mitogenicity.
PCNA immunostaining was clearly evident as granular or homogeneous
nuclear staining. Staining intensity was variable. Dark red staining nuclei depict
S-phase cells, light staining nuclei represent G1-S and G2 cells while nonstaining
nuclei represent quiescent (GO) cells (Dietrich, 1993). Every stained nucleus was
considered positive regardless of intensity. Cells in tumor tissue appeared to be
disorganized and more frequently labeled than normal cells (Fig. 2.2).
Temperature markedly influenced the incidence of AFB1-induced liver
cancer in rainbow trout (Curtis et al., 1995).
PCNA immunohistochemistry
indicated that temperature had no effect on cell proliferation. The number of
labeled hepatocytes were not different between fish acclimated, exposed and
reared at 11.0, 14.5 or 18.0°C whether they were exposed to AFB1 at
concentration that yields equivalent organ dose or exposed to AFB1 at the
same concentration (Fig. 2.3). Similarly acclimation temperature had no effect
56
Figure 2.1
Number of cells per well in fish cell culture (CHSE-214)to which
trout plasma from 10.0 or 18.0°C acclimated fish was added. The
ratio of trout plasma to LDF medium was 0.3 and 1.0%. Results are
means ± SE for cells cultured with plasma from five individual fish.
57
///
20
0% Plasma
0.3% Plasma
1.0% Plasma
1
10
18
Temperature Regimen (°C)
58
Figure 2.2 Number of labeled cells in normal and tumor tissue in the livers of
rainbow trout exposed to AFB1 at concentrations that yield
equivalent organ dose. Temperature regimen is depicted by three
numbers under each treatment. The first number represents
acclimation temperature, the second exposure temperature and the
third rearing temperature. Results are means ± SE for three
individual fish except for tumor tissue at 11-11-11 represents one
fish. Temperature 14.5°C was rounded to 14.0 in the figure.
Asterisks depict significant differences from normal tissue.
59
Normal
V/, Tumor
1000
*
800
*
600
400
200
11-11-11
14-14-14
18-18-18
Temperature Regimen (°C)
1
60
on number of labeled hepatocytes (Fig. 2.4), since the fish in these treatments
were acclimated at different temperatures, either at 10.0 or 18.0°C, but were
exposed and reared at same temperature (14.5°C). Two-way analysis of variance
showed that although the number of labeled hepatocytes in tumor tissue were
significantly increased over normal tissue, temperature had no effect on the
number of labeled hepatocytes in either tissue (Fig. 2.2).
61
Figure 2.3
Number of labeled cells in the livers of rainbow trout acclimated at
11.0, 14.5 or 18.0°C then exposed to 0.1 ppm AFB1 and reared at
their respective acclimation temperature. Temperature regimen is
depicted by three numbers under each treatment. The first number
represents acclimation temperature, the second exposure
temperature and the third rearing temperature. Results are means
± SE for three individual fish. Temperature 14.5°C was rounded to
14.0 in the figure.
62
800
T
600
1
400
0
11-11-11
14-14-14
18-18-18
Temperature Regimen (°C)
63
Figure 2.4 Number of labeled cells in the livers of rainbow trout acclimated at
11.0, 14.5 or 18.0°C then exposed to 0.1 ppm AFB1 and reared at
14.5°C. Temperature regimen is depicted by three numbers under
each treatment. The first number represents acclimation
temperature, the second exposure temperature and the third
rearing temperature. Results are means ± SE for three individual
fish. Temperature 14.5°C was rounded to 14.0 in the figure.
64
800
rirn
(1)
C.)
600
I
I
400
--1
a)
4
200
Z
0
1
11-14-14
14-14-14
18-14-14
Temperature Regimen (°C)
65
DISCUSSION
Cell proliferation was implicated in chemical carcinogenesis and cell
transformation (Cayama et al, 1978). Cultured fish cells were used in this study
to investigate the effects of acclimation temperature on plasma mitogens. Plasma
definitely stimulated CHSE-214 cell proliferation. However, plasma obtained from
fish acclimated at warm or cold temperature was equally mitogenic (Fig. 2.1).
This suggested plasma mitogens were not effected by temperature.
In this study, immunohistochemical localization of PCNA estimated cell
proliferation and assessed the role of temperature as a promoter in chemical
carcinogenesis. Within the context of the cell cycle, cells can be grouped into
three populations: proliferating cells involved in growth and division (G1, S, G2
and M-phase), nonproliferating quiescent cells (GO-phase) and dying cells. Cells
in the GO-phase either differentiate and lose their ability to divide or are triggered
to enter the cell cycle, while dying cells have permanently lost their capacity to
divide (Hall and Levison, 1990). PCNA is considered as a reliable and sensitive
indicator of proliferation because it is present in all phases of proliferating cells.
Several studies (reviewed by Dietrich, 1993) showed that the concentration of
PCNA initially increased in late G1-phase, reached its maximum during S-phase
and then gradually decreased throughout G2-phase and mitosis. For this reason,
staining intensity was variable among the labeled cells.
66
Curtis et al. (1995) demonstrated that temperature profoundly influenced
incidence of AFB1-initiated cancer in rainbow trout. When waterborne
concentrations produced equivalent target organ dose, fish acclimated, exposed
to AFB1, and reared at 18°C developed liver tumors much more frequently than
those at 10°C. Conversely hepatic [3H]AFB1 DNA adducts were slightly higher
at 10 than 18°C (Zhang et al., 1992). Among the possible mechanisms for
[3H]AFB1 DNA adduct loss is tritium exchange. However, higher tritium exchange
was an unlikely explanation for the lower persistence of hepatic DNA adducts at
warmer temperature (Bailey et al., 1988; Zhang et al., 1992). Since neither PCNA
immunohistochemistry nor plasma induced mitogenesis of cultured cells
suggested temperature acclimation altered cell proliferation, differences in adduct
persistence suggested another mechanism. DNA synthesis repair increased with
temperature in rainbow trout primary hepatocytes (Miller et al., 1989). If loss of
DNA adducts reflected error prone repair, then a negative correlation between
adduct persistence and tumor incidence across temperature was expected (Fig.
2.5).
The effects of temperature on AFB1 carcinogenicity were related to both
the level of DNA binding and the relative persistence of adducts (Zhang et al.,
1992; Curtis et al., 1995), which reflects the balance between the formation and
repair of DNA adducts and the extent of cell replication. Since temperature had
no effect on cell replication, the accuracy of DNA repair should be addressed.
Major DNA repair pathways in prokaryotes and eukaryotes were classified as:
direct repair, excision repair, recombination (postreplication repair) and mismatch
67
Figure 2.5
Liver tumor incidence plotted against percent AFB1 DNA adduct
persistence. Data from Zhang et al., 1992 and Curtis et al., 1995
used with permission.
68
70
60
50
Y = 229.3
2.6 X
r 2 = 0.67
40
30
20
10
0
60
I
I
I
I
1
65
70
75
80
85
DNA Adduct Persistence (7)
i
90
69
repair (reviewed by Myles and Sancar, 1989; Sancar, 1994). These mechanisms
were not studied extensively in fish cells. However of the repair systems
investigated, photoreactivation (direct repair) appeared to be the most prominent,
followed by excision repair (Regan et al., 1982; Mano et al., 1980, and 1982).
Excision repair, the main repair system responsible for removing bulky DNA
adducts, was deficient in fish cells (Regan et at, 1983; Walton et al., 1983; Shima
et al., 1981). Defects in DNA repair mechanisms could lead to cancer (Modrich,
1994; Sancar, 1994). In humans for example, a hereditary disease, Xeroderma
pigmentosum (XP), was associated with defects in nucleotide excision repair
(Cleaver, 1968; Tanaka and Wood, 1994). Patients with XP develop fatal skin
cancers when exposed to sunlight. In analogy, the higher liver tumor incidence
in fish at warmer temperature (Curtis et al., 1995), where DNA adducts were less
persistent (Zhang et al., 1992), could be explained by error-prone repair.
Differences in DNA adduct persistence between fish acclimated, exposed and
reared at 10°C and 18°C may not be the only factors contributing to differences
in tumor incidence. Since the proportion of DNA lesions which progress
eventually to produce tumors may depend on hormonal influence, immune status,
or other post-initiative factors which could differ between fish subjected to
different temperature regimens.
Results form this study showed that cellular proliferation was more
vigorous in tumor tissue (Fig. 2.2). This was expected since tumor promotion
involves the clonal expansion of mutants. Preferential growth of mutants may
involve stimulation of their growth or growth inhibition of normal cells. The current
70
understanding of the relative roles of these of mechanisms is not well developed
(Popp and Marsman, 1991). Despite this uncertainty there is substantial evidence
that stimulation of mutant growth may be quite important. Preneoplastic cells
were found to be more sensitive to the mitogenic effects of various tumor
promoters than were normal hepatocytes (Schulte-Hermann et al., 1981).
In conclusion, several studies in fish implicated water temperature as a
modulating factor in carcinogenesis (Hendricks et al., 1984; Kyono-Hamaguchi,
1984; Curtis et al., 1995), while others documented the importance of cell
proliferation in tumor promotion (Solt and Farber, 1976; Schulte-Hermann et al.,
1981). These studies accompanied with circumstantial evidence from DNA
adducts and tumor incidence results (Zhang et al., 1992; Curtis et al., 1995)
suggested that warmer temperature perhaps promoted tumor formation. This
study however, discounted the role of mitogenesis in temperature-modulated
chemical carcinogenesis in rainbow trout and provided a possible explanation for
higher tumor incidence at warmer temperature.
71
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Popp, J.A, and D.S. Marsman. 1991. Chemically induced cell proliferation in liver
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Risk Assessment, ed. B.E. Butterworth, T.J. Slaga, W. Far land, and M.
McClain, 389-395. New York: Wiley-Liss.
Rabes, H.M., R. Kerler, C. Schuster, G. Rode, M. Legner, L. Mueller, and T.
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Regan, J.D., R.D. Snyder, A.A. Francis, and B.L. 01 la
.
1983. Excision repair of
ultraviolet and chemically induced damage in the DNA of fibroblasts
derived from two closely related species of marine fishes. Aquat. Toxicol.
4: 181-188.
Reynolds, S.H., S.J. Stowers, R.M. Patterson, R.R. Maronpot. S.A Aaronson, and
M.W. Anderson. 1987. Activated oncogenes in B6C3F1 mouse liver
tumors: Implications for risk assessment. Science 11: 1309-1316.
Sancar, A 1994. Mechanisms of DNA Excision repair. Science 266: 1954-1956.
Schulte-Hermann, R., G. Ohde, J. Schuppler, and I. Timmermann-Trosiener.
1981. Enhanced proliferation of putative preneoplastic cells in rat liver
following treatment with the tumor promoters phenobarbital,
hexachlorocyclohexane, steroid compounds and nafenopin. Cancer Res.
41: 2556-2562.
Schulte-Hermann, R., I. Timmermann-Trosiener, and J. Schuppler. 1983.
Promotion of spontaneous preneoplastic cells in rat liver as a possible
explanation of tumor production by nonmutagenic compounds. Cancer
Res. 43: 839-844.
Shima, A., M. Ikenaga, 0. Nikaido, H. Takebe, and N. Egami. 1981.
Photoreactivation of ultraviolet light-induced damage in cultured fish cells
as revealed by increased colony forming ability and decreased content of
pyrimidine dimers. Photochem. Photobiol. 33: 313-316.
Solt, D.B., and E. Farber. 1976. New principle for the analysis of chemical
carcinogenesis. Nature 263: 702-703.
Tanaka, K., and R.D. Wood. 1994. Xeroderma pigmentosum and nucleotide
excision repair of DNA. Trends Biochem. Sci. 19: 83-86.
74
Walton, D.G., AB. Acton, and H.F. Stich. 1983. DNA repair synthesis in cultured
mammalian and fish cells following exposure to chemical mutagens.
Mutation Res. 124: 153-161.
Williams, D.E., and D.R. Buhler. 1983. Purified form of cytochrome P-450 from
rainbow trout with high activity toward conversion of aflatoxin B1 to
aflatoxin B1-2,3-epoxide. Cancer Res. 43: 4752-4756.
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Zhang, Q., K. Suorsa-Supper, and L.R. Curtis. 1992. Temperature modulated
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75
SUMMARY
Environmental variables play an important role in an organisms ability to
respond to toxic chemicals in the aquatic environment. Several studies have
implicated environmental temperature as a modulating factor in carcinogenesis,
with warmer temperature being associated with higher cancer incidence
(Hendricks et aL, 1984; Kyono-Hamaguchi, 1984). The mechanisms of thermal
modulation of chemical carcinogenesis are not clear, but potentially temperature
may act at many levels including uptake, metabolism, elimination and adduct
formation. Therefore, this study investigated temperature role in DMBA
genotoxicity and assessed it's promotional effects on carcinogenesis in rainbow
trout. A model was presented by which environmental temperature of rainbow
trout was conveniently manipulated before, during or after exposure to DMBA,
which allowed us to selectively modulate different steps of the multistage process
of chemical carcinogenesis. The physiological and biochemical adaptations that
occurred in the tissue of temperature acclimated fish provided the mechanistic
basis for this model.
This investigation was conducted by acclimating rainbow trout (2 g) at 10,
14, or 18°C for one month and then exposing them to 1.0 ppm DMBA in their
water for 20 hr. Exposures to DMBA were conducted at respective acclimation
temperatures or 10 and 18°C acclimated fish were shifted to 14°C for exposures.
Results showed that [3H]DMBA disposition in livers of rainbow trout was effected
76
by temperature. Elevated temperatures increased the liver burden of total
[3H]DMBA equivalents at 4 hr after starting waterborne exposure in both fish
exposed at their acclimation temperature and those subjected to acute
temperature shift. These results were attributed to greater gill uptake and
distribution of [3H]DMBA to livers at warmer temperature. Similarly temperature
effected modulation of DMBA metabolism.
For example, when fish were
acclimated and exposed to the genotoxin at the same temperature similar
metabolism at 10 and 18°C was expected due to ideal temperature compensation
(Precht, 1958). However, different rates of metabolizing DMBA were observed
when fish were acclimated at one temperature and exposed to the genotoxin at
another. Because under these conditions there was not sufficient time for
biochemical adaptations to maintain similar
MFO activity. The effects of
temperature of excretion were also documented since data from this study clearly
demonstrated increased hepatic elimination of [3H]DMBA with temperature.
Formation of [3H]DMBA-DNA adducts were affected by DMBA bioactivation
rates and correlated with the concentration of [3H]DMBA at the target organ,
which in turn was influenced by temperature. These adducts were less persistent
at warm temperature in livers of fish exposed to DMBA at their respective
acclimation temperature, probably due to lower rate of DNA repair synthesis at
cooler temperatures (Miller et al., 1989).
Tumor incidence in stomach, liver and swim bladder increased with
temperature increases in rainbow trout, in spite of higher concentration of
[3H]DMBA -DNA adducts which persisted in livers of fish acclimated and exposed
77
at 10°C compared to 18°C. Initially, increased cell turnover with higher
temperature was thought to underlie these results, since bulky DNA adducts may
be lost at higher rate at 18°C than at 10°C. In order to test this hypothesis, a
retrospective analyses of fish livers from a tumor study initiated with AFB1 (Curtis
et at, 1995) was conducted to evaluate cell proliferation at different temperature
regimens using antibodies to endogenous proliferating nuclear antigen (PCNA).
Immunohistochemistry for PCNA demonstrated no significance increase in
number of labeled hepatocytes with temperature. Furthermore, plasma obtained
from fish acclimated at warm or cold temperature was equally mitogenic when
assessed in cultured chinook salmon embryo cells (CHSE-214). These results
indicated a decreased importance of warm water as mitogenic agent in
promoting tumor formation. An alternative mechanism suggested was DNA error
prone repair because a negative correlation between adduct persistence and
tumor incidence was reported in this study.
Tumor incidences were also affected by both target organ dose and extent
of adduction at measured tissue. In support, results have shown that fish
subjected to 4°C upwards shift during exposure had higher hepatic concentration
of
total
[3H]DMBA
equivalents
and
[3H]DMBA -DNA
adducts
and
consequently resulted in higher tumor incidence.
In summary this study demonstrated that chemically induced tumors in
rainbow trout were modulated by temperature through genotoxin disposition and
adduct formationt, and also through the persistence of DNA adducts. It also,
discounted the role of mitogenesis in temperature-modulated chemical
78
carcinogenesis in rainbow trout and provided a possible explanation for higher
tumor incidence at warmer temperatures. Greater understanding of DNA repair
processes in rainbow trout is necessary to explore the role of conversion of
adducts to mutations in temperature-modulated cancer incidence.
79
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Amdur, J. Doull, and C.D. Klaassen, 127-200. New York: Pergamon Press.
87
You, M., U. Candrian, R.R. Maronpot, G.D. Stoner, and M.W. Anderson. 1989.
Activation of the Ki-rasproto-oncogene in spontaneously occurring and
chemically induced lung tumors of the strain A mouse. Proc. Natl. Acad.
Sci. 86: 3070-3074.
Zhang, Q., K. Suorsa-Supper, and L.R. Curtis. 1992. Temperature modulated
aflatoxin B1 hepatic disposition, and formation and persistence of DNA
adducts in rainbow trout. Toxicol. Appl. Pharmacol. 113: 253-259.
88
APPENDICES
89
APPENDIX A
Cell Proliferation Assessment
by Immunohistochemical Methods
Carcinogenesis is a complex process involving sequential mutations in
normal cellular growth control genes with subsequent clonal growth of the
preneoplastic cells (Williams and Weisburger,
1991).
Several studies
documented the importance of cell proliferation in tumor promotion (Cayama et
al., 1978; Schulte-Hermann, 1985) while others, implicated water temperature as
a modulating factor in carcinogenesis with warmer temperature being associated
with higher tumor incidence in fish (Hendricks et aL, 1984; Kyono-Hamaguchi,
1984). These studies provided an indication, that warm temperature perhaps
promoted tumor formation through it's actions on cell proliferation. Therefore the
role of temperature on cell proliferation in fish livers was estimated in this study
using two methods. One method employed antibodies to endogenous antigen
(proliferating cell nuclear antigen, PCNA), implemented to fish livers from a tumor
study initiated with aflatoxin B1. The second used antibodies to exogenously
provided 5-bromo-2-deoxyuridine (BrdU). BrdU is a non-radioactive thymidine
analogue which is incorporated into DNA-synthesizing cells ( Schutte et al., 1987)
and has been used instead of [3H]thymidine to label a variety of tissue in vivo
(Wynford-Thomas and Williams, 1986; Lanier et al., 1989). A monoclonal
90
antibody against BrdU has been developed (Gratzner, 1982) which allowed for
the rapid detection of BrdU-laden cells with immunohistochemical techniques
(Sugihara et al., 1986).
Retrospective
analyses
of
the
tumor
study
and
PCNA
immunohistochemistry techniques were described in detail in chapter 2. Results
indicated that temperature had no effect on cell proliferation and that the number
of labelled hepatocytes in tumor tissue were significantly increased over normal
tissue. PCNA immunostaining was clearly evident as granular or homogeneous
nuclear staining. Staining intensity was variable. Dark red staining nuclei depict
S-phase cells, light staining nuclei represent G1-S and G2 cells while nonstaining
nuclei represent quiescent (GO) cells (Fig. A.1). Cells in tumor tissue appeared
to disorganized and more frequently labelled than normal cells (Fig. A.2).
For labeling with BrdU rainbow trout were held at either 10.0 or 18.0°C for
at least four weeks. Several experiments were conducted to administer BrdU, in
an attempt to achieve maximum labelling. The protocols tested ranged from a
single dose to daily intraperitoneal injections (30 mg/kg) that lasted for 10 days.
Fish were sacrificed 24 hr after the last injection. Livers were fixed in 70% ethanol
for 24 hr before paraffin embedding. Five to seven-micron sections were cut and
mounted on slides. Tissue sections were then deparaffinized and rehydrated in
graded alcohols. BrdU labels in tissue were detected immunohistochemically
using the BrdU labeling and detection kit II (Boehringer Mannheim, Indianapolis,
IN). Briefly, tissue sections were washed three times with PBS and then flooded
with the primary antibody, monoclonal mouse anti-BrdU (clone BMC 9318, IgG1)
91
Figure A.1
Liver tissue of rainbow trout acclimated, exposed to 0.1 ppm AFB1
and reared at 10°C. This section was fixed with Bouin's solution,
embedded in paraffin, and stained with PCNA (PC10 antibody,
biotin -streptavidin, Fast -RedTM chromogen) for assessment of cell
proliferation.
Red staining nuclei depict dividing cells.
(Magnification x 400).
Figure A.2
Liver tumor of rainbow trout acclimated, exposed to 0.1 ppm AFB1
and reared at 18°C. This section was fixed with Bouin's solution,
embedded in paraffin, and stained with PCNA (PC10 antibody,
biotin -streptavidin, Fast-Reel chromogen) for assessment of cell
proliferation.
Red staining nuclei depict dividing cells.
(Magnification x 400).
92
Figure A.1
Figure A.2
93
diluted 1:10 in PBS contaning nucleases for DNA denaturation. The tissue with
the primary antibody were incubated for 30 min at 37°C in a humid atmosphere.
Following a wash in PBS, tissue sections were flooded with anti-mouse-Ig­
alkaline phosphatase for 30 min at 37°C. This secondary antibody was diluted
1:10 with PBS. BrdU incorporation was visualized after a final incubation for 15
to 30 min in color-substrate solution containing nitroblue terrazolium and 5­
bromo-4-chloro-3-indoly1 phosphate in demethylformamide, followed by a rinse
in PBS.
Cells that have incorporated BrdU were identified as having dark brown to
black pigments in their nuclei. Results from these experiments were consistent
in that, few hepatocytes if any were labelled in the warm acclimated fish (Fig.
A3) while in comparison, many liver cells from cold acclimated fish were labelled
(Fig. A.4). The discrepancy in labelling was attributed to the difference in BrdU
metabolism at the temperatures tested. In fish acclimated to warm water BrdU
probably was metabolized rapidly before it had a chance to be incorporated in the
cells. Similarly rapid metabolism of BrdU in vivo by the liver has been reported
in other species (Kriss and Revez, 1962). This necessitates continuous exposure
to achieve prolonged labelling. Continuous labelling can be achieved by
implanting osmotic pumps intraperitoneally. Another advantage to continuous
labelling is increased sensitivity. Enhanced sensitivity of continuous labelling
compared to pulse labelling was documented in a study by Marsman et a/. (1988).
Careful consideration should be given to labelling procedures, because the use
of pumps for label delivery was not suitable under certain experimental
94
Figure A.3
Liver tissue of rainbow trout reared at 18°C for at least four weeks
and then injected intra peritoneally with BrdU (30 mg/kg). This
section was fixed with 70% ethanol, embedded with paraffin and
stained with BrdU (BMC 9318, IgG1 antibody) for assessment of
cell proliferation. Dark brown staining nuclei depict dividing cells.
(Magnification x 400).
Figure A.4
Liver tissue of rainbow trout reared at 10°C for at least four weeks
and then injected intra peritoneally with BrdU (30 mg/kg). This
section was fixed with 70% ethanol, embedded with paraffin and
stained with BrdU (BMC 9318, IgG1 antibody) for assessment of
cell proliferation. Dark brown staining nuclei depict dividing cells.
(Magnification x 400).
95
Figure A.3
Figure A.4
96
conditions. For example, minipump implantation was impractical for small fish. To
this end, Moore and his colleagues (1994) recommended the use of bath
exposure to BrdU for small fish and aquatic invertebrates. Furthermore, using
osmotic pumps to determine the rate of cell proliferation at different temperatures
was not appropriate because their rate of label delivery was influenced by
temperature. On the other hand, a critical issue in pulse labelling was knowing
when to sacrifice the animals in relation to the time the label was administered to
achieve maximum labelling. Therefore, experiments designed to determine rate
of cell proliferation should be planned carefully and all possible aspects of the
experiment should be considered. Because the quality of data depends on the
procedures and protocols followed for these labor-intensive studies.
97
REFERENCES
Cayama, E., H. Tusda, D.S.R. Sarma, and E. Farber. 1978. nitiation of chemical
carcinogenesis requires cell proliferation. Nature 257: 60-62.
Gratzner, H.G. 1982. Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine:
New reagent for detection of DNA replication. Science 218: 474-475.
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. Monogr. 65: 129-137.
Kriss, J.P, and L. Revez. 1962. The distributio and fate of bromodeoxyuridine and
bromodeoxycytidine in the mouse and rat. Cancer Res. 22: 254-265.
Kyono-Hamaguchi, Y. 1984. Effects of temperature and partial hepatectomy on
the induction of liver tumors in Oryzias latipes. Natl. Cancer. Inst. Monogr.
65: 337-344.
Lanier T. L., E. K. Berger , and P. I. Eacho. 1989. Comparison of
5-bromodeoxyuridine and [3H]thymidine in rodent hepatocellular
proliferation studies. Carcinogenesis 10: 1341-1343.
Marsman D.S., R.C. Cattley, J.G. Conway, and J.A. Popp. 1988. Relationship of
hepatic peroxisome proliferation and replicative DNA synthesis to the
hepatocarcinogenicity
of the
peroxisome
proliferators
di(2­
ethylhexyl)phthalate and [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthiojacetic
acid (Wy-14,463)in rats. Cancer Res. 48: 6739-6744.
Moore, M.J., D.F. Leavitt, A.M. Shumate, PI Alatalo, and J.J. Stegeman. 1994. A
cell proliferation assay for small fish and aquatic invertebrates using bath
exposure to bromodeoxyuridine. Aquat. Toxicol. 30: 183-188.
Schulte-Hermann, R. 1985. Tumor promotion in the liver. Arch. Toxicol. 57: 147­
154.
Schutte, B., M.M.J. Reynders, F.T. Bosman, G.H. Blijham. 1987. Studies with
antibromodeoxyuridine antibodies: II. Simultaneous immunocytochemical
detection of antigen expression and DNA synthesis by in vivo labeling of
mouse intestinal mucosa J. Histochem. Cytochem. 35: 371-380.
Sugihara, H., T. Hattori, M. Fukuda. 1986. Immunohistochemical detection of
bromdeoxyuridine in formalin-fixed tissues. Histochemistry 85: 193-195.
98
Wynford-Thomas, D., and E.D. Williams. 1986. Use of bomodeoxyuridine for cell
kinetic studies in intact animals. Cell Tissue Kinet. 19: 179-182.
Williams, G.M. and J.H. Weisburger. 1991. Chemical Carcinogenesis. In Casarett
and Doull's Toxicology: The Basic Science of Poisons, 4th ed, ed. M.O.
Amdur, J. Doull, and C.D. Klaassen, 127-200. New York: Pergamon Press.
99
APPENDIX B
Temperature-Modulated Incidence of Aflatoxin B1-Initiated
Liver Cancer in Rainbow Trout'
Lawrence R. Curtis, Quan Zhang, Camille EI-Zahr, Hillary M. Carpenter,
Cristobal L. Miranda, Donald R. Buhler, Daniel P. Selivonchick, Daniel N.
Arbogast and Jerry D. Hendricks
'Published in Fundamental and Applied Toxicology, Volume 25, pp. 146-153,
1995.
100
ABSTRACT
Rainbow trout (initial weight of 4-5 g) were acclimated at a cool, 11.0°C(C);
a warm, 18.0°C(W); or an intermediate temperature, 14.5 C(I) for one month.
There was a slight difference in hepatic microsomal content of one of six
cytochrome P-450 isozymes between acclimation groups. Monounsaturated fatty
acids in hepatic phosphotidyl-ethanolamine but not phosphotidylcholine
increased at lower acclimation temperatures.
Saturated fatty acid content
decreased with temperature for both phospholipid classes. Fish were exposed
to 0.08 - 0.12 ppm water-borne aflatoxin B1 (AFB1) for 30 min at respective
acclimation temperatures or after acute temperature shifts (24 hr) and reared for
9 months at C, I or W. With exposure concentrations which delivered equivalent
target organ doses, trout acclimated, exposed and reared at C, I or W had liver
tumor incidences of 4, 35 and 61% respectively. The average number of tumors
per liver increased from 1.25 - 1.34 at C to 2.46 - 2.66 at W. There were no
temperature-dependent differences in tumor diameter.
When C and W
acclimated fish were AFB1 exposed and reared at I, tumor incidence was 12.5%
for W-I shifted fish and 26.5% for C-I shifted fish. This was consistent with
previous work which demonstrated acute downward temperature shift reduced
[3H]AFB1 adduction to hepatic DNA. Tumor incidence and multiplicity data
suggested manipulation of temperature permitted selective modulation of cancer
initiation and promotion in rainbow trout.
101
INTRODUCTION
Temperature modulates xenobiotic disposition in fish and underlying
mechanisms vary with exposure conditions. Hepatic accumulation of waterborne
AFB1 increases with exposure temperature in rainbow trout (Zhang et al., 1992).
This is probably explained by increases in gill ventilation and cardiac output that
are associated with higher oxygen consumption at warmer temperature (Prosser
and Heath, 1991). Biliary excretion of benzo[a]pyrene metabolites (Curtis et al.,
1990), phenolphthalein metabolites (Curtis, 1983) and exogenously provided
taurocholate (Kemp and Curtis, 1987) increases with temperature in rainbow
trout. This stimulation of biliary excretion by warm temperature occurs both in fish
maintained for at least one month at a given temperature (acclimation) or those
subjected to an acute temperature shift (1-24 hr).
Interactions between
temperature and xenobiotic metabolism are more complex. Hepatic microsomes
from cold and warm acclimated rainbow trout exhibit similar mixed-function
oxidase activities when assayed at their respective acclimation temperatures
(Blanck et al., 1989; Koivusaari, 1983). This is a common phenomenon for
various biochemical pathways in poikilotherms and generally referred to as ideal
temperature compensation (Precht, 1958). However, when assayed at a common
intermediate temperature hepatic microsomes from cold acclimated fish have
markedly higher aryl hydrocarbon hydroxylase activity than those from warm
acclimated fish (Carpenter et al., 1990; Curtis et al., 1990). In yjyg metabolism
of benzo[a]pyrene exhibits a similar pattern (Curtis et al., 1990). These results
102
reflect biochemical adaptations which underlie ideal temperature compensation
(White and Somero, 1982).
Temperature modulated the incidence of chemically-induced cancer in fish
in previous laboratory work.
Kyono-Hamaguchi (1984) exposed Japanese
medaka to water-borne diethylnitrosamine for 8 weeks at 22-25°C or 5-8°C. All
fish exposed at warm temperature, and then reared for an additional 4 weeks in
water without diethylnitrosamine, developed liver tumors. No tumors occurred in
fish exposed and reared at low temperature. Shifting warm temperature-exposed
fish to low temperature for rearing modestly reduced tumor development. Shifting
low temperature-exposed fish to warm temperature for rearing produced tumors
in about 5% of fish. Hendricks et al. (1984a) exposed rainbow trout embryos to
aflatoxin B1 (AFB1) for 15 min at 12, 15 or 18°C and then returned the fish to
12°C for rearing. At 9 months after exposure, liver tumor incidence approximately
doubled in fish exposed at 18 as compared to 12°C. This difference was not
evident 12 months after exposure (liver cancer incidence ranged from 62-68%
among groups at that time). Taken together, these results suggested modulated
genotoxicity was an underlying mechanism for temperature-induced differences
in cancer incidence in fish.
AFB1 genotoxicity is associated with AFB1-8,9-epoxide binding to DNA
(Croy et al., 1980). This reactive intermediate is the product of rainbow trout liver
cytochrome P450 LMC2 (Williams and Buhler, 1983) recently reclassified as
cytochrome 450 2K1 (Buhler et al., 1994). A major detoxication pathway for
AFB1 is the conversion to aflatoxin M1 that is catalyzed by hepatic cytochrome
103
P450 LM4B (Williams and Buhler, 1983), reclassified as cytochrome P450 1A1
(Heilman et al., 1988). A cytosolic dehydrogenase converts AFB1 to aflatoxicol
(AFL) which may be reconverted to AFB1 or be glucuronidated and readily
excreted in bile (Schoenhard et al., 1976). When 10°C and 18°C-acclimated trout
are exposed at an intermediate temperature (14°C), there is significantly more
hepatic [H]AFB1 DNA adduction after an acute temperature increase (Zhang et
al., 1992). Acclimation and exposure at 10 and 18°C results in slightly higher
adduction at the lower temperature at equivalent target organ dose (Zhang et al.,
1992).
Our recent results suggest that a higher activity of the cytosolic
dehydrogenase at 18°C at least partially explains this (Carpenter et al., 1995).
While temperature clearly modulates AFB1 genotoxicity, the contribution
of this mode of action to cancer incidence remains unclear. Temperature is a
major environmental variable and may influence cancer incidence in feral fish.
Characterization of the relationship between temperature and carcinogenesis in
fish may improve our ability to explain cancer incidences in natural populations
exposed to genotoxins. Further, temperature modulation of chemically-induced
cancer in fish can provide novel approaches to understanding fundamental
aspects of carcinogenesis. The following studies assess temperature modulation
of AFB1-induced liver cancer in rainbow trout within the context of the multistage
process model of initiation and promotion/progression.
104
MATERIALS AND METHODS
Animals
Shasta strain rainbow trout (Qjagabyngtma mykiss) were provided by
Oregon State University's Marine and Freshwater Biomedical Center core facility.
Fish weighed 4-5 g at the outset of a 1 month temperature acclimation period at
a cool temperature, 11°C(C); a warm temperature, 18°C(W) or an intermediate
temperature, 14.5°C(I). Fish were fed Oregon Test Diet (Lee et al., 1991) at
rations adjusted to maintain similar growth rates at each temperature (Kemp and
Curtis, 1987). Fish were housed in flow-through tanks under a 12 hour light: 12
hr dark photoperiod.
Chemicals
Purity and concentration of AFB1 (Sigma Chemical Co., St. Louis, MO)
were verified by measuring its absorbance in ethanol at 362 nm using an
extinction coefficient of 21,800 and TLC followed by scanning spectrophotometry
from 220 to 440 nm as previously described (Zhang et al., 1992). Working AFB1
solutions were prepared in ethanol and the ethanol concentration in AFB1
waterborne exposures was 0.1%.
105
Liver Lipids and Cytochromes P450
Hepatic lipids were extracted from 3 replicates of 10 pooled livers of fish
from each temperature acclimation group as described by Bligh and Dyer (1959).
Phospholipids were separated by two-dimensional TLC on silica gel 60A
(Whatman Ltd., England) with chloroform: methanol:28% aqueous ammonia
(65:35:5) and chloroform:acetone:methanol: glacial acetic acid:water (10:4:2:2:1).
Plates were sprayed with 2,7-dicholorfluoroscein, lipids were visualized with a UV
lamp and spots removed for quantitation. Methyl esters were prepared with
sulfuric acid-methanol (Morrison and Smith, 1964) and analyzed with a HewlettPackard 5890 gas chromatograph equipped with a flame ionization detector, a 30
m, 0.25 mm ID fused silica Supelco 2330 capillary column and a 3393A
integrator.
Column temperatures were programmed as follows:
the initial
temperature of 175°C was held for the first 7 min, increased to 210°C in steps of
5°C per min, and the final temperature of 210 C was held for 25 min. Each
sample was run in duplicate. Identification of the fatty acid methyl esters was
accomplished through comparisons with standard mixtures of known methyl
esters (20A and 68A NuChek Prep., Elysian, MN and PUFA2, Supelco,
Bellefonte, PA). Phospholipids for phosphorous content determination were
separated by one-dimensional TLC using silica gel plates and a solvent system
of chloroform:methanol:water (70:30:4). Lipids were stained with iodine vapor.
Phosphorous content was determined calorimetrically by the procedures of Eng
and
Noble
(1968).
Total
cholesterol
was
determined
using
the
106
spectrophotometric method of Zak et al. (1957) with purified cholesterol as a
standard (Sigma Chemical Co., St. Louis, MO). Aliquots of lipids were dried
under a stream of nitrogen and 6 ml of glacial acetic acid and 4 ml of a stock
ferric chloride solution were added to each sample. The solutions were allowed
to stand for 10 min and the absorbance was measured at 550 nm.
Hepatic microsomes were isolated by differential centrifugation from 4
replicates of 50 pooled livers from each temperature acclimation group
(Carpenter et al., 1990). Briefly, livers were homogenized in 4 volumes of 0.1M
tris-acetate, pH 7.4; 0.1 M KC1, 1mM EDTA; 20 pM butylated hydroxytoluene;
and 0.1 mM phenylmethylsulfonylfluoride. Homogenates were centrifuged at
10,000 g for 30 min and the resultant supernatant at 100,000 g for 90 min. The
microsomal pellet was resuspended in 0.1 M phosphate buffer, pH 7.25 with 20%
glycerol and 1 mM EDTA for storage at -80°C.
Microsomal protein was determined as described by Lowry et al. (1951).
Total cytochrome P450 was calculated from difference spectra of CO vs CO-
sodium dithionite reduced microsomes (Estabrook et al., 1972). Cytochrome
P450 reductase activity was determined at 20°C (Imai, 1976). Antibodies to 6
rainbow trout cytochrome isozymes were previously characterized (Miranda et al.,
1989).
Immunoquantitation of each isozyme from acclimation groups was
conducted as described by Miranda et al. (1989).
107
Tumor Study
Waterborne exposures to AFB1 were conducted as described in a
previous study of [3H]AFB1 hepatic disposition and DNA adduction (Zhang et al.,
1992). Briefly, temperature acclimated trout were transferred to aerated tanks
containing AFB1 at respective acclimation temperatures I for acute temperature
shift experiments. Waterborne AFB1 concentrations ranged from 0.08 (rounded
from nominal concentration of 0.075 ppm) to 0.12 ppm.
Fish were then
transferred to uncontaminated water in tanks equipped with submersed charcoal
filters and maintained at respective exposure temperatures for 1 day. Fish were
then reared for 9 months at their (1) acclimation and exposure temperature (C-C­
C, I -I -I or W-W-W), (2) acclimation temperature after acute temperature shift for
exposure (C-I-C or W-1-W), or (3) the acute shift temperature (C -I -I or W-I-I).
Tumor incidence and multiplicity was assessed by gross examination at
necropsy followed by histopathological verification (Hendricks et al., 1984b).
Livers were fixed in Bouin's solution. After at least 48 hr of fixation, livers were
hand sliced into 1 mm slices to locate any internal tumors and to verify the grossly
observable surface tumors. Trout liver tumors show up distinctly as clear yellow
areas after Bouin's fixation. Tumor numbers were determined from hand slicing
results. Tumor identification was based on the number of tumors that could be
included in one tissue slice per tumor-bearing liver for economy purposes. For
groups with few tumored fish and few tumors per liver, the sampling was
108
exhaustive. In higher response groups, it was not but should not have treatly
effects the results. Only microscopic tumors (<0.5 mm) are missed by this
procedure. Tumors sizes were grouped as detectable to 0.5mm (0.5), > 0.5
> 1.5 - 2.5mm(2.0), > 2.5 - 3.5mm(3.0), >3.5 - 4.5mm(4.0) and >
4.5mm(5.0).
Statistics
All data were analyzed by analysis of variance and the Tukey test for
multiple comparisons of means. Percentage data were arcsine transformed prior
to statistical analyses (Sokal and Rohlf, 1981). Significance was set at P < 0.05.
Results were expressed as means + SEM.
109
RESULTS
At the outset of tumor studies groups of fish from each acclimation
temperature were killed for examination of hepatic lipids and cytochrome P-450
system components (Fig.
B.1).
There were no acclimation temperature-
dependent differences in body weight (overall average, 10.9 ± 0.1 g) or liver as
percent body weight (overall average, 1.15 ± 0.04 %) in fish sampled for liver
constitutive analysis. There were no acclimation temperature related differences
in the amounts of microsomal protein, cytochrome P-450 reductase, or in the total
amounts of cytochrome P-450 (Table B.1). Immunoquantitation of the individual
cytochrome P-450 isozymes by a Western blotting technique showed no
differences in the amounts of LMC1, LMC2, (2K1), LMC3, LMC4, or LM4B (1A1)
but a slight decrease in LMC5 from C fish (Table B.2).
Liver total lipid, phospholipid and cholesterol content were significantly
decreased with increased temperature (Table B.3). There were no acclimation
temperature-dependent differences in percent of total phospholipid recovered as
phosphatidyicholine (overall mean of 66.8 ± 1.0%), phosphatidylethanolamine
(overall mean of 16.3 ± 0.6%), sphingomyelin (overall mean of 3.2 + 0.7%) or
phosphatidylserine (overall mean of 1.3 + 0.4%). The percent of total saturated
fatty acids in phosphatidyicholine was significantly higher at W than C or I (Table
B.4). Monosaturated fatty acid content was lower at I than C or W. There was
also a lower percentage of total saturated fatty in phosphatidylethanolamine in
110
Figure B.1
Schematic for temperature acclimation/aflatoxin B1 exposure
regimens. *Temperature shifted fish were either returned to their
acclimation temperature 24hr after aflatoxin B1 exposure or
maintained at the shift temperature throughout the rearing period.
111
Constitutive Liver Analyses
Tumor
Quarnitation
Or
Aflatoxin B1 exposure
10°C
1,t
14°C
14°C
14 °C
18°C
Acclimation
(1 month)
til
Rearing
(9 months)
112
Table B.1
Absence of an effect of temperature acclimation on general hepatic
microsomal parameters in rainbow trout'.
Acclimation
temperature
(°C)
Microsomal
protein
(mg/9)
Cytochrome P450 reductase
(nmol/min/mg)
Cytochrome P450
(nmol/g)
11
6.7+1.0
37.9+5.9
0.25+0.03
14.5
7.8+0.4
34.5+1.0
0.31+0.01
18
6.8+0.9
26.9+0.8
0.31+0.01
'Values are means + SE, n = 4 tissue pools of approximately 50 fish each.
113
Table B.2
Absence of an effect of temperature acclimation on hepatic
microsomal content of cytochrome P-450 isozymes in rainbow
trout'.
Acclimation
Cytochrome P-450 isozymes (pmol/g)
Temperature
LMC5
LMC1
LMC2°
LMC3
LMC4
11
0.008+0.000
0.030+0.008
0.028+0.009
0.016+0.002
0.023+0.002* 0.001+0.001
14.5
0.011+0.001
0.036+0.006
0.021+0.007
0.018+0.000
0.035+0.003
0.004+0.002
18
0.011+0.002
0.032+0.004
0.009+0.002
0.014+0.002
0.035+0.004
0.003+0.001
LM413°
(°C)
Values are mean + SE, n = 4 tissue pools of approximately 50 fish each.
°Current nomenclature for this isozyme is cytochrome P450 2K1 (Buhier et al., 1994).
`Current nomenclature for this isozyme is cytochrome P450 1A1.
*Indicates significant difference from other temperature acclimation groups.
114
Table B.3
Quantitative extraction data for liver lipids from temperature
acclimated rainbow trout.
Temperature °C
11
14.5
18
Total liver lipid, mg/g liver
49.84+0.53*
37.72+0.07
33.20+0.21
Phospholipid, mg/g liver
28.16+0.66*
23.77+0.60
20.33+0.24
Cholesterol, mg /g liver
6.34+0.12*
4.68+0.16
4.21+0.09
Parameter measured
Values are means± SE of three replicates of 10 pooled fish livers each.
Indicates significant difference from other temperature acclimation groups.
115
Table B.4
Fatty acid composition of phosphatidylcholine from livers of
temperature acclimated rainbow trout.
Acclimation Temperature (°C)
PHOSPHATIDYLCHOLINE
11
14.5
18
14:0
2.41 + 0.04
2.90 ± 0.18*
2.11 + 0.24
16:0
27.56 + 0.20
29.33 ± 0.94
32.27 + 1.16*
16:1n-7
2.19 + 0.02*
1.50 ± 0.13
1.33 + 0.04
18:0
1.64 + 0.06*
2.13 ± 0.04
2.77 + 0.02
18:1n-9
9.50 + 0.09
8.38 ± 0.39
10.74 + 0.07*
18:1n-7
1.73 + 0.01
1.62 ± 0.03
1.70 + 0.04
18:2n-6
0.45 + 0.01*
0.51 ± 0.01
0.57 + 0.01
20:1n-9
0.88 + 0.09*
0.61 ± 0.05
0.42 + 0.04
20:4n-6
0.55 + 0.02
0.61 ± 0.02
0.65 + 0.03
22:1n-11
0.06 + 0.05
0.15 ± 0.06
0.12 ± 0.05
22:1n-9
0.41 + 0.01
0.48 ± 0.02*
0.43 + 0.02
20:5n-3
4.39 + 0.04*
5.09 ± 0.09
5.87 + 0.06
22:5n-3
0.74 + 0.03*
1.20 ± 0.04
1.43 + 0.09
22:6n-3
38.90 + 0.45
36.59 ± 1.24
32.74 + 0.65*
Saturated
31.60 + 0.28
34.36+ 1.12
37.14 + 1.41*
Monounsaturated
14.77 + 0.10
12.75 + 0.26*
14.74 + 0.14
Polyunsaturated
45.02 + 0.40
43.99 + 1.35
41.26 + 0.80
0.53 + 0.01
0.61 +0.03
0.66 + 0.04*
Fatty Acid
Total
SFA: UFA
Values are means + SE of three replicates.
*represents significant values at different temperatures.
116
livers from C than I or W fish (Table B.5). Monosaturated fatty acid content was
lower at W than C or I. The percentage content of a number of individual fatty
acids also differed with temperature acclimation (Tables B.4 and B.5).
Temperature markedly influenced the incidence of AFB1-induced liver
cancer in rainbow trout (Fig. B.2). When fish were acclimated, exposed to AFB1
concentrations which delivered equivalent target organ dose (Zhang et al., 1992)
and reared at a constant temperature (Fig. B.2, upper panel) tumor incidence
increased from 4% at C, to 35% at I, and 61% at W. Tumor incidences after
AFB1 exposure concentrations were held constant across exposure temperatures
also increased with temperature. Tumor incidence was lower in fish temperature
down-shifted for AFB1 exposure and rearing (W-1-1) than in those temperature
upshifted for exposure and rearing (C -I -I) (Fig. B.2, lower panel). Equivalent
AFB1 concentrations occurred in rainbow trout liver immediately after these
exposure regimens (Zhang et al., 1992). Fish exposed to a W-I-W regimen were
lost in an equipment failure (a blocked set of water delivery lines).
This
eliminated the appropriate comparison to C-I-C for evaluation of temperature shift
only during AFB1 exposure.
There were more tumors per liver in tumor bearing W-W-W than C-C-C
fish (Fig. B.3, upper panel). There were no differences in tumor number for
temperature shifted fish (Fig.
B.3, lower panel).
There was little effect of
temperature on cell type of rainbow trout liver tumors (Table B.6).
117
Table B.5
Fatty acid composition of phosphatidylethanolamine from livers of
temperature acclimated rainbow trout.
Acclimation Temperature (°C)
PHOSPHATIDYLETHANOLAMINE
11
14.5
18
14:0
0.25 + 0.10
0.33 + 0.14
0.67 + 0.54
16:0
16.68 + 0.26
17.81 +0.16
16.97 + 0.50
16:1n-7
1.31 + 0.00*
1.10 + 0.05
0.74 + 0.02
18:0
5.58 + 0.05
7.31 ±0.06
9.31 ±0.02*
18:1n-9
17.77 + 0.29
17.83 + 0.29
16.40 + 0.15*
18:1n-7
6.15 + 0.13
5.49 + 0.20
4.55 + 0.13*
18:2n-6
1.23 + 0.28
1.21 ±0.02
1.45 + 0.03
20:1n-9
3.61 +0.02*
2.00 + 0.34
1.84 + 0.04
20:4n-6
0.99 + 0.02
1.11 + 0.03
1.39 + 0.13*
22:1n-11
trace
trace
trace
22:1n-9
0.30 + 0.12
trace
trace
20:5n-3
3.98 + 0.23
4.20 + 0.01
5.04 + 0.10*
22:5n-3
0.18 + 0.15
trace
0.92 + 0.04
22:6n-3
38.51 + 1.20
37.37 + 0.32
33.66 + 0.78*
Saturated
22.52 + 0.20*
25.46 + 0.34
26.95 + 0.64
Monounsaturated
29.12 + 0.52
26.42 + 0.19
23.53 ± 0.21*
Polyunsaturated
44.89 + 1.23
43.88 + 0.33
42.46 + 0.75
SFA:UFA
0.30 + 0.00
0.36 + 0.01
0.41 ±0.01*
Fatty Acid
Total
Values are means + SE of three replicates.
*represents significant values at different temperatures.
118
Figure B.2
Percent of rainbow trout bearing liver tumors 9 months after 30 min
waterborne AFB1 exposures at the concentrations specified on the
ordinant. Duplicate tanks of 100 fish were used for regimens with
an error bar. A single tank of 100 fish was used for those regimens
without an error bar. Regimens without duplication were omitted
from statistical analyses. *Indicates significant difference from
other duplicated regimens in that panel.
119
80
80
*
40
0
0.10 0.12
C-C-C
0.08 0.10
0.10
I-I-I
W-W-W
Ann (ppm)
TEMPERATURE
*
40­
IIIMIII
20
_L.
*IIMMI1 IP
0
0.10 0.12
C-I-C
0.10
0.12
C-I-I
0.10
0.12
W-I--I
A7/31 (ppm)
TEMPERATURE
120
Figure B.3
Liver tumor multiplicity in rainbow trout 9 months after 30 min
waterborne AFB1 exposures at the concentrations specified on the
ordinant. Other details were presented in Figure B.2. A diamond
indicates significant difference from the C-C-C group.
121
3.0 ­
2.5 ­
2.0 ­
1.5 ­
0.5 ­
rg
0.0
0.10
0.12
0.08 0.10
0.10
W-W-W
C-C-C
3.5 -­
A121 (ppm)
TEMPERATURE
IIM11
3.0
E-4
2.5
IMIINI=1
2.0 ­
1.5 ­
1.0 ­
0.5 ­
0.0
0.10 0.12
C
C
0.10
0.12
C-I-I
0.10
0.12
W-I-I
A181 (ppm)
TEMPERATURE
Table B.6
Percent' of tumor bearing fish possessing particular tumor typesb 9 months after AFB1 exposures.
Temperature regiment
MC
HCC
HCA
CCC
Ch
(012 ppm)
0.0 + 0.0*
41.6 + 58.9
58.4 + 58.9
0.0 + 0.0
0.0 + 0.0
(0 10 npm)
26.6 + 9.4
57.8 + 3.1
15.6 + 11.1
0.0 + 0.0
0.0 + 0.0
47.1 + 19.5
32.0 + 4.0
14.7 + 7.4
2.1 + 3.0
4.2 + 5.9
(0 08 opm)
50.4 + 2.2
29.7 + 10.6
11.4 + 1.2
6.7 + 6.2
1.8 + 2.6
(0 10 npm)
46.3 + 20.5
34.2 + 12.5
14.5 + 7.5
3.0 + 4.2
2.0 + 2.8
(0.10 ppm)
23.1
53.8
23.1
0
0
(0 12 npm)
16.7
58.3
20.9
0
4.2
(0 10 npm)
19.6 + 10.0
45.8 + 17.7
29.1 + 5.9
0.0 + 0.0
5.4 + 1.8
(0 12 ppm
34.8
56.5
8.7
0
0
22.2 + 31.4
54.8 + 46.2
17.4 + 4.4
0.0 + 0.0
5.6 + 7.8
143
71,4
143
0
0
(AFB1 concentration)
C-C-C
I-I-I
(010 ppm)
W-W-W
C-I-C
C-I-I
W-I-I
(010 ppm)
(0.12 nnml
_
Results are means + SEM for duplicate determinations of tanks with 100 fish. In regimens which were not duplicated the percentage
for a single tank of 100 fish was alculated, thus no SEM was available.
°MC, mixed hepatocellular-cholangiocellular carcinoma; HCC, hepatocellular carcinoma; HCA, hepatcellular adenoma; CCC,
cholangiocellular carcinoma; Ch, cholangioma.
Indicates statistical difference for percentage of this tumor type between temperatures.
N
123
There were no mixed hepatocellular-chloangiocellular carcinomas in C-C-C fish
exposed to 0.12 ppm AFB1 and this was significantly different from other groups.
No other significant differences in tumor cell type were detected. There were no
significant differences in sizes of liver tumors between temperature groups (data
not shown). Tumors of 2 mm or smaller diameter composed 93% of the total
number.
124
DISCUSSION
Increased tumor incidences in feral fish is associated with heavy sediment
polycyclic aromatic hydrocarbon and organochlorine contamination (Koza et al.,
1993; Myers et al., 1987). While the extent of sediment contamination and other
estimates of exposure correlate with occurrence of preneoplastic and neoplastic
lesions, other environmental factors may influence tumor response. Temperature
is a predominant variable in aquatic ecosystems that is influenced by the season,
land use practices (e.g., amount of shade provided by terrestrial vegetation) and
direct thermal pollution.
The present work demonstrates that temperature
profoundly influences incidence of AFB1-initiated cancer in rainbow trout (Fig.
B.2). After procarcinogen exposures which produce equivalent target organ
doses (Zhang et al., 1992), fish acclimated, exposed and reared at W
temperature develop tumors much more frequently than those at C. Tumor
multiplicity is also temperature dependent (Fig. B.3). These results indicate a
role of temperature either in promotion/progression processes or conversion of
adducts into mutations in rainbow trout. It is important to emphasize that the
temperature regimens used here produce no evidence of temperature-induced
stress and that all groups grow at the same rate (Results). There are at least two
general applications for an increased understanding of the role of temperature in
fish tumor response. (1) An appreciation for the potency of temperature as a
modulator of chemically-induced cancer is important for interpreting site-specific
differences in tumor occurrences in feral fish. (2) The potential of temperature
125
to independently modulate genotoxic events (adduct formation and conversion
of adducts into mutations) and/or cancer promotion/progression provides a novel
model for mechanistic studies.
Constitutive analyses of rainbow trout hepatic microsomes reveal no
significant adaptive response of general protein parameters to temperature
acclimation (Table B.1).
Carpenter et al. (1990) reported no differences in
hepatic microsomal cytochrome P450 LMC2 (2K1) and only a slight difference
(30%) in P450 LM4B (1A1) in juvenile rainbow trout acclimated at 10 or 18°C.
Reconstitution and antibody inhibition studies provide limited characterization of
catalytic function of the various forms of trout P450 (Miranda et al., 1989; Miranda
et al., 1991). LMC1 is one of the major catalysts for the hydroxylation of lauric
acid (Miranda et al., 1989) and this hydroxylation occurs at the co-6 position of the
fatty acid (D.R. Buhler, unpublished data). LMC2 is the major catalyst for the o -1
hydroxylation of lauric acid and the bioactivation of aflatoxin B1 (Miranda et al.,
1989; Williams and Buhler, 1983). Based on antibody inhibition studies, LMC3
and LMC4 were recently found to contribute to the microsomal conversion of
progesterone to metabolites that are presently unidentified (Miranda et al.,
unpublished data).
LMC5 is a major catalyst for the 60-hydroxylation of
progesterone (Miranda et al., 1989; Miranda et al., 1991). P450 LM4B, an
ortholog of rat P450 1A1 (Heilmann et al., 1988), is the major catalyst for the
hydroxylation of benzo(a)pyrene (Williams and Buhler, 1984) and converts AFB1
to AFM1, a detoxication reaction (Williams and Buhler, 1983). Of specific
126
relevance to AFB1 bioactivation in rainbow trout, microsomal cytochrome P450
LMC2 and LM4B specific content are similar in all acclimation groups (Table B.2).
Consistent with most work on temperature acclimation in fish (Prosser and
Heath, 1991; White and Somero, 1982), hepatic lipid changes occur across the
temperature acclimation range for this study. Differences in percent liver total
lipid, phospholipid and cholesterol (Table B.3) are not easily explained by
increased metabolic rate at higher temperature since adjustment of ration
maintained similar growth in all groups (Results). Decreases in membrane lipid
saturated-to-unsaturated fatty acid ratios (Tables B.4 and B. 5) are associated
with maintenance of membrane fluidity in low temperature acclimated fish
(Prosser and Heath, 1991; White and Somero, 1982). This lipid-mediated
regulation of membrane fluidity is broadly recognized as a predominant
mechanism of ideal temperature compensation (Precht, 1958).
Acute temperature shifts significantly influence [3FI]AFB1 adduction to
hepatic DNA of temperature acclimated rainbow trout (Zhang et al., 1992). At a
common target organ dose, hepatic DNA from 10°C-acclimated fish exposed at
14°C contained almost twice the adduct concentration as that frdm 18 Cacclimated fish exposed at 14°C. This probably reflects increased procarcinogen
bioactivation by cytochrome P450 LMC2.
Hepatic microsomes from cold
acclimated fish metabolize benzo[a]pyrene more rapidly than those from warm
acclimated fish when assays are conducted at a common in vitro temperature
(Carpenter et al., 1990, Curtis et al., 1990). While hepatic microsomal content
of six cytochrome 450 isozymes are similar after C, I and W acclimation (Table
127
B.2), lipid composition differences (Tables B.3- B.5) likely underlie stimulation of
biotransformation after acute temperature increase. Phospholipid fatty acid
composition is an important determinant of activity for cytochrome P450 1A1
(Eberhart and Parkinson, 1991). Higher tumor incidence in C -I -I than W-1-I fish
is probably influenced by enhanced AFB1 bioactivation, which is probably
regulated by increased membrane fluidity, during temperature upshift relative to
temperature downshift. Initiation is therefore an important event in modulation of
AFB1-induced cancer in this protocol. Since acclimation temperature-dependent
changes in fish tissue lipid composition are complete within 4 weeks (White and
Somero 1982), and there is no difference in 9 month rearing temperatures
between C-1-I and W-1-I groups, an influence on promotion/progression events
seems unlikely for these regimens.
Tumor incidence and multiplicity (Fig. B.2 and B.3) for C-C-C, 1-1-1 and W­
W-W do not positively correlate with results from previous j3HJAFB1 DNA adduct
formation data (Zhang et al., 1992). In fact, at common target organ dose there
are fewer adducts in W than C-acclimated and exposed fish. Significantly higher
conversion of AFB1 to AFL (readily glucuronidated and excreted) by cytosolic
dehydrogenase at W relative to C partially explains this (Carpenter et al., 1995).
Hepatic cytosol from W acclimated fish converts about 50% more AFB1 to AFL
than that from C acclimated fish when assays are conducted at respective
acclimation temperatures. At constant acclimation and exposure temperature, an
important role for temperature/ activity relationships for membrane bound in
128
bioactivationldetoxication pathways is less likely in light of evidence for ideal
temperature compensation (Blanck et al., 1989; Koivusarri et al., 1983).
Hepatic [3H]AFB1 DNA adducts are less persistent at warm than cold
temperature (Zhang et al., 1992). Higher tritium exchange at warm temperature
is an unlikely explanation (Bailey et al., 1988; Zhang et al., 1992). If mitotic rate
increases with temperature, bulky AFB1 adducts may be lost at a higher
frequency at W than C. Recent immunohistochemical results with proliferating
cell nuclear antigen do not support a mitogenic role for increased temperature in
rainbow trout liver (El Zahr et al., 1994). An increase in death rate of AFB1
damaged cells with temperature cannot be ruled out, however. Greater loss of
adducts at 18 than 10°C may reflect higher rates of spontaneous depurination or
DNA repair. In vitro DNA synthesis repair of rainbow trout hepatocytes more than
doubled with a 10°C temperature increase (Miller et al., 1989). Our tumor results
(Fig. B.2 and B.3) negatively correlate with adduct persistence (Zhang et al.,
1992) and the number of liver tumors increase at W (Results). This suggests an
increase in probability that a mutation and ultimately a tumor results from adduct
loss. Greater understanding of DNA repair processes in rainbow trout liver is
necessary to explore the role of conversion of adducts to mutations in
temperature-modulated cancer incidence.
Modulation of promotion/progression events may underlie differences in
liver tumor incidence in regimens with constant acclimation, exposure and rearing
temperature (Fig. B.2). While there is no evidence for increase of normal liver
proliferation with temperature (El Zahr, 1994), this does not rule out selective
129
stimulation (i.e., clonal expansion) of mutated cells at W. No increase in tumor
diameter at W is not consistent with promotion however, there was a significant
reduction in percentage of mixed hepatocellular-cholangiocelluar carcinomas in
the C-C-C relative to other temperature regimens (Table B.6). We have observed
a consistent pattern of hepatic tumor types in rainbow trout over a broad range
of initiating carcinogens and modulating factors (Fong et al., 1993; Kelly et al.,
1993; Nunez et al. 1988). Typically, mixed hepatocellular/cholangiocellular
carcinomas predominate (> 30%), and the remainder of the tumors are pure
cholangiocellular forms.
In the current study, we have obsered a higher
percentage of pure hepatocellular tumors particularly at the lower temperatures.
This may indicate that reduced temperatures select for the hepatocellular
phenotype, but more data are needed to verify this. Screening tumors composed
of different cell types for mutant oncogene or cancer suppressor gene alleles
could provide molecular level understanding of such data.
Tumor results (Fig. B.2 and B.3) demonstrate temperature markedly
influences AFB1 carcinogenicity in rainbow trout. This is not readily explained
by modulation of [31-1]AFB1 adduct formation in hepatic DNA in another study of
the same design (Zhang et al., 1992). Manipulation of temperature during the
multistage cancer process may permit selective modulation of initiation and
promotion/progression events in fish.
130
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134
APENDEX C
In vitro and In vivo Temperature Modulation of Hepatic
Metabolism and DNA Adduction of Aflatoxin B1
in Rainbow Trout'
Hillary M. Carpenter, Quan Zhang, Camille El Zahr,
Daniel P. Selivonchick, Daniel E. Brock and Lawrence R. Curtis
'Published in Journal of Biochemical Toxicology, Volume 10, pp. 1 - 10, 1995.
135
ABSTRACT
Alterations in membrane lipid composition during temperature acclimation
of poikilotherms is hypothesized to compensate for direct effects of temperature
on membrane fluidity. Temperature also influences disposition and actions of
some xenobiotics. This suggests the potential for complex interactions between
temperature and metabolism of chemical carcinogens. Whole livers and hepatic
microsomes from rainbow trout acclimated at 18°C have more saturated fatty
acids and less mono- and polyunsaturated fatty acids than those from fish
acclimated at 10°C. Such changes are consistent with a role for membrane lipid
fluidity in temperature compensation. When 10 and 18°C acclimated fish are ip
injected with 0.4 mg/kg [3H]aflatoxin B1 (AFB1) at their respective acclimation
temperatures hepatic disposition of AFB1, DNA adduction, and biliary metabolites
are similar. An acute shift of 18°C acclimated trout to 14°C reduces [3H]AFB -DNA
adduct formation, while
acclimated
fish
to
[3H]AFB1
adduction
after acute shift of 10 C
14°C is no different than in non-shifted fish. Hepatic
microsomes isolated from 10 or 18°C acclimated trout, incubated with 10 NM
[3H]AFB1 and calf thymus
DNA between 6 and 22°C exhibit no differences in the "break points" of
Arrhenius plots (16°C in both groups). There is, however, more in vitro DNA
adduction of [3H]AFB1
by microsomes
from ° 18 C acclimated
fish,
a
difference abolished by 0.5 mM a-naphthoflavone. These results suggest that
temperature acclimation of trout differentially modifies activities of cytochrome
136
P450 isozymes.
When assayed at respective acclimation temperatures
hepatic cytosol
from 18°C fish produces more aflatoxicol, a detoxication
product of AFB1, than cytosol from 10°C fish. Therefore, this soluble enzyme
does not exhibit ideal temperature compensation. Such temperature-induced
differences
in
microsomal
cytochrome
P450
isozymes and cytosolic
dehydrogenase partially explain temperature-modulated AFB1 genotoxicity.
137
INTRODUCTION
Temperature acclimation of poikilotherms frequently results in an "ideal
temperature compensation", maintenance of similar rates of metabolism by
membrane-bound systems across a temperature range (1). This compensation
also applies to xenobiotic metabolism. In vitro metabolism of various substrates
of the cytochrome P-450 system is similar when hepatic microsomes from cold
and warm acclimated trout are assayed at their respective acclimation
temperatures (2,3).
Therefore, one might predict acclimation temperature
independent incidences of chemically-induced tumors in fish for carcinogens that
require metabolic activation. This is clearly not the case. A direct relationship
exists between temperature and the incidence of some chemically-induced
tumors in fish (4-6). Further, temperature significantly influences formation of
[3H]aflatoxin B1 (AFB1) hepatic DNA adducts at equivalent target organ dose (7).
Apparently, temperature interactions with
activation and/or detoxification
pathways for carcinogens are incompletely explained by ideal temperature
compensation.
Temperature is a major variable in aquatic ecosystems and may influence
cancer incidence in feral fish. Our previous work investigated the influence of
incubation or exposure temperature on the in vitro metabolism, or exposure
temperature on in vivo toxicity and disposition of benzo[a]pyrene and AFB1 in
temperature acclimated rainbow trout (4, 7-9).
In rainbow trout AFB1 is
metabolized to AFB1-8,9-epoxide by cytochrome P-450 2K1 which binds to DNA,
138
a prerequisite for the carcinogenicity of AFB1 (10). A second isozyme, P-450
1A1, produces aflatoxin M1 (AFBM1), a non-genotoxic metabolite (11). Cytosolic
dehydrogenase converts AFB1 to aflatoxicol (AFL) (12). AFL may either be
reconverted to AFB1 or conjugated with a glucuronide and excreted in the bile.
The production of AFL results in a detoxication pathway if a glucuronide is
produced and excreted or, a storage depot for AFB1 from which additional AFB1­
8,9- epoxide may be generated. Modification of these pathways by acclimation
and/or exposure temperature could alter AFB1 metabolism and carcinogenicity.
The present study examined the differential effects of temperature
acclimation on the activities of membrane-bound cytochrome P-450s and
cytosolic dehydrogenase. Homeviscous adaptation, predominantly alteration of
membrane fatty acid saturation and unsaturation to change phase transition
temperature,
is
a widely proposed mechanism for ideal temperature
compensation (1, 13). For this reason lipid compositions of whole liver and
hepatic microsomes are reported here for temperature acclimated rainbow trout.
Arrhenius plots examine the physical state of hepatic microsomal membranes
after temperature acclimation.
We also demonstrate in vivo temperature
modulation of [3H]AFB1 hepatic DNA adduction and metabolism was studied in
fish of the same size and age as those from in vitro work.
139
MATERIALS AND METHODS
Animals
Shasta strain rainbow trout (Oncorhyncus mykiss, 150-230 g) were
provided by the Marine Freshwater Biomedical Center core facility at Oregon
State University. Fish were acclimated for one month at 10 or 18°C. Rations of
Oregon Test Diet were adjusted to maintain similar growth rates between
acclimation groups (14). During the experiments, fish were held in flow-through
tanks and kept under a 12-hr light-dark cycle. Fish were sexually immature and
not fed for 48 hr before in vivo [3H]AFB1 exposures.
For in vitro studies
acclimated trout were killed, their livers removed, and microsomal and cytosolic
fractions were prepared as previously described (8).
Chemicals
Uniformly labeled [3H]AFB1 (24 mCVmmol in methanol) was obtained from
Moravek Biochemicals, Inc. (Brea, CA) and purity was determined by TLC on
Silica gel G with benzene:acetone:ethylacetate (55:15:30) and radioscanning.
Unlabeled AFB1 was obtained from either Sigma Chemical Co. (St. Louis, MO)
or Aldrich Chemical Co. (Milwaukee, WI). Stock AFB1 solutions were prepared
and quantitated as previous reported (7). Calf thymus DNA was obtained from
Sigma Chemical Co. All other reagents were of the highest grade commercially
available.
140
Arrhenius Plots of in vitro AFB1-DNA Binding
Hepatic microsomes from trout acclimated to 10 or 18°C were incubated
with rHjAFB1 and calf thymus DNA as follows. A 2 ml incubation mixture
contained: 45 mM Tris-HCI (pH 7.2 at 14°C), 0.35 mg of microsomal protein per
ml, 1 mM NADPH, 1 mg calf thymus DNA per ml, and 10 pM [3H]AFB1. The
reaction, initiated by the addition of NADPH, was run in the dark for 30 min at 6,
8, 10, 12, 14, 16, 18, 20, or 22°C. A second series of incubations contained the
cytochrome P-450 1A1 inhibitor a-naphthoflavone (ANF, 0.5 mM). Reactions
were terminated by addition of 850 pl of a 4 M NaCI solution and 90 pl a 5% SDS
solution. DNA was extracted and the [3H]AFB1-DNA adduction was determined
by liquid scintillation counting (LSC) previously described (7).
DNA
concentrations were determined by the Burton (15) method.
In vitro Metabolism of AFB1
Trout liver microsomes or cytosol (4 mg/ml) were added to a medium which
contained 50 mM phosphate buffer (pH 7.4), 1 mM NADPH, 5 mM MgC12, 60 mM
KCI and 0.5 nM [31-1]AFB1 (10.22 mCi/mmol) in a final volume of 2 ml. The
mixtures were incubated at 18°C for 60 min in the dark. The reaction was
stopped with 2 ml of cold methanol and samples were extracted with two 4 ml
volumes of chloroform. The chloroform extracts were pooled and evaporated to
dryness under a stream of nitrogen. Residues were redissolved in methanol and
analyzed by high pressure liquid chromatography as previously described (7).
141
A second set of experiments used hepatic cytosol from 10 and 18°C acclimated
fish. Here the mixtures were incubated in the dark at 10, 14 or 18°C for 60 min.
In vivo Adduction and Metabolism of r3HJAFB1
In vivo binding of 0.4 mg [3H]AFB1/kg fish to liver microsomal protein and
nuclear DNA were determined 16 hr after an ip injection. Trout were either
injected and held at their acclimation temperature or acutely shifted (1 hr prior to
injection) to an intermediate temperature of 14°C and then held at 14°C
for 16 hr
after injection. After overdose in MS-222 (150 mg/ml), a blood sample was taken
and livers and gallbladders were removed. Bile was diluted with an equal volume
of methanol, centrifuged and analyzed using a model 8800 Spectra-Physics
HPLC, a C18 Phenomenex column and a linear gradient solvent system of
methanol:ethylacetate (10 to 60 percent methanol in 45 min). Fractions were
collected and analyzed by LSC. Metabolites were identified by comparison with
authentic material (kindly provided by Ms. Pat Loveland, Dept of Food Science,
OSU). Covalent binding of [3H]AFB1 to microsomes was determined using a
dialysis method (16). Nuclear DNA was extracted and adducted [31-1]AFB1 as
determined by LSC (7).
Hepatic membrane fatty acid composition
The fatty acid compositions of liver whole homogenates or microsomes
from 10 and 18°C acclimated trout were determined as previously reported (17).
142
Fractions
were
immediately
extracted
with
chloroform:methanol
and
phospholipids for fatty acid determination were separated using two-dimensional
TLC. Silica gel coated plates (Whatman Ltd., England) were activated at 90°C
for
45
min.
The
solvent
system
for the
first
dimension
was
chloroform:methanol:28% aqueous ammonia (65:35:5) and for the second
dimension
was
chloroform:acetone:methanol:glacial
acetic
acid:water
(10:4:2:2:1). Plates were sprayed with 2,7-dichlorofluorescein and lipids were
visualized using a UV lamp. Methyl esters were prepared with sulfuric acidmethanol (18) and analyzed with a Hewlett-Packard 5890 gas chromatograph
equipped with a flame ionization detector, 3393A integrator and a 9122 dual disc
drive and a 30 m, 0.25 mm ID fused silica Supelco 2330 capillary column.
Column temperature was 175°C for the first 7 min, increased to 210°C in steps of
5°C per min and the final temperature of 210°C was held for 25 min. Each sample
was run in duplicate. Fatty acid methyl esters were identified by comparison with
standard mixtures of known methyl esters (20A and 68A NuChek Prep., Elysian,
MN and PUFA2, Supelco, Bellefonte, PA).
Phospholipids for phosphorous
content determination were separated by one-dimensional TLC using silica gel
plates and a solvent system of chloroform:methanol:water (70:30:4). Lipids were
stained with iodine vapor. Phosphorous content was determined colorimetrically
(19).
143
Total cholesterol
Total cholesterol was determined spectrophotometrically (20) with purified
cholesterol as a standard (Sigma Chemical Co., St Louis, MO). Subsamples of
lipid extract were dried under a stream of nitrogen and 6 ml of glacial acetic acid
and 4 ml of a stock ferric chloride solution were added to each sample. After 10
min absorbance was measured at 550 nm.
Plasma enzyme analysis
Blood was centrifuged and plasma subsamples analyzed by LSC.
Activities of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic
transaminase (GPT) in plasma were determined using commercially available kits
(Sigma Chemical Co., St. Louis MO.).
Statistical analysis
Data are expressed as mean t S.E. and were analyzed by either a twoway analysis of variance, followed by point comparison using the Tukey test, or
by comparisons of means using the Student's t-test. P s 0.05 was used as the
criterion of significance.
144
RESULTS
Arrhenius plots
Adduction of [3H]AFB1 to calf thymus DNA was higher with hepatic
microsomes from fish acclimated at 18°C than 10°C when incubated between 6
and 22°C (Fig. C.1, upper panel).
However, the break point in both lines
(apparent phase transition temperature) was 16°C for both acclimation groups
(Fig. C.1, upper panel). a-Naphthoflavone (ANF), an inhibitor of cytochrome P­
450 1A1 and 1A2: (1) eliminated differences in [3H]AFB1 -DNA adduction
catalyzed by microsomes from 10 and 18°C acclimated fish, (2) increased the
amounts of DNA adduction in both groups, and (3) caused a shift in the break
point of the line(s) to 14°C (Fig. C.1, lower panel).
In vitro metabolism of AFB1 by the hepatic cytosolic fraction
When cytosol from 10 and 18°C fish were incubated at 10, 14 or 18' C
there were significant differences in the amounts of AFB1 converted to AFL (Fig.
C.2). Approximately 50% of the AFB1 present in the system was converted to
AFL by the cytosol from 10°C acclimated fish when assayed at 10 C while
approximately 70% of the AFB1 was converted to AFL by cytosol from 18°
assayed at 18°C. More AFL was produced by cytosol from 10°C fish than those
from 18°C fish when they were-assayed at an intermediate temperature of 14°C.
145
FIG. C. 1
Upper panel. Arrhenius plots for in vitro [3FIJAFB1-DNA binding to
calf thymus DNA catalyzed by hepatic microsomes from rainbow
trout acclimated to 10 or 18°C. Symbols are mean ± S.E., n = at
least 3. ANOVA showed that the data from 10 and 18°C acclimated
groups were significantly different.
Lower panel. Arrhenius plots of a-naphthoflavone modulated in
vitro [3HIAFB1-DNA binding. Symbols are mean ± S.E., n = at least
3. Data from the 10 and 18°C acclimated groups are no longer
different when subjected to ANOVA.
146
Incubation Temperature (°C)
22
20
18
16
14
12
10
8
1000.0
zA
00
al 100.0
O
5
0 1000
-1
100
3.41
3.45
3.49
3.53
3.57
Incubation Temperature ( 1/k x 1000)
147
FIG. C. 2
The effect of temperature acclimation on the in vitro production of
AFL from [3H]AFB1 by the cytosol from rainbow trout acclimated to
10 or 18°C. Assays conducted at 10, 14 or 18°C. Symbols are mean
± S.E., n = at least 3.
148
70
ACCLIMATION TEMPERATURE
II.
60
0 - 10 °C R 2= 0.70
18 °C R2= 0.91
:§
40
1
i
30
8
1
1
i
i
I
I
10
12
14
18
18
20
INCUBATION TEMPERATURE (°C)
149
In vivo adduction and metabolism of rHAFB1
Adduction of [3H]AFB1 to hepatic DNA was a linear function of total liver
[3H]AFB1 equivalents in
fish acclimated
and exposed at 10 or
18°C
(Fig. C.3). There was no significant difference between the slopes of these lines
for these two groups. In addition, there were no differences in the amounts of
[3H]AFB1 covalently bound to microsomal proteins between the acclimation
groups (data not shown). When 10 and 18°C acclimated fish were acutely shifted
to an intermediate temperature of 14°C, the slopes of the plots of liver
concentration/DNA adducts were different (Fig. C. 4).
[3H]AFB1
to
hepatic
DNA of the
Here, adduction of
18°C acclimated fish
14°C was not correlated with the tissue r H1AFB1
shifted
equivalents
to
and
the slope of the line was not different from zero.
Gallbladders of fish treated with AFB1 were distended, indicating a
treatment related alteration of bile flow. The two primary biliary metabolites of
AFB1 were AFL glucuronide and AFL M1 glucuronide (Fig. C.5).
AFL
glucuronide made up about 50% of the recovered material while AFL M1
glucuronide averaged about 25% of the recovered amount. There were no
apparent acclimation temperature dependent differences in the amounts of these
metabolites (Fig. C.6).
Typically about 15% of the recovered radioactivity
occurred in an unidentified peak.
150
FIG. C.3
The effect of temperature acclimation on in vivo [3H]AFB1-DNA
binding versus total hepatic rHAFB1 concentration. Fish received
a single ip dose of 0.4 mg/kg [3H]AFB1 16 hr prior to killing. Each
value is from an individual fish. The upper panel represent results
from fish acclimated and tested at 18°C and the lower panel those
acclimated and tested at 10°C. Slopes calculated by least squares
estimation were not statistically different.
151
1000
800 _
18-18
y=25X+69
r =0.73
0
600
:14
Z 400
0
al
CD.­
200
U­
4
0
oo
I
t
1
10
20
30
E
c
......
as
800
10-10
c
0c
in 600
0
y = 35X -18
r = 0.87
00
0
44
Z
0 400
0
200
0
0
0
i
1
I
10
20
30
Liver Concentration (nmol AFE31 /g liver)
152
FIG. C.4
In vivo [3F]AFB1-DNA binding versus total hepatic AFB1
concentration in temperature shifted fish. The upper panel is data
from fish acclimated at 18°C and shifted to 14°C one hr prior
to injection. The lower panel is data from fish acclimated to 10°C
and shifted to 14°C one hr prior to injection. Slopes calculated by
least squares estimation were statistically different.
153
1200
18-14
y=22x+100
r=0.34
c
c
c3
0
0
800
400
0
4
20
10
Liver concentration
30
(nrnol AFBi/g liver)
1200
10-14
y=39x-52
r=0.95
1:f
c
c
3 800
e
I It
0
E400
00
4
0
20
Liver concentration
(nmol AFIBi /g liver)
10
30
154
FIG. C.5
Representative HPLC chromatogram of the biliary [3H1AFB1
metabolite profile from rainbow trout acclimated and exposed at
14°C. One min fractions were collected and quantitated by LSC.
155
L glucuronide
....
ro
25
0
x 20
E
a.
v 15
L-M1 glucuronide
I
0
y..
3
._ 10
tao
Unknown
5
*-13
a
ct
14
21
28
Fraction Number
35
42
156
FIG. C.6
The percentage of [3FIJAFB1 biliary metabolites produced by
rainbow trout acclimated to 10 or 18°C. Values are the mean ± S.E.
(n = at least 4) of the percent of total radioactivity recovered during
the analysis. The first number in each pair is the acclimation
temperature. The second number is the temperature the fish were
placed in one hr prior to injection.
157
100
Unknown
O Aflatoxicol M1 glucuronide
80 _
l Aflatoxicol glucuronide
60
.0
0
2 40
0
20
0
10-14
18-14
18-18
10-10
Temperature Regimen (°C)
158
TABLE C.1 Fatty Acid Composition of Lipid Extracts from Whole Livers of
Temperature Acclimated Trout'
Total Lipids
Fatty
10°C
Phosphotklylcholine
18°C
18°C
10°C
Phosphotidylethanolamine
10°C
18°C
Acid
Whole Liver Extracts
+ 0.3
2.7 ± 0.1
1.4 ± 0.1
1.7 + 0.1
26.6 ± 0.6
29.1 ± 0.2b
26.0 ± 0.8
27.2 ± 0.7
16:1n-7
2.0 ± 0.1
2.0 ± 0.0
1.3 ± 0.1
1.1
18:0
6.7
± 0.2
± 0.2
2.9 + 0.1
3.8 ± 0.1
12.1
± 0.1
11.9 + 0.1
10.2
+ 0.4
10.4
± 0.2
18:1n-7
2.5 + 0.0
2.5 + 0.0
1.7
± 0.0
1.6
+ 0.0
5.5
± 0.2
4.8 + 0.2b
18:2n-6
0.9 + 0.0
0.8 ± 0.0b
0.6
± 0.0
0.5 + 0.0b
1.7
± 0.0
1.3
20:1n-9
0.8 + 0.0
0.9
0.5
± 0.0
0.6 + 0.0b
2.0 ± 0.1
2.3 ± 0.1
20:4n-6
3.1
1.9
+ 0.2
2.4 ± 0.1
22:1n-11
0.4 ± 0.1
22:1n-9
0.6
± 0.0
0.5
± 0.0
0.5
20:5n-3
6.7 ± 0.1
7.2
22:5n-3
1.9 ± 0.1
1.5 + 0.1
22:6n-3
25.7 ± 0.5
23.8 ± 0.2
14:0
16:0
18:1n-9
2.1
± 0.1
7.2
± 0.0
± 0.0
0.3 + 0.0
14.6
± 0.0
± 0.5 13.7 ± 0.4
0.9 + 0.0
9.8
0.3
0.8
± 0.0
+ 0.5 10.6 ± 0.0
17.0 + 0.1
16.2
± 02b
+ 0.0
0.9 ± 0.1
0.9
± 0.0
0.1
± 0.0
0.1
+0.0
± 0.0
0.6
± 0.0
0.5
+ 0.0
0.4
± 0.3
9.0
± 0.5
9.7
± 0.6
6.9 ± 0.3
6.7 ± 0.1
+ 0.0
1.2 ± 0.1
2.9 ± 0.1
± 0.0
2.3 ± 0.1
1.9 ± 0.1
35.9
± 12
32.7 + 0.4
30.7 + 0.6 32.5 + 0.8
30.3
± 1.0
32.7
± 0.7
24.7 + 1.1 24.6 ± 0.4
± 0.5
14.4
+ 0.2
25.8 + 0.3 24.5 ± 0.3
45.7 + 1.0
46.4 + 1.1 44.1 ± 0.9
1.3
Totals
± 1.0 39.0 + 0.0b
Saturated
35.4
Mono
18.3 ± 0.1
17.7 ± 0.1b
14.5
Poly
38.3 ± 1.1
36.2 + 0.3
48.7 ± 1.6
± 0.0
0.7 ± 0.0b
0.5 + 0.0
SFA:UFAc
0.6
0.6
+ 0.0
0.4 + 0.0
0.4
± 0.0
'Values were expressed as mean ± S.E., for weight percentage of total lipids. Each
mean was calculated for duplicate gas chromatographic analyses from three individual
fish.
bValue was significantly different from that of 10°C acclimated group at P < 0.05.
°Saturated fatty acid-to-unsaturated fatty acid ratio.
159
TABLE C.2 Fatty Acid Composition of Lipid Extracts from Hepatic Microsomes
of Temperature Acclimated Trout'
Phosphotidylcholine
Total Lipids
Fatty
10°C
18°C
10°C
Phosphotidylethanolamine
10°C
18°C
0.3 ± 0.0
0.5 ± 0.0b
28.9 + 1.0b 10.9 ± 0.2
12.8 ± 0.2b
18°C
Acid
Microsomai Fatty Acids
14:0
1.8 ± 0.1
16:0
26.7 ± 0.6
2.5 + 0.2b
28.8 + 0.8
± 0.0
16:1n-7
1.7 ± 0.1
1.5
18:0
6.5 ± 0.2
7.5 ± 0.1°
18:1n-9
11.9
± 0.3 11.5 ± 0.2
1.2 + 0.1
24.4 + 0.7
2.0 + 0.1
1.2
± 0.0
1.2
± 0.0
0.8
± 0.0
3.2
± 0.2
42 ± 0.3
9.1
± 0.3 10.4 ± 0.2b
9.5 ± 0.1
9.9 ± 0.1
17.9
± 0.4 17.9 ± 0.5
± 0.0
1.5 ± 0.1
4.9 ± 1.0
5.0
1.0 ± 0.1
± 1.0
18:1n-7
2.4 ± 0.1
2.2 ± 0.0
1.7
18:2n-6
0.8 ± 0.0
0.7 ± 0.0
0.5 ± 0.1
0.6
± 0.0
1.9 ± 0.1
1.9 + 0.1
20:1n-9
0.8 + 0.0
0.7 ± 0.0b
0.5
± 0.0
0.6
± 0.3
2.1
+ 0.1
2.5 ± 0.2
20:4n-6
3.3 + 0.1
3.3 ± 0.1
1.0 ± 0.1
0.8
± 0.0
1.9
± 0.2
2.4 + 0.3
0.1
+ 0.0
0.1
± 0.0
0.6
± 0.0
0.5 ± 0.0b
0.5
+ 0.0
0.5 ± 0.0
± 0.3
8.9
± 0.2
9.4
1.7 ± 0.1
22:1n-1
22:1n-9
0.6
± 0.0
0.5 + 0.0b
20:5n-3
7.3
± 0.2
7.9
22:5n-3
1.8 + 0.1
1.5 ± 0.1
2.4
± 02
22:6n-3
27.0 ± 0.6
25.8 + 0.8
38.5
± 1.0
32.1
± 0.5
7.8 ± 0.1
7.4
+ 0.3
± 0.0
1.1
± 0.1b
1.5
± 1.0 33.6 + 0.4 31.8 ± 1.2
Totals
Saturated
Mono
± 0.7 38.8 ± 0.9 28.8 ± 1.0
17.3 ± 0.4 162 ± 0.3 13.6 ± 0.1
35.1
35.1 ± 1.0b 20.2
+ 0.5 23.7 ± 0.4b
13.8 ± 0.1
± 0.6 26.6 + 0.2
26.1
402 ± 0.9 39.2 + 0.9 51.4 + 1.0 44.5 + 1.0b 46.7 + 0.2 44.6 ± 0.6
0.6 ± 0.0b 0.28 + 0.0 0.33 ± 0.1b
0.4 ± 0.0
0.7 ± 0.0
SFA:UFAc 0.6 ± 0.0
Poly
aValues were expressed as mean ± S.E. for weight percentage of total lipids. Each mean
was calculated for duplicate gas chromatographic analyses from three individual fish.
bValue was significantly different from that of 10°C acclimated group at P < 0.05.
°Saturated fatty acid-to-unsaturated fatty acid ratio.
160
Hepatic lipid contents
Temperature acclimation altered fatty acid composition of whole livers (Table
C.1) and hepatic microsomes (Table C.2). Saturated fatty acids were higher and
polyunsaturated fatty acids were lower in 18 than did the 10°C acclimated fish.
Ratios of saturated-to-unsaturated fatty acids also differed for microsomal
phospholipids and total lipids of whole liver between acclimation groups. There
were no acclimation temperature-related differences in cholesterol of whole liver (­
5 mg/g liver) or hepatic microsomes (- 0.9 mg/g liver).
Plasma enzyme activities
Plasma GOT and GPT activities were elevated following AFB1 treatment
(Table C.3); however, no temperature effects were apparent. This was the case for
both acutely shifted fish and those that were injected at their acclimation
temperature.
161
TABLE C. 3
The effect of temperature and [3H]AFB1 on the activities of
plasma GOT and GPT.'
Enzyme
GOT
Control
121
* 13 (6)
GPT
10
*2 (6)
33
22
27
28
* 9c (7)
* 3c (6)
* 1c (6)
* 4c (7)
[3H]AFB Treated'
18-18°C
10-10°C
10-14°C
18-14°C
188 * 12c (9)
195 * 14c (5)
208 * 5` (9)
206 * 5c (7)
a Values were expressed as mean * S.E., sample size in parenthesis.
"The first temperature was acclimation temperature and the second was the temperature
the fish were held at following injection. Fish were killed 16 hr after injection and plasma
was prepared within 15 min.
c Value significantly different from control at P s 0.05.
162
DISCUSSION
Temperature acclimation-dependent differences in tissue lipid composition
are thought modulate the activities of membrane-associated enzymes (13, 21-22).
One hypothesized mechanism is that enzyme activity is affected by local
membrane fluidity. Increased concentrations of unsaturated fatty acids at colder
temperatures are proposed to decrease lipid phase transition temperature and
maintain membrane fluidity (1, 21-25). This presumes an important role for lipid
fluidity in regulation of membrane enzyme activities (13).
Acclimation
temperature-dependent differences in saturated and unsaturated fatty acids
(Tables C.1 and C.2) were consistent with previous work (21,22). Standard
errors were often zero for low weight percent fatty acids due to rounding of low
variability data. The saturated fatty acid-to-unsaturated fatty acid ratio for hepatic
microsomal phosphotidylcholine was 50 percent higher in 18 than 10°C
acclimated fish. This difference was only about 15% for phosphotidylethanoline
and statistically insignificant for total lipids.
Arrhenius plots (reciprocal of absolute temperature vs. log enzyme activity)
portray temperature effects on the thermodynamics of enzyme activation. Such
plots describe the number of molecules that exist in an activated state and
capable of reaction. Typically these are straight lines throughout the range of
activity. However, for membrane bound enzymes, breaks in the slopes of these
lines are associated with transitions in the membrane physical state (23).
Specifically, for membrane enzymes, break points in Arrhenius plots are thought
163
to reflect a phase transition in the lipids or a structural transition in the proteins
(13).
Homeoviscous adaptation typically results in measurable differences
between temperature acclimation groups in the break points of the Arrhenius
plots for membrane-bound enzymes (13). Here, no differences in the break
points of the Arrhenius plots for microsomes from 10 and 18°C fish (Fig. C.1)
indicates no major role of membrane physical state in temperature-modulated
metabolic activation of AFB1.
When [H]AFB1 -DNA adduction was plotted versus incubation temperature
for microsomes from 10 or 18°C acclimated trout there was more total binding for
the 18°C group. These in vitro results contrasted the results of Zhang et al. (7)
where in vivo rates of [3H]AFB1 -DNA adduction in 10°C acclimated trout
were slightly higher than those in 18°C acclimated fish. Such conflicting results
reinforced the necessity of evaluating multiple metabolic pathways in vitro (e.g.,
microsomal and cytosolic) to explain in vivo results. Despite the apparent
differences in [3H]AFB1 binding to DNA, there were no temperature acclimation-
related differences in the break points of the Arrhenius plots (Fig. C.1, upper
panel). This suggested temperature acclimation caused no gross lipid phase
transition differences or structural transition in the enzymes responsible for the
adduction of [3H]AFB1 to DNA of trout. However, ANF significantly increased
[3H]AFB1 -DNA adduction catalyzed by microsomes from both the 10 and le C
acclimation groups (Fig. C.1, lower panel). Inhibition of cytochrome P450 1A1
(which produces AFM1) by ANF perhaps increased AFB1 available for
metabolism to the epoxide by the 2K1 form. Alternatively, perhaps ANF directly
164
stimulated 2K1 activity. Previous studies showed that ANF stimulated the in vitro
formation of AFB1-8,9-epoxide by human microsomes (25) and human
cytochrome P450 3A4 (26). Since microsomal fatty acid composition differed
between 10 and 18°C acclimated fish (Table C.2), ANF action was possibly
mediated via interaction with membrane lipids. The sequence of full-length cDNA
for cytochrome P450 2K1 was novel enough that the gene was placed in a new
subfamily (27). Limited homology restricted inference of data from the human
3M to trout 2K1 form. ANF abolished the temperature dependent differences in
[3H]AFB1 binding and shifted the break points in the Arrhenius plots from 16 to
14°C. These results were also consistent with temperature acclimation-induced
changes in cytochrome P-450 isozymes. In fish of a similar size to those used
here, hepatic microsomes from 10°C acclimated rainbow trout contained one-third
more cytochrome P450 1A1 than those from 18°C acclimated fish while 2K1
content was the same (8). However, hepatic microsomal cytochromes P450 1A1
and 2K1 were not different between 10 and 18°C-acclimated 10 g rainbow trout
(4).
Temperature modulation of AFB1 genotoxicity (7) and carcinogenicity (4,5)
likely involved detoxication pathways that compete with the cytochrome P450 2K1
production of AFB1-8,9-epoxide. In vitro production of AFL by cytosol from 18°C
acclimated fish assayed at 18°C was higher than that of cytosol from
10°C acclimated fish assayed at 10°C (Fig. C.2).
When the incubation
temperature of the assay was varied, the activity of this enzyme increased with
temperature. Given the importance of formation of AFL with its subsequent
165
glucuronidation as a detoxication pathway, stimulated activity of the cytosolic
dehydrogenase perhaps increased AFL detoxication and reduced DNA
adduction. These results were consistent with, and at least partially explained the
slightly higher levels of in vivo adduct
formation in 10°C acclimated and
exposed than in 18°C acclimated and exposed fish (7).
In vivo temperature modulation of AFB1-DNA adduction in 150-250 g fish
(Fig. C.3) was generally consistent with that seen with 2-3 g fish (7). That is,
when 10 or 18°C acclimated fish were exposed at their acclimation temperature
similar amounts of DNA adduction were observed (Fig. C.3). However, acutely
shifting 18°C acclimated fish to 14°C for exposure to [3H]AFB reduced adduction
(Fig. C.4).
Here, binding no longer occurred in direct relationship to tissue
concentration of AFB1. The relative activity rates of cytochrome P450 1A1 and
2K1 (though not addressed here) may be of particular importance in the
protection provided by lowering the exposure temperature of the fish.
Elevated plasma enzyme activities indicate that the dose of AFB1
administered to the trout was cytotoxic (Table C.3). The increased levels of both
GPT and GOT indicated damage to the liver and probably kidney, respectively.
Evidence for acute AFB1 nephrotoxicity was provided by distended gallbladders
in the AFB1 treated groups, this indicated disrupted osmoregulation (i.e, water
gain). Incomplete bile retention by the gallbladder at high bile flows may have
been responsible for the apparent lack of effect of temperature on in vivo rates
of AFL production. Accurate estimates of biliary excretion were only possible
166
under conditions where the gallbladder released little or no bile over the course
of an experiment (28).
Temperature effects on AFB1 genotoxicity are complex. When acclimation
and exposure temperatures were the same, similar in vivo adduction of [3H]AFB1
occurred at 10 and 18°C.
In vitro experiments with hepatic microsomes
suggested a greater potential for AFB1-8,9-epoxide formation at 18°C (Fig. C.1)
while those with hepatic cytosol supported higher AFL formation at 18°C (Fig.
C.2). In vivo competition of these pathways at least partially explained why AFB1
adduction to hepatic DNA (Fig. C.3) was not significantly higher at 18 than 10°C.
In addition to modulation of genotoxicity, temperature may also affect
conversion of DNA adducts to mutations, tumor promotion and/or progression (4).
Such effects might provide an explanation for the poor correlations between
adduct concentrations in liver and liver tumor incidence in our temperature
experiments (4,7). Temperature modulation of carcinogenesis in trout may be a
valuable model for examining the various stages of cancer development.
167
ACKNOWLEDGMENTS
We thank Dr. David E. Williams and Dr. Duncan J. Gilroy for providing
valuable reviews of this paper. This investigation was supported by research
Grants ES05543 and ES03850 from the National Institutes of Environmental
Health Sciences, National Institutes of Health.
168
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