AN ABSTRACT OF THE THESIS OF
Melanie L. Barnhill for the degree of Master of Science in Toxicology presented on
March 25, 2002. Title: Dieldrin Stimulates Biliary Excretion of [14C1BenzoIa1pvrene
Polar Metabolites but Does Not Change the Metabolite Profile in Rainbow Trout
(Oncorhyncus mykiss).
Redacted for Privacy
Abstract approved:
Lawrence R.
Feeding rainbow trout 0.3-0.4 mg dieldrinlkg/d for 9-12 weeks stimulated the biliary
excretion of a subsequent dose of [14Cjdieldrin by 500% and [3H]7,12-
dimethylbenz[a]anthracene (DMBA) by 240%.
In vitro
work demonstrated that this
interaction occurred without induction of the cytochrome P450 system, or other
hepatocellular proteins involved in metabolism. The present research examined the
effects of dieldrin pretreatment on the disposition of the polycyclic aromatic
hydrocarbon, benzo[a]pyrene (BaP). The study assessed whether increased biliary
excretion of DMBA in dieldrin-fed fish also occurred with BaP, a closely related
compound. This substrate, which undergoes complex metabolism, characterized the in
vivo state of the cytochrome P450 system, UDP-glucuronyltransferases, and
sulfotransferases. Rainbow trout were fed control or dieldrin diets (0.324 mg
dieldrinlkgld for 9 weeks or 0.162 mg dieldrinlkg/d for 11 weeks), followed by an ip
challenge dose of [14C]BaP (10 pmols/kg). In experiment 1, dieldrin pretreatment
significantly elevated the concentration of ['4C]BaP in bile (142% and 200% at 9 and 12
weeks, respectively) but not liver or fat. In experiment 2, the concentration of ['4C]BaP
was elevated in bile (223%), liver (232%), and fat (268%), however, the difference was
not significant relative to controls. Extraction of bile sub-samples confinned dieldrin
pretreatment significantly stimulated total biliary excretion of [14CIIBaP polar
metabolites (244% and 221% at week 9 and 12, respectively in experiment 1; 197% in
experiment 2). Bile was extracted and then hydrolyzed by fl-glucuronidase and
arylsulfatase to regenerate BaP metabolites conjugated by phase II enzymes. Evaluation
of biliary polar metabolite profiles of ['4C]BaP revealed no significant differences
between control and dieldrin-fed fish. There was no indication of selective enhancement
of any particular peak or induction of a novel biotransformation pathway with dieldrin
pretreatment. General increases in many of the biliary metabolite fractions from
dieldrin-fed fish were observed, suggesting that a particular P450 was not altered. This
study confirmed that enhanced biliary excretion, following chronic dieldrin exposure, is
not explained by induction of xenobiotic metabolizing enzymes. The results are
consistent with induction of hepatic intracellular trafficking proteins in dieldrin-fed fish.
Dieldrin Stimulates Biliary Excretion of [14C]Benzo[a]pyrene Polar Metabolites but
Does Not Change the Metabolite Profile in Rainbow Trout (Oncorhyncus mykiss)
by
Melanie L. Bamhill
A ThESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Presented March 25, 2002
Commencement June 2002
Master of Science thesis of Melanie L. Barnhill presented on March 25, 2002.
APPROVED:
Redacted for Privacy
Major Professor, representing Toxicology
Redacted for Privacy
Chair of the Department of Environmental and Molecular Toxicology
Redacted for Privacy
Dean of th
iraduate 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
Melanie L. Bamhill, Author
ACKNOWLEDGMENTS
I offer my deepest appreciation and gratitude to my advisor, Dr. Lawrence R.
Curtis, who provided continued guidance and valuable support during my graduate
studies, while also encouraging independent thought and research along the way. In
particular, I thank him for his contributions, especially his input and revisions, which
made completion of this document possible.
I am grateful to the following people who provided their expertise, assistance,
time, or support during this project: Dr. William M. Baird, Dr. Philip Whanger, Rob
Chitwood, Sam Bradford, Corwin Willard, Tamara Musafija-Jeknic, Magalie Rosemond
and staff at the Marine/Freshwater Biomedical Sciences Center. I'd also like to thank
the members of my graduate committee for their input and participation: Dr. David E.
Williams, Dr. Jeffrey J. Jenkins, and Dr. Kermit Cromack. The Oregon Agricultural
Experiment Station provided support for this research.
Most importantly, I'd like to acknowledge my family for their support. I extend
my heartfelt appreciation to my parents, who believed in me and offered their
compassion, understanding, and encouragement. Special thanks, to Clint for his endless
patience and ability to bring me back from the brink of insanity. Thanks to my friends,
especially Jennifer for your encouragement. I dedicate this thesis to Treston, Braydon,
and Bethany. Thank you for the happy memories we have made together. I hope that
someday I will have inspired you to pursue your own dreams and aspirations.
TABLE OF CONTENTS
Pag
1.
2.
3.
4.
INTRODUCTION ............................................................................................
1
1.1
Chlorinated cyclodienes and polycycic aromatic hydrocarbons ........... 1
1.2
Rainbow trout adaptation to chronic dieldrin exposure ........................ 2
1.3
Induction of hepatocellular proteins involved in metabolism ............... 3
1.4
Induction of non-enzymatic hepatocellular proteins ............................. 5
1.5
Research objective ............................................................................... 7
MATERIALS AND METHODS ...................................................................... 8
2.1
Materials ..............................................................................................8
2.2
Treatment and sampling protocol ......................................................... 8
2.3
[14CIBenzoIa]pyrene disposition following dieldrin pretreatment ........ 10
2.4
Biliary ['4C]benzo[a]pyrene polar metabolite profile ........................... 11
2.5
Statistical analysis ................................................................................ 13
RESULTS ......................................................................................................... 14
3.1
Animals ................................................................................................14
3.2
[14CIBenzoIIa]pyrene disposition following dieldrin pretreatment........ 14
3.3
Biliary ['4C]benzo[a]pyrene polar metabolite profile ........................... 19
3.4
Polar metabolites of [14C]benzo[alpyrene excreted into bile ................ 22
3.5
Percentage of polar metabolites excreted into bile ............................... 36
SUMMARY AND CONCLUSION .................................................................. 38
TABLE OF CONTENTS, Continued
Page
BIBLIOGRAPHY........................................................................................................ 44
APPENDICES .............................................................................................................. 47
AFpendixA
C]Benzo[alpyrene polar metabolite profile (Experiment 1)
48
III
ApendixB ....................................................................................................... 50
[1
CjBenzo[alpyrene polar metabolite profile (Experiment 2)
AppendixC ....................................................................................................... 52
Stimulation of [3HjBenzo[a]pyrene Metabolism Following Dieldrin
Pretreatment in the Rainbow Trout Liver Cell Line, RTL-Wl
LIST OF FIGURES
Figure
Page
3.1.
Weight gain in rainbow trout (initial wt -2 g) following dieldrin
exposure for 9 wks (A) and body weight (initial wt -88 g) following
dieldrin exposure for 11 wks (B) ....................................................................... 15
3.2.
Tissue concentrations of lL'4Cjbenzo[a]pyrene in liver (A) and fat (B)
following dieldrin pretreatment for 9 wks ......................................................... 17
3.3.
Concentration of I14CIbenzo[alpyrene in bile (A) and total biliary
excretion (B) following dieldrin pretreatment for 9 wks ................................... 18
3.4.
Tissue concentrations of ['4Clbenzo[ajpyrene in liver (A) and fat (B)
following dieldrin pretreatment for 11 wks....................................................... 20
3.5.
Concentration of ['4Cjbenzojajpyrene in bile (A) and total biliary
excretion (B) following dieldrin pretreatment for 11 wks ................................. 21
3.6.
II14CjBenzo[allpyrene parent and unconjugated oxidized metabolite
profile in gallbladder/bile at week 9 (A) and week 12 (B), following
dieldrin pretreatment in rainbow trout (initial wt -2 g) for 9 wks..................... 23
3.7.
[14C]Benzo[a]pyrene metabolite profile of cleaved glucuronide
conjugates in gallbladder/bile at week 9 (A) and week 12 (B), following
dieldrin pretreatment in rainbow trout (initial wt -2 g) for 9 wks..................... 25
3.8.
[14C]Benzo[aljpyrene metabolite profile of cleaved sulfate conjugates in
gallbladder/bile at week 9 (A) and week 12 (B), following dieldrin
pretreatment in rainbow trout (initial wt -2 g) for 9 wks .................................. 27
3.9.
['4C]Benzo[a]pyrene parent and unconjugated oxidized metabolite
profile in gallbladder/bile, following dieldrin pretreatment in rainbow
trout (initial wt -88 g) for 11 wks ..................................................................... 29
3.10.
['4C] Benzo[alpyrene metabolite profile of cleaved glucuronide conjugates
in gallbladder/bile, following dieldrin pretreatment in rainbow trout (initial
wt--88g)forllwks ......................................................................................... 30
LIST OF FIGURES, Continued
Figure
Page
3.11.
[14C]Benzo[allpyrene metabolite profile of cleaved sulfate conjugates in
gallbladder/bile, following dieldrin pretreatment in rainbow trout (initial
wt-88g)forllwks ......................................................................................... 31
3.12.
Polar metabolites of II14Cjbenzo[a]pyrene excreted into bile, following
dieldrin pretreatment in rainbow trout (initial wt -2 g) for 9 weeks:
phase 1 metabolites (A), cleaved glucuronide conjugates (B), cleaved
sulfate conjugates (C), and residual poiar material (D) ..................................... 33
3.13.
Polar metabolites of [14CIIbenzo[a]pyrene excreted into bile, following
dieldrin pretreatment in rainbow trout (initial wt -88 g) for 11 weeks:
phase 1 metabolites (A), cleaved glucuronide conjugates (B), cleaved
sulfate conjugates (C), and residual polar material (D) ..................................... 35
Dieldrin Stimulates Biliary Excretion of [14CJBenzo[a}pyrene Polar
Metabolites but Does Not Change the Metabolite Profile in Rainbow
Trout (Oncorhyncus
mykiss)
CHAPTER 1
INTRODUCTION
1.1
Chlorinated cyclodienes and polycyclic aromatic hydrocarbons
Dieldrin is one member of the chlorinated cyclodiene class of insecticides, which
also includes aidrin, chiordane, endrin, heptachior, and mirex. These compounds all
have similar chemical structures. They are non-planar molecules and contain several
carbon-chlorine (C-Cl) bonds that contribute to their stability in the environment.
Because dieldrin is highly persistent and tends to bioaccumulate in fish, wildlife, and
humans the Environmental Protection Agency (EPA) banned all uses in the 1970s,
except for termite control, which was discontinued in 1987 [1,2]. However, dieldrin is
still detected in all environmental media, including air, soil, sediment, water, fish, and
wildlife because it breaks down slowly and because of its continued use into the late
1980s [3].
The U.S. Geological Survey (USGS) National Water Quality Assessment
Program (NAWQA) detected chlordane and dieldrin in the environment at
concentrations that exceeded guidelines, set to protect aquatic life [3]. Elevated levels
were most commonly found in sediment and fish [3]. Because cyclodiene insecticides
remained at levels that represented a potential concern, particularly at sites of
contamination, the EPA listed aldrin, dieldrin, chlordane, and mirex as priority level-i
2
pollutants that are persistent, bioaccumulative, and toxic (PBTs) [4]. National action
plans, aimed at reduction of risk to human health and the environment, were initiated on
each of these pollutants [4].
Benzo[a]pyrene (BaP) belongs to a group of over 100 different chemicals,
classified as polycyclic aromatic hydrocarbons (PAHs). The key structural feature of
PAHs is two or more fused benzene rings [5,6]. These compounds are widely
distributed in the environment, and form during the incomplete combustion of organic
material
[5,6].
The EPA also listed BaP as a priority level-i pollutant that is
bioaccumulative, persistent, and toxic (PBT) [4]. Assessing the interaction that occurs
between organochlorines and PAHs is toxicologically relevant because these chemicals
are present in the environment as mixtures and not just as one single chemical.
1.2
Rainbow trout adaptation to chronic dieldrin exposure
Bioaccumulation of dieldrin was measured in rainbow trout following
waterborne and dietary exposures for 16 weeks [7]. Fish were expected to accumulate a
steady state body burden of the chemical, yet assimilation efficiency decreased during
chronic exposure. When corrected for lipid content, whole body dieldrin concentrations
(expressed as total dieldrinlmg total body lipid) increased through 8 weeks. However, at
week 16 dieldrin concentrations dropped to levels comparable to those observed in trout
exposed for 2 weeks. During a preliminary disposition experiment, rainbow trout were
exposed to dieldrin in the diet for 2,4, or 6 weeks, followed by a challenge dose of
[14C]dieldrin [7]. Distribution of the challenge dose to bile increased as the length of
dieldrin pretreatment increased. In addition, the concentration of IL'4C]dieldrin peaked in
certain tissues, including liver, following 4 weeks of dieldrin exposure.
A subsequent study confirmed that chronic exposure of rainbow trout to dieldrin
changed tissue distribution [81. Feeding rainbow trout 0.4 mg dieldrinlkgld for 12 weeks
stimulated the biliary excretion of a [14C]dieldrin challenge dose by 500%. In addition,
[14C]dieldrin was significantly elevated in liver (200%) and mesenteric fat (500% at 10
weeks, 1200% at 12 weeks) but was decreased in carcass lipid, suggesting redistribution
of the challenge dose.
Chronic dieldrin exposure also altered disposition of a subsequent dose of
[3H17, 1 2-dimethylbenz[a] anthracene (DMBA), a polycydic aromatic hydrocarbon [9].
Feeding rainbow trout 0.1 or 0.3 mg dieldrinlkg/d for 9 weeks stimulated the biliary
excretion of [3H]DMBA by 155% and 240%, respectively. In addition, the
concentration of [3H]DMBA increased in the liver by 118% and 166%, respectively. In
a separate study, precision cut liver slices were prepared from rainbow trout fed 0.4 mg
dieldrinlkg/d for 10-12 weeks [1011. Pretreatment stimulated a two-fold increase in the
uptake of [14C]dieldnn by liver slices. In addition, an increase in the uptake and efflux
of [3H]DMBA by liver slices was observed. This demonstrated that altered disposition,
following dieldrin pretreatment, was not specific to a subsequent dose of dieldrin.
1.3
Induction of hepatocellular proteins involved in metabolism
Fish adapt to chronic dieldrin exposure with a decrease in bioaccumulation of
this compound, or another lipophilic chemical. Increased biliary excretion of a
xenobiotic is often interpreted as direct stimulation of metabolism. However, fish are
refractory to induction of hepatic monooxygenases by non-planar organochlorines, like
dieldrin [111. Cytochrome P4501A (CYP1A) enzymes, regulated by the
aryihydrocarbon receptor (AhR), are not induced because non-planar compounds are
unable to activate this receptor 1111. In addition, many CYP2B inducers (e.g.,
phenobarbital and non-planar OCs) do not induce CYP2B enzymes in fish [11]. This
indicates that other processes, rather than induction of xenobiotic metabolizing enzymes,
are responsible for this adaptation.
In rainbow trout pretreated with dieldrin for 10 weeks, no changes in microsomal
cytochrome P450 proteins or enzyme activities were observed [101. Microsomes from
control and dieldrin-fed fish were incubated with [14C]BaP or [3HJDMBA, in vitro.
Hepatic microsomes from dieldrin pretreated and control fish contained equivalent aryl
hydrocarbon hydroxylase (AHH) activities towards both substrates. In addition, the
levels of six cytochrome P450 isozymes, determined by Western blot analysis, were not
altered in microsomes from dieldrin-fed fish. Both of these experiments provided
evidence that microsomal oxidative metabolism in the liver was not being changed by
dieldrin pretreatment. In a separate study, hepatic xenobiotic metabolizing activity was
not altered in rainbow trout pretreated with dieldrin for 12 weeks [8]. No differences in
the following oxidative or conjugative enzyme activities were observed: total
cytochrome P-450 or exthoxyresorufin-O-deethylase (EROD), pentoxyresorufin-Odeethylase (PROD), glutathione S-transferase (GST), or UDP glucuronosyltransferase
(UDPGT). Recent research also demonstrated epoxide hydrolase activities in hepatic
microsomes and cytosol were similar in control and dieldrin-fed rainbow trout [121.
Epoxide hydrolases are potentially active towards dieldrin and PAll epoxide
metabolites.
1.4
Induction of non-enzymatic hepatocellular proteins
Since dieldrin pretreatment did not alter the in vitro activity of various
xenobiotic-metabolizing enzymes, stimulation of biliary excretion is not readily
explained by increased hepatocellular content of proteins that catalyze these reactions.
However, biliary excretion of xenobiotics is complex and involves hepatic processes
other than metabolism [91. At least two events precede hepatic metabolism of
xenobiotics: 1) uptake across the sinusoidal membrane, and 2) intracellular trafficking
to sites of metabolism [91. In addition, there are events that follow metabolism which
include: 1) intracellular trafficking to sites of elimination and 2) excretion into the bile
[9]. Therefore, increased performance of hepatic proteins involved in dieldrin uptake,
intracellular trafficking, or elimination may underlie stimulated biliary excretion in
rainbow trout, following chronic exposure to low levels of dieldrin [8,10,131.
Accumulation of [3H] fl-estradiol and [3H]cholic acid, as well as [14C]dieldrin
and [3H]DMBA, increased in precision cut liver slices from rainbow trout pretreated
with dieldrin for 10-12 weeks, compared to controls [101. This suggested binding
proteins responsible for the uptake of these substances into the cell, or trafficking
proteins that move them through the aqueous environment of the cell to sites of
metabolism, were increased [131. To examine the interaction between dieldrin and the
increased biliary excretion of DMBA observed in earlier studies, hepatic cytosol was
isolated from control and dieldrin-fed trout after 10 weeks [131. Increased hepatic
cytosolic binding of 113H]DMBA occurred in pretreated fish. The increased binding
capacity of hepatic cytosol suggests that trafficking proteins are being stimulated inside
the cell, rather than uptake proteins located on the plasma membrane. There is no
evidence that dieldrin pretreatment is altering the plasma clearance rate, therefore
accumulation of lipophilic compounds are most likely occurring during fatty acid uptake
by the cell, as part of a lipoprotein complex. With an increase in the binding capacity of
hepatic cytosol stimulation of downstream events like metabolism and elimination will
occur.
Induction of hepatic proteins involved in the excretion of xenobiotics into bile
could still potentially explain the increase in elimination [13]. Multidrug resistance
(MDR) proteins, belong to a superfamily of membrane proteins, called ATP-binding
cassette (ABC) transporters that are involved in the transport of endogenous and
exogenous substrates across membranes 114]. These proteins were first discovered
because of their ability to confer resistance to cancer cells when exposed to
chemotherapeutic agents [14]. In the liver, the multidrug resistant (MDR) pglycoproteins (MDR1 and MDR2IMDR3), belonging to the subfamily ABCB, and the
multiple organic anion transporter (MOAT) system in the ABCC subfamily are
particularly important for the excretion of xenobiotics into bile 114,15]. These ATPdependent proteins are located in the canalicular membrane of hepatocytes and pump
high molecular weight compounds and their metabolites from liver to bile [13,15].
Imniunohistochemistry of liver sections, incubated with multidrug resistance protein
antibodies, revealed that the hepatic content of MOAT and MDR proteins were similar
in rainbow trout fed a control or dieldrin diet for 10 weeks [131. However, the sequence
similarity of these proteins are high so the antibodies to one protein will cross react with
another protein [131. Therefore, the antibodies did not allow distinction of subtle
changes among different forms of these proteins [131.
1.5
Research objective
This research examined the effects of dieldnn pretreatment on the disposition of
the polycycic aromatic hydrocarbon, BaP. The study assessed whether an increase in
biliary excretion of DMBA in dieldrin-fed fish also occurs with BaP, a closely related
compound. Earlier in
vitro
work demonstrated that the increase in biliary excretion,
observed following chronic dieldrin exposure, was not explained by induction of the
P450 system, or other hepatocellular proteins involved in metabolism [8,10,12]. Tn this
study, investigation of a substrate that undergoes complex metabolism was used to
characterize the
in vivo
state of the P450 system, UDP-glucuronyltransferases, and
sulfotransferases. Therefore, the biliary polar metabolite profile of [14C]BaP was
compared between control and dieldrin-fed fish.
CHAPTER 2
MATERIALS AND METHODS
2.1
Materials
Dieldrin was supplied by ChemServices (West Chester, PA; 98.8% pure).
Unlabeled BaP was purchased from Sigma Chemical Co. (St. Louis, MO; 97% pure).
Radiolabeled ['4C]BaP was obtained from ChemSyn Laboratories (Lenexa, KS; 51.6
mCilmmol, >98% purity by TLC). The enzymes ,B-glucuronidase (type H-3 from Helix
pomatia) and arylsulfatase (type V from keyhole limpets, Patella vulgata; 1lGlucuronidase activity <2 Sigma units per mg solid) were purchased from Sigma
Chemical Co. (St. Louis, MO). The following standards were obtained from the
National Cancer Institute (NC!) Chemical Carcinogen Reference Standard Repository
(distributed by Midwest Research Institute, Kansas City, MO): 3hydroxybenzo[alpyrene (?98% pure by HPLC), benzo[a]pyrene-3-sulfate potassium salt
(>99% pure by HPLC), and 3-benzo(a)pyrenyl fl-d-glucopyranosiduronic acid (99%
pure by HPLC). NCS II Tissue Solubilizer was supplied by Amersham Corporation
(Arlington Heights, IL). All additional material and reagents were purchased from
Sigma Chemical Co. (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA).
2.2
Treatment and sampling protocol
Animals. Shasta strain rainbow trout (Oncorhynchus mykiss) were provided by the
Marine/Freshwater Biomedical Center core facility at Oregon State University
(Corvallis, OR). Fish were held in continuous-flow (approximately 6 L'min; 13°)
circular tanks (88.9 L; 80 fish/tank) and kept on a 15-h light/9-h dark photoperiod during
experiment 1 and an 11-h Jight/13-h dark photoperiod during experiment 2.
Dieldrin exposure. In the first experiment small fish (-2 g) were fed a growth ration
(4% dry weight diet/dry weight fish) of Oregon Test Diet [16] with or without dieldrin
(15 ppm; 0.324 mg dieldrinlkg/d) for 9 weeks. Dieldrin was then removed from the diet
at this time due to signs of toxicity and all fish were provided a 2% maintenance ration.
All fish were fed control diet for an additional 3 weeks. In the second experiment large
fish (-88 g) were fed a maintenance ration (2% dry weight diet/dry weight fish) of
Oregon Test Diet [16] with or without dieldrin at the same concentration (15 ppm; 0.162
mg dieldrin/kg/d) for 11 weeks.
[14C}BaP exposure. Fish were not fed for 24 h prior to [14C]BaP exposure. Fish were
transferred to polypropylene buckets, containing clean well-water (static conditions) and
submersed charcoal filters at weeks 9 and 12 (9 control and 9 treated) during experiment
1 and at week 11 (4 control and 4 treated) during experiment 2. To help maintain
optimal temperature ice was added as needed in the first experiment, while cold running
water surrounded the buckets during the second experiment. Trout were then injected
(ip) with 10 pmol ['4C]BaP/kg (in 10 mI/kg menhaden oil during experiment 1 and in 2
nil/kg menhaden oil during experiment 2). The large fish were anesthetized with MS222 (100 mgJL) prior to injection. Fish were killed 24 h later with an overdose of MS222 (300 mg/L) and gallbladder/bile, liver, and dissectible visceral fat were removed.
IDI
23
['4C]Benzo[a]pyrene disposition following dieldrin pretreatment
Gallbladder/bile. Gallbladder/bile was placed in amber microcentrifuge tubes or amber
vials without solvent to prevent spontaneous oxidation to quinone metabolites. During
experiment 2, vials were not pre-weighed so 20 ul duplicates were removed and
analyzed for radioactivity by a liquid scintillation counter (LSC) to determine the
number of dpms per ul. The remaining contents were transferred to pre-weighed vials.
Original vials were rinsed with acetone and analyzed for radioactivity by LSC. Using
dpms/ul the volume left behind in the original vial was determined. The total
gallbladder/bile volume was then calculated.
Sub-samples of bile were removed during both experiments and the remainder
was transferred to 20°C for metabolite analysis. Polar and non-polar metabolites in the
sub-samples were separated using the methanollwater-chlorofonn extraction system
described by Bligh and Dyer 117]. The aqueous and chloroform fractions were each
transferred to scintillation vials and evaporated overnight. Cytoscint ESTM was added to
each vial and radioactivity was analyzed by LSC.
Liver and fat. In experiment 1 liver and fat tissues were placed whole into scintillation
vials and digested with NCS II Tissue Solubilizer at 40°C for 48 h. During the second
experiment liver and fat tissues were homogenized. Sub-samples (200 ul duplicates) of
the homogenate were digested with NCS H Tissue Solubilizer at 40°C overnight.
Cytoscint ESTM was added following the digestions and radioactivity analyzed by LSC.
11
2.4
Biiary ['4C]benzo[a]pyrene polar metabolite profile
Biiary extractions. Bile samples frozen for metabolite analysis were extracted using
the method described by Willet et al. [18], with the following modifications.
Gallbladder/bile samples from the small fish were combined with 1 ml of buffer, while
samples taken from the large fish were brought up to -1 ml with buffer [potassium
phosphate buffer with 1.0% (w/v) bovine serum albumin, pH 6.8 @ 37°C]. Parent and
unconjugated oxidized metabolites were extracted with 2 ml ethyl acetate. Vials were
vortexed for 1 minute and transferred to -20°C overnight to freeze the aqueous phase.
The ethyl acetate phase was then transferred to amber vials and returned to -20°C, after
fifty microliter duplicates were removed. The 50 ul duplicates were analyzed for
radioactivity by LSC.
The aqueous phase was then incubated with /J-glucuronidase for 6 h at 37°C
[1000 units (8.4 ul) for small fish and 2000 units (16.8 ul) for large fishi. The cleaved
glucuronide conjugates were extracted with 2 ml ethyl acetate and vials were processed
as described above. The remaining aqueous phase was frozen while the metabolite
profile in the organic fractions was analyzed using reverse-phase high performance
liquid chromatography (ITPLC). Afterwards, the aqueous phase was extracted with 2 ml
ethyl acetate to remove residual parent compound or oxidation products. The vials were
again processed as described above.
The pH of the remaining aqueous phase (buffer) was adjusted from 6.8 to 5
(using HC1) and incubated with 19 units arylsulfatase (dissolved in sodium chloride
immediately before use; negligible fl-glucuronidase activity) for 6 h at 37°C. Cleaved
12
sulfate conjugates were extracted with 2 ml ethyl acetate and vials were processed as
described above. Fifty microliter duplicates were also removed from the aqueous phase
and analyzed by LSC to determine residual polar material (e.g., glutathione conjugates
and sulfate or glucuronide conjugates refractory to cleavage).
The metabolite profile in the organic fraction was analyzed using reverse-phase
I-IIPLC. A blank and standard (40 pmol each of 3-hydroxybenzo[alpyrene and 3benzo(a)pyrenyl fl-d-glucopyranosiduronic acid) were included in each HPLC run and
were processed with the samples through each series of incubations.
High-pressure liquid chromatography (HPLC). The organic fractions, containing
BaP and unconjugated metabolites, were removed from the freezer and transferred to
amber microcentrifuge tubes. The ethyl acetate was evaporated to dryness under a
nitrogen stream and then resuspended in 50 ul methanol [191. Twenty microliters were
injected onto a C-18 reverse-phase HPLC column (Vydac 21 8TP54, Vydac, Hesperia,
CA) and the rest was placed in the freezer for additional analysis if necessary. A twostep gradient was used to separate BaP and its metabolites, as described by Willet et al.
[181 with the following modifications: methanol:water:acetic acid (50:49.5:0.5) to
methanol:water (83.5:16.5) in 30 minutes and then to 100% methanol in 60 minutes.
Final conditions were maintained for an additional five minutes before the gradient was
returned to initial settings. The flow-rate was lowered (0.4 mls/min) for the first two
minutes during sample injection and then held at 1 mI/mn.
Eluent from the column was analyzed by fluorescence spectrophotometry, using
a Shimadzu RF-55 1 fluorescence detector (excitation, 370 nm; and emission, 430 nm
13
[20,2 1]). After passing the fluorescence detector 3-minute HPLC fractions were
collected by a fraction collector (FC 203, Gilson Inc., Middleton, WI). Three hundred
microliter duplicates from each fraction were pipetted into Deep-Well LumaPlateTM
microplates that contained solid scintillator [22]. The plates were evaporated under the
hood, sealed, and placed in the TopCount® microplate scintillation and luminescence
counter (Packard Instrument Company, Meriden, CT) [22]. Radioactivity provided a
more sensitive method of detection and was used for data analysis.
2.5
Statistical analysis
Statgraphics Plus 5.0 was used for all statistical analyses. Two-way analysis of
variance compared multiple means and determined significant time or treatment effects.
A two-sample comparison, or t-test, was used when comparing two means for significant
treatment effects. Significance was determined using a 95.0% confidence level
(p<O.O5). If assumptions of either test were violated and transformation of the data did
not correct the problem then non-parametric methods (e.g., Kruskal-Wallis test) were
used to compare medians rather than means.
14
CHAPTER 3
RESULTS
3.1
Animals
In the first experiment (initial fish weight -2 g) dieldrin-fed fish exhibited signs
of toxicity between 6 and 9 weeks of treatment. Although the percent cumulative
mortality was fairly low (2.1% in controls and 3.8% in dieldrin-fed fish over 12 weeks),
the body weight of dieldrin-fed fish was 75% of control fish at week 9 (figure 3.1A).
Therefore, dieldrin was removed from the diet and fish were switched from a 4% growth
ration to a 2% maintenance ration after 9 weeks. All fish received control diet for an
additional 3 weeks. At week 12, there was not a significant difference in body weight
between control and dieldrin-fed fish (figure 3.1A). The estimated daily dose of dieldrin
up to 9 weeks was 0.324 mg dieldrinlkg body weight (15 ppm).
hi the second experiment (initial fish weight -88 g) no mortality occurred in
control or dieldrin-fed fish (2% maintenance ration) and no significant difference in
body weight was observed at week 11 (figure 3.1B). The estimated daily dose of
dieldrin for 11 weeks was 0.162 mg dieldrinlkg body weight (15 ppm).
3.2
[14C]Benzo[a]pyrene disposition following dieldrin pretreatment
In the first experiment, two-way analysis of variance revealed no evidence of a
significant interaction between time and treatment effects on the tissue concentrations of
[14C]BaP in liver or fat. The concentration of [14C]BaP in pretreated fish was lower in
15
-0-- Control
e Dieldrin
16
'' 14
E
12
'.-' 10
6
3
12
9
Week
0 Control
Dieldrin
250.00
:1::
100.00
50.00
Control
Dieldrin
Treatment
Figure 3.1. Weight gain in rainbow trout (initial wt 2 g) following dieldrin
exposure for 9 wks (A) and body weight (initial wt 88 g) following dieldrin
exposure for 11 wks (B). The body weight of dieldrin-fed fish was 75% of control
fish at wk 9 in the first experiment. Therefore, dieldrin was removed from the diet and
fish were switched from a 4% growth ration to a 2% maintenance ration. All fish
received control diet for an additional 3 wks. Values are means ± SE. (Experiment 1
wk 3 and 6: n=9; wk 9: n=18; wk 12: n=33. At wk 12 the SE is based on a sample size
of 9. Individual weights of 24 fish were not available because their weights were
pooled. Experiment 2 n=4).
16
liver (figure 3.2A) and fat (figure 3.2B) compared to controls, however there was no
significant treatment effect. There was a significant difference in the mean
concentrations of [14CJBaP between week 9 and 12, averaged over treatment. After 12
weeks there was a significantly higher liver concentration (nmols/g) of [14C]BaP, in
both control and dieldrin-fed fish, than after 9 weeks (figure 3.2A). However, the
concentration of ['4C]BaP in visceral fat was significantly higher in control and dieldrin-
fed fish at 9 weeks compared to 12 weeks (figure 3.2B).
There was no evidence of a significant interaction between time and treatment
effects on the tissue concentration or total biliary excretion of [14C]BaP in bile.
However, the main effect, treatment, was significant. The mean concentration of
[14C]BaP in bile, averaged over time, was significantly different between control and
pretreated fish (1 outlier removed from 12 week control). Dieldrin-fed fish had a
significantly higher concentration (nmols/g) of ['4CIBaP in bile compared to control fish
(figure 3.3A). Polar and nonpolar metabolites were then extracted with methanol/waterchloroform. Stimulation of total biliary excretion (total nmols) was more pronounced
compared to the concentration of ['4C]BaP in bile (figure 3.3A and 3.3B). Residuals
from the polar metabolite data were not normally distributed; therefore the response was
transformed (log 10). On average, polar metabolites of [14CIBaP in dieldrin-fed fish
were significantly elevated (2.4-fold higher at 9 weeks and 2.2-fold higher at 12 weeks)
compared to control fish (figure 3 .3B) regardless of data transformation. No difference
was observed in nonpolar metabolites between control and pretreated trout (figure 3.3B).
17
0 Control
Dieldrin
16
.
8
9
12
Week
30
R (1't
25
2O
.
15
10
9
12
Week
Figure 3.2. Tissue concentrations of [14C]benzo[a]pyrene in liver (A) and fat (B)
following dieldrin pretreatment for 9 wks. Rainbow trout (initial wt -2 g) were fed
control or dieldrin (0.324 mg dieldrin/kg/d) diets for 9 wks. Dieldrin was removed from
the diet at this time due to overt toxicity. At wk 9 and 12 trout were injected (ip) with
10 pmol [14C]BaP/kg. Fish were sacrificed 24 h later and gallbladder/bile, liver, and
visceral fat were removed for analysis. Values are means ± SE (9 wks: control n=8,
treated n=5; 12 wks: control n=9, treated n=8). At wk 9, 1 control fish died (reason
unknown) and 4 treated fish were lost due to temperature control. At wk 12, 1 treated
fish was injected twice and did not recover. fSignificant time effect: significantly
different from wk 9 or wk 12, p< 0.05.
II1
0 Control
70
Dieldrin
A (D,\
E 60
50
0
30
20
10
9
12
Week
3
E
Polar
Nonpolar
Polar
12
Nonpolar
Week
Figure 3.3. Concentration of [14C]benzo[a]pyrene in bile (A) and total biliary
excretion (B) following dieldrin pretreatment for 9 wks. Rainbow trout (initial wt -2
g) were fed control or dieldrin (0.324 mg dieldrinlkgld) diets for 9 wks. Dieldnn was
removed from the diet at this time due to overt toxicity. At wk 9 and 12 trout were
injected (ip) with 10 pmol [14C] BaP/kg. Fish were sacrificed 24 h later and
gallbladder/bile, liver, and visceral fat were removed for analysis. Values are means ±
SE (9 wks: control n=5, treated n=3; 12 wks: control n=9, treated n=8). Wk 9, 1 control
died: reason unknown (3 inadequate bile volume); 4 treated lost due to temperature
control (2 inadequate bile volume). At wk 12, 1 treated fish was injected twice and did
not recover. *Sigrlificant treatment effect: significantly different from control, p<O.O5.
19
In experiment 2, the concentration of [14C]BaP in dieldrin-fed fish was 2.3-fold
higher in liver (figure 3.4A) and 2.7-fold higher in fat (figure 3.4B). However, the
concentrations were not significantly different from control fish (determined using a
two-sample comparison, or t-test).
The concentration (nmols/g) of [14CIIBaP in bile of dieldrin-fed fish was elevated
compared to controls (figure 3.5A), however, the difference was not significant (p-value,
0.0584). Although there was marginal evidence of a treatment effect a larger sample
size may have allowed us to detect a significant effect at the 95% confidence level.
Dieldrin pretreatment stimulated total biliary excretion (total nmols). Polar and nonpolar
metabolites were extracted with methanol/water-chloroform. Polar metabolites of
[14C]BaP in dieldrin-fed fish were significantly higher on average (2.0-fold higher)
compared to control fish (figure 3.5B). Two-sample comparison revealed a significant
difference between the means of control and dieldrin-fed fish; however, the test was
violated because of a significant difference between the standard deviations. Using a
non-parametric method to compare the medians rather than the means revealed that the
difference was still significant. No significant difference was observed in nonpolar
metabolites between control and pretreated trout (figure 3 .5B). Residuals were not from
a normal distribution so the response was transformed (10gb).
3.3
Biiary [14C]benzo[a]pyrene polar metabolite proffle
After metabolites were separated, HPLC fractions were collected and analyzed
for radioactivity using the TopCount® microplate scintillation and luminescence
20
0 Control
200.00
A
Dieldrin
IT
E
150.00
I
Control
Dieldrin
Treatment
200.00
100.00
50.00
0.00
Control
Dieldrin
Treatment
Figure 3.4. Tissue concentrations of [14C]benzo[a]pyrene in liver (A) and fat (B)
following dieldrin pretreatment for 11 wks. Rainbow trout (initial wt -88 g) were fed
control or dieldrin (0.162 m dieldrin/kg/d) diets for 11 wks. At wk 11 trout were
injected (ip) with 10 pmol 4C]BaP/kg. Fish were sacrificed 24 h later and
gallbladder/bile, liver, and visceral fat were removed for analysis. Values are means ±
SE (control n=3, treated n=4). One control fish died (airline knocked off).
II
21
0 Control 0 Dieldrin
A /TI1\
100.00
E
60.00
.
40.00
20.00
0.00
Control
Dieldrin
Treatment
30
B (Total Biiary Excretion)
25
'20
15
E
10
E5
Polar
Treatment
Nonpolar
Figure 3.5. Concentration of [14C]benzo[a]pyrene in bile (A) and total biliary
excretion (B) following dieldrin pretreatment for 11 wks. Rainbow trout (initial wt
-88 g) were fed control or dieldrin (0.162 mg dieldrin/kg/d) diets for 11 wks. At wk 11
trout were injected (ip) with 10 pmol [14CIBaP/kg. Fish were sacrificed 24 h later and
gallbladder/bile, liver, and visceral fat were removed for analysis. Values are means ±
SE (n=3). One control fish died (airline knocked off) and one treated fish had
inadequate bile volume. *significantly different from control, p< 0.05.
22
counter. This enabled us to look at peak intensity and observe the metabolite profile in
the first and second experiment for the parent and unconjugated oxidized metabolites
pulled out in the first extraction (figure 3.6 and 3.9, respectively), the cleaved
glucuronide conjugates following the ,B-glucuronidase reaction (figure 3.7 and 3.10,
respectively), and the cleaved sulfate conjugates following the arylsulfatase reaction
(figure 3.8 and 3.11, respectively). Please see appendix A (first experiment) and
appendix B (second experiment) for the mean cpms per fraction ± SE for each
chromatogram.
Induction of a novel biotransformation pathway did not occur in dieldrin-fed fish
(e.g., a unique metabolite was not created). In addition, there was no evidence that an
enzyme expressed at a low level constitutively was being induced (e.g., elevated levels
of a specific metabolite in pretreated fish were not observed). In both experiments, there
was no indication of a selective enhancement of any particular peak with dieldrin
pretreatment. However, in the first experiment there was a general increase in many of
the biliary metabolite fractions from dieldrin-fed fish, which suggests that a particular
P450 was not altered (figure 3.6, 3.7, and 3.8). In both experiments the biliary
metabolite profile of ['4C]BaP, did not differ significantly between control and dieldrinfed fish.
3.4
Polar metabolites of [14C]benzo[a]pyrene excreted into bile
Only a small percentage of ['4C]BaP would not be metabolized and excreted
unchanged into the bile. For that reason, parent ['4C]BaP on HPLC chromatograms was
23
Figure 3.6. [14C}Benzo[a]pyrene parent and unconjugated oxidized metabolite
profile in gallbladder/bile at week 9 (A) and week 12 (B), following dieldrin
pretreatment in rainbow trout (initial wt -2 g) for 9 wks. Bile was extracted with
ethyl acetate and run on reverse-phase HPLC to separate the metabolites. I-IPLC
fractions were collected at 3-minute intervals and 300 ul duplicates of each fraction were
placed in the TopCount system to analyze radioactivity. Values are mean cpms per
fraction (see appendix 1 for the mean cpms per fraction ± SE for each chromatogram).
Of the total radioactivity in the major peaks the percentage of parent ['4CIBaP (-63
minutes) was 64% (control) and 77% (treated) at 9 wks (A) and 75% (control) and 66%
(treated) at 12 wks (B).
eD
-.
45-48
''
:
''
51-54
48-51
45-48
42-45
36-39
39-42
21-24
24-27
27-30
30-33
33-36
0-3
3-6
6-9
9-12
12-15
15-18
18-21
78-81
81-84
84-87
87-90
78-81
81-84
84-87
87-90
72-75
75-78
72-75
69-72
66-69
69-72
75-78
63-66
66-69
57-60
60-63
57-60
60-63
54-57
C
C" C
54-57
51-54
48-51
C
C
C
63-66
''
''
27-30
30-33
33-36
36-39
39-42
42-45
21-24
24-27
12-15
15-18
18-21
912
0-3
3-6
6-9
C
Average cpmslfraction
-
Average cpms/fraction
-
eD
-.
0
.
25
Figure 3.7. [14C]Benzo[a]pyrene metabolite profile of cleaved glucuronide
conjugates in gallbladder/bile at week 9 (A) and week 12 (B), following dieldrin
pretreatment in rainbow trout (initial wt -2 g) for 9 wks. Metabolites remaining in
the aqueous phase, following the lS extraction with ethyl acetate, were hydrolyzed with
fl-glucuronidase to regenerate phase-i metabolites prior to their conjugation. Cleaved
glucuronide conjugates were extracted with ethyl acetate and run on reverse-phase
HPLC to separate the metabolites. HPLC fractions were collected at 3-minute intervals
and 300 ul duplicates of each fraction were placed in the TopCount system to analyze
radioactivity. Values are mean cpms per fraction (see appendix 1 for the mean cpms per
fraction ± SE for each chromatogram). Of the total radioactivity in the major peaks the
parent ['4C]BaP (-63 minutes) not removed in the initial extraction was 19% (control
and treated) at 9 wks (A) and 15% (control) and 11% (treated) at 12 wks (B). The 3hydroxy peak (-46 minutes) was confirmed using a standard.
4548
87-90
84-87
8 1-84
78-8 1
63-66
66-69
69-72
72-75
75-78
60-6 3
54-57
57-60
5 1-54
a 48-51
E
:
24-27
27-30
30-33
33-36
36-39
39-42
42-45
2 1-24
12-15
15-18
18-21
9-12
0-3
3-6
6-9
4
C
L1
Co
99
Average Cpms/fraction
U
C
(J
C
C
C
V
63-66
60-63 &
57-60
51-54
54-57
87-90
84-87
8 1-84
78-8 1
75-78
72-75
69-72
66-69
''
21-24
24-27
27-30
30-33
33-36
36-39
39-42
42-45
45-48
48-51
12-15
15-18
18-21
9-12
0-3
3-6
6-9
C
C
C
C
9
C
9
Average cpms/fraction
eD
C
UI
1
27
Figure 3.8. ['4C]Benzo[a]pyrene metabolite profile of cleaved sulfate conjugates in
gallbladder/bile at week 9 (A) and week 12 (B), following dieldrin pretreatment in
rainbow trout (initial wt 2 g) for 9 wks. Metabolites remaining in the aqueous phase,
following the 2' extraction with ethyl acetate, were hydrolyzed with arylsulfatase to
regenerate phase-i metabolites prior to their conjugation. Cleaved sulfate conjugates
were extracted with ethyl acetate and run on reverse-phase HPLC to separate the
metabolites. HPLC fractions were collected at 3-minute intervals and 300 ul duplicates
of each fraction were placed in the TopCount system to analyze radioactivity. Values
are mean cpms per fraction (see appendix 1 for the mean cpms per fraction ± SE for each
chromatogram).
oc
1
,
57-60
75-78
78-81
81-84
84-87
87-90
75-78
78-8 1
8 1-84
84-87
87-90
69-72
72-75
69-72
63-66
66-69
63-66
60-63
57-60
60-63
"'
51-54
54-57
5 1-54
54-57
45-48
48-51
66-69
'
21-24
24-27
27-30
30-33
33-36
36-39
39-42
42-45
72-75
D
-.
21-24
24-27
27-30
30-33
33-36
36-39
39-42
42-45
45-48
15-18
18-21
18-2 1
9-12
12-15
0-3
3-6
6-9
9-12
12-15
15-18
0-3
3-6
6-9
P
Average cpms/fraction
-
©
Ll
LS
4
Average cpms/fraction
4
C
C
0
-.
-
-.
Dieldrin 0 Control
1200
1000
800-
600
400
200
Figure 3.9. [14C]Benzo[a]pyrene parent and unconjugated oxidized metabolite
profile in gallbladder/bile, following dieldrin pretreatment in rainbow trout (initial
wt 88 g) for 11 wks. Bile was extracted with ethyl acetate and run on reverse-phase
HPLC to separate the metabolites. HPLC fractions were collected at 3-minute intervals
and 300 ul duplicates of each fraction were placed in the TopCount system to analyze
radioactivity. Values are mean cpms per fraction (see appendix 2 for the mean cpms per
fraction ± SE for each chromatogram). Of the total radioactivity in the major peaks the
percentage of parent II14CIBaP (-63 minutes) was 80% (control) and 76% (treated).
30
Dieldrin 0 Control
LIII]
600500
400
300
200
100
Figure 3.10. ['4C]Benzo[a]pyrene metabolite profile of cleaved glucuronide
conjugates in gallbladder/bile, following dieldrin pretreatment in rainbow trout
(initial wt -88 g) for 11 wks. Metabolites remaining in the aqueous phase, following
the extraction with ethyl acetate, were hydrolyzed with fl-glucuronidase to regenerate
phase-i metabolites prior to their conjugation. Cleaved glucuronide conjugates were
extracted with ethyl acetate and run on reverse-phase HPLC to separate the metabolites.
HPLC fractions were collected at 3-minute intervals and 300 ul duplicates of each
fraction were placed in the TopCount system to analyze radioactivity. Values are mean
cpms per fraction (see appendix 2 for the mean cpms per fraction ± SE for each
chromatogram). Of the total radioactivity in the major peaks the parent [14CIBaP (-63
minutes) not removed in the initial extraction was 15% (control) and 13% (treated). The
3-hydroxy peak (-46 minutes) was confirmed using a standard.
31
Dieldrin 0 Control
120
Figure 3.11. ['4C]Benzo[a]pyrene metabolite profile of cleaved sulfate conjugates
in gallbladder/bile, following dieldrin pretreatment in rainbow trout (initial wt -88
g) for 11 wks. Metabolites remaining in the aqueous phase, following the 2nd extraction
with ethyl acetate, were hydrolyzed with arylsulfatase to regenerate phase-i metabolites
prior to their conjugation. Cleaved sulfate conjugates were extracted with ethyl acetate
and run on reverse-phase HPLC to separate the metabolites. HPLC fractions were
collected at 3-minute intervals and 300 ul duplicates of each fraction were placed in the
TopCount system to analyze radioactivity. Values are mean cpms per fraction (see
appendix 2 for the mean cpms per fraction ± SE for each chromatogram).
32
assumed to be contamination on the outside of the gallbladder, as a result of the IF
injection (e.g., BaP that was not processed by the liver and excreted into bile).
Therefore, to provide an estimate of the biliary systems performance (e.g., the amount
transported from liver to bile) parent compound was subtracted from the total recovered.
This yielded the total amount (nmols) of II'4C]BaP polar metabolites excreted into bile
(figure 3.12 for experiment 1 and figure 3.13 for experiment 2).
Fifty microliter duplicates of each organic fraction (containing parent BaP and
phase 1 metabolites, cleaved glucuronide conjugates, or cleaved sulfate conjugates) and
the remaining aqueous phase were analyzed for radioactivity by LSC. This determined
the total nmols present prior to HPLC separation. TopCount analysis of the HPLC
effluent allowed us to determine what percentage of the total radioactivity in the major
peaks was parent ['4CIBaP. The total number of nmols loaded onto HPLC was
multiplied by that percentage to calculate the nmols of parent [14C]BaP. Parent
compound was then subtracted from the total to obtain the amount of polar metabolites
present in each fraction.
In the first experiment two-way analysis of variance revealed no evidence of a
significant interaction between time and treatment effects on the level of polar
metabolites excreted into bile (figure 3.12 A, B, C, D). At week 12, the amount in
dieldrin-fed fish was elevated for unconjugated oxidized metabolites (figure 3.1 2A),
cleaved glucuronide conjugates (figure 3.1 2B), cleaved sulfate conjugates (figure
3.12C), and residual polar material (figure 3.12D). However, the levels in dieldrin-fed
fish were not significantly different from controls.
33
Figure 3.12. Polar metabolites of [14C]benzo[a]pyrene excreted into bile, following
dieldrin pretreatment in rainbow trout (initial weight -2 g) for 9 weeks: phase 1
metabolites (A), cleaved glucuronide conjugates (B), cleaved sulfate conjugates (C),
and residual polar material (D). Values are means ± SE (9 wks: control n=3, treated
n=4; 12 wks: control and treated n=3). At wk 9, 1 control fish died for unknown reasons
(4 had inadequate bile volume, 1 not used in this analysis) and 4 treated fish were lost
due to temperature control (1 not used in this analysis). At wk 12, 5 control fish had
inadequate bile volume (1 not used in this analysis) and 1 treated fish was injected twice
and did not recover (4 had inadequate bile volume, 1 not used in analysis). There were
no significant time or treatment effects.
-.
eD
©
0
O\
00
t)
o
4
-
b,
bo
o
bo
nmols residual polar material
.
0 0 © 0
p'.)
b o bopo P
nmols cleaved sulfate conjugates
-I
P
©
P
© -
i'_)
00
LQ
© 00000000
b
0
Lu
©
nmols cleaved glucuronide conjugates
©
nmols phase 1 metabolites
'-1
'
U
35
El
2
Control
Dieldrin
A
10
1.8
1.6
1.4
V
1.2
1
0.6
0.4
0.2
E
0
r
Control
Dieldrin
Control
Dieldrin
16
1.8
14
10
0.8
0.6
V
0.4
: 0.2
E
[]
Control
Dieldrin
Control
Dieldrin
Figure 3.13. Polar metabolites of ['4C]benzo[a]pyrene excreted into bile, following
dieldrin pretreatment in rainbow trout (initial wt -88 g) for 11 wks: phase 1
metabolites (A), cleaved glucuronide conjugates (B), cleaved sulfate conjugates (C),
and residual polar material (D). Values are means ± SE (control and treated n=3).
One control fish died (airline knocked off) and 1 treated fish had inadequate bile volume.
36
In the second experiment, the level of polar metabolites were lower in dieldrin-fed fish,
but were not significantly different from control fish (figure 3.13 A, B, C, D).
3.5
Percentage of polar metabolites excreted into bile
The nmols in each fraction were divided by the total nmols recovered in bile for
each fish. This yielded the percent radioactivity in each fraction containing phase 1
metabolites, cleaved glucuronide conjugates, cleaved sulfate conjugates, and residual
polar material (table 3.1). In the first experiment, statistical analysis revealed no
significant time or treatment effects. Therefore, the averages of control and treated fish
at week 9 and 12 were pooled into one average. In the second experiment, no significant
treatment effects were identified so the averages of control and treated fish were also
pooled into one average.
37
Table 3.1. Percentage of ['4C]benzo[a]pyrene polar metabolites excreted into bile
following dieldrin pretreatment in rainbow trout.
Phase 1
Cleaved
Metabolites Glucuronide
Conjugates
Cleaved
Sulfate
Conjugates
Residual Polar
Material
*Parent
Exp. 1
6% ± .92
24% ± .93
2% ± .31
43% ± 1.3
25% ± 1.4
Exp. 2
5% ± .27
23% ± .05
4% ± .18
42% ± 4.8
26% ± 4.4
Experiment 1: Rainbow trout (initial wt -2 g) were pretreated for 9 wks. Statistical
analysis revealed no significant time or treatment effects. Therefore, the averages of
control and treated fish at week 9 and 12 were pooled into one average. The early peak
on all chromatograms was 7.36% ± .25 of the total radioactivity.
Experiment 2: Rainbow trout (initial wt -88 g) were pretreated for 11 wks. Statistical
analysis revealed no significant treatment effects so the averages of control and treated
fish were pooled into one average. The early peak on all chromatograms was 6.8 1% ±
.50 of the total radioactivity.
*To provide an estimate of the biliary systems performance parent compound was
subtracted from the total recovered to determine the total amount (nmols) of [1 4cj BaP
polar metabolites excreted into bile.
CHAPTER 4
SUMMARY AND CONCLUSION
Feeding rainbow trout 0.3-0.4 mg dieldrin/kgld for 9-12 weeks stimulated the
biliary excretion of a subsequent dose of ['4C]dieldrin by 500% and [3H]7,12-
dimethylbenz[a]anthracene (DMBA) by 240% [8,9]. The same exposure significantly
increased the disposition of ['4C]dieldrin to liver (200%) and mesenteric fat (500% at 10
weeks, 1200% at 12 weeks) and elevated the levels of [3H]DMBA in liver (166%) [8,9].
These results demonstrated that altered disposition, following dieldrin pretreatment, is
not specific to a subsequent dose of dieldrin. Increased biliary excretion of a xenobiotic
is often interpreted as direct stimulation of metabolism. However, in
vitro
work
demonstrated that this interaction occurred without induction of the cytochrome P450
system, or other hepatocellular proteins involved in metabolism [8,10,12]. The present
research examined the effects of dieldrin pretreatment on the disposition of the
polycyclic aromatic hydrocarbon, benzo[a]pyrene (BaP). The study assessed whether
increased biliary excretion of DMBA in dieldrin-fed fish also occurred with BaP, a
closely related compound. This substrate, which undergoes complex metabolism,
characterized the in
vivo
state of the cytochrome P450 system, UDP-
glucuronyltransferases, and sulfotransferases.
Rainbow trout were fed control or dieldrin diets (0.324 mg dieldrinlkg/d) for 9
weeks, followed by control diet for an additional 3 weeks. At week 9 and 12 trout
received an ip challenge dose of [14C]BaP (10 pmols/kg). In a subsequent study, trout
were fed control or dieldrin diets (0.162 mg dieldrin/kg/d) for 11 weeks, also followed
39
by an ip challenge dose of ['4C]BaP (10 pmolsfkg). Gallbladder/bile, liver, and visceral
fat were removed 24 h later and analyzed for radioactivity. Dieldrin pretreatment altered
[14C]BaP disposition, which is consistent with earlier work using DMBA. hi experiment
1, dieldrin pretreatment significantly elevated the concentration of [14C]BaP in bile
(142% and 200% at 9 and 12 weeks, respectively) but not liver or fat. In experiment 2,
the concentration of [14CJBaP was elevated in bile (223%), liver (232%), and fat (268%),
however, the difference was not significant relative to controls. Extraction of bile subsamples with methanol/water-chloroform confirmed dieldrin pretreatment significantly
stimulated total biliary excretion of ['4CIBaP polar metabolites (244% and 221% at
week 9 and 12, respectively in experiment 1; 197% in experiment 2).
In the first experiment, the liver concentration of [14CIIBaP was significantly
higher after 12 weeks in both control and dieldrin-fed fish, than after 9 weeks. However,
the concentration of [14CJBaP in visceral fat was significantly higher in control and
dieldrin-fed fish at 9 weeks, compared to 12 weeks. The difference in these two tissues,
between week 9 and 12, may be due to the feeding ration, which in turn influences
[14CIBaP tissue distribution. Triacyiglycerols (or fat) leaving the liver are packaged into
very low-density lipoproteins (VLDLs) for transport through the circulation to various
tissues including muscle, heart, and adipose tissue [23]. The triacylglycerols are then
stored for future use or utilized to generate energy via oxidation [23]. The liver is the
main regulator of lipid homeostasis and therefore modulates tissue distribution of
[14C]BaP, which is incorporated into these lipoproteins. During the first 9 weeks when
fish received a growth ration energy was stored as fat. The retention of [14C]BaP by
liver decreased while more was distributed to adipose tissue. After 9 weeks, dieldrin
was removed from the diet due to signs of toxicity. All fish were provided a
maintenance ration and fed control diet for an additional 3 weeks. As a result of the
lower food ration, synthesis of fat decreased by week 12 because more energy was being
stored for the production of lean muscle mass. Therefore, the amount of VLDLs
exported by the liver to transport fat to adipose tissue also decreased. As a result,
accumulation of II14CIBaP by liver increased, while distribution to fat decreased.
Consistent with previous research, the present study demonstrates the ability of
rainbow trout to adapt to chronic dieldrin exposure [7-91. Bioaccumulation of dieldrin,
or another lipophilic chemical, decreases apparently by increasing elimination of the
xenobiotic via biliary excretion [7-9]. Although there is a marked difference in the
ability of the liver to excrete dieldrin or DMBA into bile, following dieldrin
pretreatment, there is comprehensive data providing evidence that increased hepatic
content of drug-metabolizing enzymes do not explain this response 18,10,121. Although
the levels of six cytochrome P450 isozymes, determined by Western blot analysis, were
not altered in microsomes from dieldnn-fed fish induction of a metabolite in the in vivo
work may suggest alteration of a particular P450, for which there was no antibody [101.
h addition, there was no difference in the amount of ['4C]BaP or [3H]DMBA turned
over by hepatic microsomes from control and dieldrin-fed fish [10]. However, the
metabolite profile was not examined leaving the possibility open that different
metabolites were produced by microsomes from dieldrin-fed fish. Therefore, the
41
metabolism of BaP was examined in control and dieldrin-fed fish to determine if
pretreatment altered the biliary polar metabolite profile.
Bile was extracted to isolate parent compound and unconjugated oxidized (or
free) metabolites. To provide an accurate representation of xenobiotic biotransformation
by the P450 system, bile was hydrolyzed by /3-glucuronidase and arylsulfatase to
regenerate BaP metabolites, conjugated by phase II enzymes (glucuronyl transferase and
sulfotransferase). Material extracted by organic solvent was separated by reverse-phase
HPLC and fractions were analyzed for radioactivity. Evaluation of biliary polar
metabolite profiles of [14C]BaP revealed no significant differences between control and
dieldrin-fed fish. There was no indication of selective enhancement of any particular
peak or induction of a novel biotransformation pathway with dieldrin pretreatment.
General increases in many of the biliary metabolite fractions from dieldrin-fed fish were
observed, suggesting that a particular P450 was not altered. Of the total radioactivity in
bile, the majority was detected as cleaved glucuronide conjugates (23-24%) while only a
small proportion of free metabolites (5-6%) and cleaved sulfate conjugates (2-4%) were
recovered. Recovery of parent compound (25-26%) was assumed to be contamination
on the outside of the gallbladder, as a result of the IP injection.
In agreement with other studies, the present results found that glucuronide
conjugates predominate, while only a small proportion of sulfate conjugates are
recovered in bile [18-21]. There may be several reasons why detection of sulfate
conjugates in bile is low. Unlike sulfate conjugates, xenobiotics conjugated with
glucuronic acid are eliminated, via the urine and bile, because of their increased water
42
solubility and recognition by organic anion transport systems [24]. Ji addition, sulfate
conjugates that are excreted into bile may be hydrolyzed by intestinal microflora,
making the xenobiotic sufficiently lipophilic for re-absorption (e.g., enterohepatic
circulation) 11241. Another potential explanation is directly related to sulfation reactions,
catalyzed by sulfotransferases, which occur during phase II biotransformation. These
enzymes transfer the sulfate group (S03) from the cofactor 3' -phosphoadenosine 5'phosphosulfate (PAPs) to hydroxyl groups on PAHs, introduced during phase 1
biotransformation [24]. The sulfate, however, is a good leaving group, which leads to
the formation of a reactive, electrophilic carbocation species [241. Therefore, the
instability of sulfate conjugates could be a significant factor contributing to the small
percentage recovered in bile.
Approximately 42-43% of the total radioactivity remained in the final aqueous
phase. This residual polar material contained water-soluble conjugates, including
glutathione and glucuronide or sulfate conjugates not hydrolyzed during the /3-
glucuronidase or arylsulfatase reactions. The following standards, 3hydroxybenzo[al pyrene, 3 -benzo(a)pyrenyl 113-d-glucopyranosiduronic acid, and
benzo[a]pyrene-3-sulfate potassium salt were chosen based on earlier studies that
demonstrated 3-hydroxy is one of the primary BaP metabolites formed, which is
consistent with our results 11 18-20]. The efficiency of hydrolysis for the glucuronide
conjugate of 3-hydroxy was 91% ± 4.7, however, the sulfate conjugate of 3-hydroxy was
resistant to hydrolysis.
43
Polar metabolites of [14C]BaP (unconjugated oxidized metabolites, cleaved
glucuronide conjugates, cleaved sulfate conjugates, or residual polar material) excreted
into bile were not significantly increased in dieldrin-fed fish. At week 12 in experiment
1, all poiar metabolites were elevated in dieldrin-fed fish. However, this response was
not apparent at week 9 in experiment 1, nor was it apparent in experiment 2. Oxidation
products created during storage and sample processing may explain why this data does
not show enhanced biliary excretion of [14C]BaP polar metabolites. Analysis of bile
sub-samples removed immediately following each experiment most likely provides a
better estimate of total biliary excretion, rather than reconstruction of data following the
metabolite analysis. Bile was not stored and no opportunity for oxidation was provided.
In conclusion, chronic dieldrin exposure stimulates biliary excretion in rainbow
trout given subsequent doses of [14C]dieldrin, [3H]DMBA, and [14C]BaP. This study
confirmed that the interaction is not explained by induction of xenobiotic metabolizing
enzymes. Therefore, the mechanism by which dieldrin pretreatment enhances biliary
excretion of lipophilic compounds requires further examination. Biliary excretion of
xenobiotics is complex and involves hepatic processes other than metabolism [9]. For
example, induction of hepatic proteins involved in uptake across the plasma membrane,
intracellular trafficking to sites of metabolism and elimination, or excretion into bile may
be responsible [8-10,13]. The current hypothesis is that chronic dieldrin exposure is
altering hepatic proteins involved in intracellular trafficking [8-10,13]. Therefore,
research is focused on isolating binding proteins for BaP and DMBA.
BIBLIOGRAPHY
U.S. Environmental Protection Agency. Cancellation of Registration Under the
FIFRA of Products Containing Aldrin or Dieldrin, PR Notice 71-4. Washington:
Government Printing Office, 1971.
2.
U.S. Environmental Protection Agency. Office of Pesticide Programs.
Suspended, Cancelled, and Restricted Use Pesticides, EPA-20-T-1002.
Washington: Government Printing Office, 1990.
3.
U.S. Department of the Interior. U.S. Geological Survey. The Quality of Our
Nation's Waters--Nutrients and Pesticides, US Geological Survey Circular 1225.
Washington: Government Printing Office, 1999.
4.
U.S. Environmental Protection Agency. Office of Pollution, Prevention, and
Toxics. 2000 PBT Program Accomplishments, EPA-742-R-0 1-003. Washington:
Government Printing Office, 2001.
5.
McElroy, A. E., Farrington, J. W., and Teal, J. M. 1989. Bioavailability of
polycycic aromatic hydrocarbons in the aquatic environment. In Metabolism of
polycyclic aromatic hydrocarbons in the aquatic environment, ed. U. Varanasi,
pp. 1-39. Boca Raton, Florida: CRC Press, Inc.
6.
Cerniglia, C. E. and Heitkamp, M. A. 1989. Microbial degradation of polycyclic
aromatic hydrocarbons (PAH) in the aquatic environment. In Metabolism of
polycyclic aromatic hydrocarbons in the aquatic environment, ed. U. Varanasi,
pp. 41-68. Boca Raton, Florida: CRC Press, Inc.
7.
Shubat, P. J. and Curtis, L. R. 1986. Ration and toxicant pre-exposure influence
dieldrin accumulation by rainbow trout. Environ. Toxicol. Chem. 5:69-77.
8.
Gilroy, D. J., Carpenter, H. M., Siddens, L. K., and Curtis, L. R. 1993. Chronic
dieldrin exposure increases hepatic disposition and biliary excretion of
[14C]dieldrin in rainbow trout. Fundam. Appl. Toxicol. 20:295-301.
45
9.
Donohoe, R. M., Thang, Q., Siddens, L. K., Carpenter, H. M., Hendricks, J. D.,
and Curtis, L. R. 1998. Modulation of 7,12-dimethylbenz[a]anthracene
disposition and hepatocarcinogenesis by dieldrin and chiordecone in rainbow
trout. J. Toxicol. Environ. Health 54:227-242.
10.
Gilroy, D. J., Miranda, C. L., Siddens, L. K., Zhang, Q., Buhier, D. R., and
Curtis, L. R. 1996. Dieldrin pretreatment alters 1'4Clldieldrin and [3H17,12dimethylbenz[allanthracene uptake in rainbow trout liver slices. Fundam. Appl.
Toxicol. 30:187-193.
11.
Vodicnik, M. J., Elcombe, C. R., and Lech, J. J. 1981. The effects of various
types of inducing agents on hepatic microsomal monooxygenase activity in
rainbow trout. Toxicol. Appl. Pharmacol. 59:364-374.
12.
Rosemond, M. Dieldrin pretreatment does not stimulate hepatic microsomal or
cytosolic epoxide hydrolase activities in rainbow trout (Oncorhyncus mykiss).
Master's thesis, Oregon State University, 2002.
13.
Curtis, L. R., Hemmer, M. J., and Courtney, L. A. 2000. Dieldrin induces
cytosolic [3H17, 1 2-dimethylbenz[a] anthracene binding but not multidrug
resistance proteins in rainbow trout liver. J. Toxicol. Environ. Health 60:275-289.
14.
Dean, M., Rzhetsky, A., and Allikmets, R. 2001. The human ATP-binding
cassette (ABC) transporter superfamily. Genome Res. 7:1156-1166.
15.
Moslen, M. T. 1996. Toxic responses of the liver. In Casarett and Doull's
Toxicology: The basic science ofpoisons, eds. C. D. Klaassen, M. 0. Amdur, and
J. Doull, pp. 403-416. New York: McGraw-Hill.
16.
Sinnhuber, R. 0., Hendricks, J. D., Wales, J. H., and Putnam, G. B. 1977.
Neoplasms in rainbow trout, a sensitive animal model for environmental
carcinogens. Ann. N.Y. Acad. Sci. 298:389-408.
17.
Bligh, E.G. and Dyer, W. J. 1959. A rapid method of total lipid extraction and
purification. Can. J. Biochem. Physiol. 37:911-917.
18.
Willett, K. L., Gardinali, P. R., Lienesch, L. A., and Di Guilio, R. T. 2000.
Comparative metabolism and excretion of benzo[a]pyrene in 2 species of
ictalurid catfish. Toxicological Sciences 58:68-76.
19.
Varanasi, U., Stein, J. E., Nishimoto, M., and Hom, T. 1982. Benzo[a]pyrene
metabolites in liver, muscle, gonads, and bile of adult English sole (Parophrys
vetulus). In Polynuclear aromatic hydrocarbons: seventh international
symposium onfonnation, metabolism and measurement, eds. M. W. Cooke and
A. J. Dennis, pp. 1221-1234. Columbus, Ohio: Battelle Press.
20.
Nishimoto, M., Yanagida, G. K., Stein, J. E., Baird, W. M., and Varanasi, U.
1992. The metabolism of benzo(a)pyrene by English sole (Parophrys vetulus):
comparison between isolated hepatocytes in vitro and liver in vivo. Xenobiotica
22:949-961.
21.
Varanasi, U., Nishimoto, M., Reichert, W. L., and Eberhart, B. L. 1986.
Comparative metabolism of benzo(a)pyrene and covalent binding to hepatic
DNA in English sole, Starry flounder, and Rat. Cancer Research 46:38 17-3824.
22.
Bomsen, K. 0. 2000. Using the TopCount® microplate scintillation and
luminescence counter and deep-well LumaPlateTM microplates in combination
with micro separation techniques for metabolic studies, pp. 1-6. Meriden,
Connecticut: Packard Instrument Company.
23.
Mathews, C. K., van Holde, K. E., and Ahern, K. G. 2000. Lipid metabolism I:
fatty acids, triacylglycerols, and lipoproteins. In Biochemistry: 3rd Edition, ed.
B. Roberts, pp. 627-666. San Francisco, California: Addison Wesley Longman,
Inc.
24.
Parkinson, A. 1996. Biotransformation of xenobiotics. In Casarett & Doull's
Toxicology: The basic science of poisons, eds. C. D. Klaassen, M. 0. Amdur, and
J. Doull, pp. 113-186. New York: McGraw-Hill.
47
APPENDICES
Appendix A. [14C]Benzo[a]pyrene polar metabolite profile (Experiment 1).
Metabolite profile in gallbladder/bile, following dieldrin pretreatment in rainbow trout
(initial weight -2 g) for 9 weeks. Bile was extracted with ethyl acetate and run on
reverse-phase HPLC to separate the metabolites. HPLC fractions were collected at 3minute intervals and 300 ul duplicates of each fraction were placed in the TopCount
system to analyze radioactivity. Values are mean cpms per fraction ± SE for 9 week
control (9C) and treated (9D) and 12 week control (12C) and treated (12D).
Time
0-3
3-6
6-9
9-12
12-15
15-18
18-21
21-24
24-27
27-30
30-33
33-36
36-39
39-42
42-45
45-48
48-51
51-54
54-57
57-60
60-63
63-66
66-69
69-72
72-75
75-78
78-81
81-84
84-87
87-90
9C
Parent and Phase 1 Metabolites
9D
7±1
50±8
23±4
20±0
9±1
75±28
24±4
21±2
22±3
23±3
23±4
15±2
19±3
21±7
21±5
23±6
23±3
21±2
17±2
18±3
15±3
32±2
27±6
67± 16
38±3
23±4
20±0
20±5
13±2
17±4
17±2
20±3
255±63
132±67
25±10
20±3
12±2
17±2
13±2
15±3
15±0
14±1
55 ±25
30±10
25±7
14±2
25±6
18±3
16±2
15±2
19±2
525±315
179±103
34±11
25±5
18±3
14±2
13±1
23±4
19±6
12C
6±2
40±8
25±5
18±2
15±3
13±2
15±3
23±2
22±7
22±4
18±6
22±2
38±7
25±2
17±2
17±2
20±3
13±3
12±4
15±3
18±3
347±111
62±18
20±3
18±4
18±2
15±3
13±2
13±3
17±2
12D
7±1
72±16
28±4
15±3
13±2
17±2
15±3
22±6
30±5
18±6
22±2
27±2
68±22
25±5
25±0
20±6
20±3
17±2
15±0
17±3
18±3
358±126
53±16
20±3
22±2
20±3
18±2
18±3
22±7
18±2
Cleaved_Glucuronide_Conjugates
9C
9±2
190±75
52±17
30±6
25±5
18±2
20±5
50±13
53±11
65±35
30±6
32±3
125± 13
83±16
42±3
70±35
165±100
42±12
23±2
25±5
18±2
107±25
48±4
32±7
18±2
17±2
15±3
15±3
17±4
15±3
9D
12±3
164±63
59±23
30±6
20±2
21±2
30±3
51±11
43±11
29±8
30±9
34±7
180±80
12C
7±1
163±67
35±8
27±6
18±2
22±4
20±5
35±5
28±4
20±0
33±6
28±6
148±42
52±17
75±20
40±15
36±10
68±46
53±19
214±90 248±190
38±14
28±8
27±7
28±6
13±2
20±4
19±4
18±2
160±77
58±7
53±13
23±2
21±4
13±2
17±3
18±4
21±4
17±3
19±2
15±3
16±3
17±4
20±2
23±2
16±2
18±6
12D
8±2
195±55
65±10
27±2
25±0
23±2
27±12
68±17
62±12
35±10
42±9
47±7
170±49
88±32
68±19
102±32
265±48
50±8
28±6
20±6
23±4
75±3
47±19
18±4
20±3
22±2
20±3
22±3
17±4
17±2
9C
Cleaved_Sulfate_Conjugates
9±3
50±3
25±3
22±4
17±2
15±3
5±3
20±5
25±3
23±6
17±2
17±4
32± 12
27±4
22±2
15±3
22±2
20±0
15±3
12±3
12±2
17±3
5±3
13±3
15±0
13±2
15±0
13±2
10±3
12±4
9D
12±3
45±12
21±9
16±2
15±2
18±3
11±2
5±3
21±2
16±2
16±1
16±3
21±3
11±1
16±2
13±3
13±1
13±1
14±2
15±2
14±1
13±3
15±2
15±2
16±1
15±2
15±0
13±1
13±3
15±3
12C
10±2
42±9
30±8
17±2
20±0
22±2
20±0
22±4
25±8
22±2
17±2
18±2
48±28
18±2
15±0
20±3
20±10
15±3
12±2
12±2
15±0
15±5
12±2
12±2
17±2
12±3
15±3
12±2
12±3
15±5
12D
9±2
60±19
45±15
22±2
18±4
15±3
17±3
25±3
23±3
17±3
18±4
22±4
43±7
23±6
18±2
17±2
32±7
15±3
17±4
13±3
22±3
15±3
13±3
12±2
12±2
12±2
18±2
13±2
13±2
13±2
50
Appendix B. [14C]Benzo[a]pyrene polar metabolite profile (Experiment 2).
Metabolite profile in gallbladder/bile, following dieldrin pretreatment in rainbow trout
(initial weight 88 g) for 11 weeks. Bile was extracted with ethyl acetate and run on
reverse-phase HPLC to separate the metabolites. HPLC fractions were collected at 3minute intervals and 300 ul duplicates of each fraction were placed in the TopCount
system to analyze radioactivity. Values are mean cpms per fraction ± SE.
Parent and Phase 1 Metabolites
Time
0-3
3-6
6-9
9-12
12-15
15-18
18-21
21-24
24-27
27-30
30-33
33-36
36-39
39-42
42-45
45-48
48-51
51-54
54-57
57-60
60-63
63-66
66-69
69-72
72-75
75-78
78-81
81-84
84-87
87-90
7±1
77± 12
38±2
20±3
18±2
12±2
20±5
20±5
28±4
22 ± 2
22 ± 4
32±3
11±4
63± 12
21±7
32±9
20±3
547±210
182±49
93±29
88±30
80±28
105±38
203±42
23 ± 2
27 ± 6
95 ± 19
28±11
18±4
17±3
15±3
18±2
20±8
147 ±28
75 ± 10
108±17
10±1
303±48
75±10
23 ± 2
30 ± 5
20 ± 5
27 ± 4
390± 16
113 ±4
78± 16
83± 15
38 ± 10
72 ± 26
30 ± 10
108±7
85±9
57±3
115±28
73±7
27 ± 2
23 ± 2
27 ± 2
267 ± 97
748 ± 342
122 ± 27
18±4
18±2
22±2
23±6
1172±635
525±233
50±14
38±11
28±3
20±0
17±2
22±6
28±6
18±4
13±2
22±2
38±14
63±11
48±13
30±6
27±3
777 ±99
375± 120
18±3
25±5
15±3
35±5
22±2
20±3
23±4
22±3
18±3
17±4
43±7
25±8
28±3
13±2
20±3
117±37
8±1
37 ± 4
52 ± 2
37±4
37±2
38±4
70±15
100±32
18 ± 6
10±1
63± 11
68± 18
83± 13
23±7
Dieldrin
107± 16
472± 137
237 ±38
35±6
23±2
Cleaved Sulfate Conjugates
control
Dieldrin
Control
Dieldrin
Control
Cleaved Glucuronide Conjugates
413 ± 107
33±3
33±4
27±6
28±6
210±90
38±4
18±2
18±3
23±2
18±3
18±6
25±3
22±7
42±3
28±3
25±8
32±4
33±9
72±16
53±6
32±4
75±8
40±13
27±6
17±3
25±0
17±2
18±2
22±4
13±2
13±2
17±4
43±26
15±3
17±4
15±3
27±3
23±3
22±3
20±3
32±6
47±4
32±2
30±5
57±9
25±8
35 ± 13
17±4
17±4
18±2
25±8
15±3
13±3
17±3
12±2
15±3
15±3
17±2
12±3
15±3
LI,
APPENDIX C
52
Stimulation of [3H]Benzo[a]pyrene Metabolism Following Dieldrin
Pretreatment in the Rainbow Trout Liver Cell Line, RTL-W1
C.1
Introduction
Feeding rainbow trout 0.3-0.4 mg dieldrinlkg/d for 9-12 weeks stimulated the biliary
excretion of a subsequent dose of ['4Cldieldrin by 500% and 13H17,12-
dimethylbenz[a]anthracene (DMBA) by 240% 11,21. This interaction occurs without
induction of the cytochrome P450 system, but could be related to increased performance
of hepatic proteins involved in dieldrin uptake, intracellular trafficking, or elimination
11,3,41. To determine if a similar response occurs in a cell line, we examined the
influence of dieldrin pretreatment on the metabolism of benzo[ajpyrene (BaP) in the
rainbow trout liver cell line, RTL-Wl which expresses cytochrome P450-dependent
monooxygenase activity [5].
C.2
Methods
In vitro BaP metabolism was examined in the rainbow trout liver cell line, RTL-Wl,
grown in control or dieldrin contaminated medium. Plates with a growth area of 28.27
cm2 were each seeded with RTL-Wl at an approximate density of 1 .6x105 cells in 5 ml
of growth medium (L- 15 supplemented with 10% fetal bovine serum and dieldrin test
concentration). Cells were exposed to six different treatments, each applied to three
cultures: control, 30 pM retinoic acid, 20 pM dieldrin + 30 pM retinoic acid, 20 pM
dieldrin, 10 pM dieldrin, and 3 pM dieldrin. Cells underwent two rounds of division
53
for approximately 6 days. At this time the medium was replaced with 5 ml of new
growth medium containing 10 pM [3H]BaP for 24 h. Polar and non-polar metabolites
were separated using the hexane/ethyl acetate solvent extraction system on the cells and
medium. Radioactivity was counted on a liquid scintillation counter. Statgraphics Plus
5.0 was used for statistical analysis. One-way analysis of variance determined
significant treatment effects.
C.3
Results
Most of the nonpolar [3H]BaP remained in the medium, however the rainbow trout liver
cell line, RTL-Wl, accumulated some material. Cells exposed to retinoic acid + 20 pM
dieldrin accumulated significantly more [3H]BaP than the control, 20 pM dieldrin, 10
pM dieldrin, or 3 pM dieldrin treatment groups (figure C.JA). The level of nonpolar
[3HIBaP remaining in the medium was significantly elevated in the 20 pM dieldrin
group (figure C. 1 A) compared to all other treatment groups (due to an analytical error
1
value was excluded as an outlier in the retinoic acid + 20 pM dieldrin treatment group;
there was a significant difference among the standard deviations so a nonparametric
method was used to compare medians rather than means). Polar [3HIBaP metabolites
were present in cells (figure C.1B); however, there was no significant difference
between treatment groups (there was a significant difference among the standard
deviations so a nonparametric method was used to compare medians rather than means).
Polar II3HIIBaP metabolites appeared in the medium but no significant differences among
the treatment groups were observed (figure C.1B).
C.4
Summary and Conclusion
55
Many ligandactivated transcription factors that regulate binding proteins
heterodimerize with the retinoid X receptor (RXR) [6-81. Therefore, retinoic acid, a
ligand for the RXR, was added because of its ability to activate these heterodimers [6-81.
Results from the present study indicated dieldrin pretreatment did not alter the mixed
function oxidase (MFO) system in RTL-W1 cells. Although no marked changes were
observed, statistical analysis revealed dieldrin significantly increased BaP accumulation
by cells, which only occurred in the presence of retinoic acid. However, this was not
associated with increased production of polar metabolites. End-product inhibition may
be one explanation for absence of altered metabolism. It is unlikely that efflux
pathways, specific to the canalicular domain, are retained by the cell-line. As a result,
elimination of material from the cell would cease leading to inhibition of metabolic
pathways. Polar metabolites were detected in the medium, which suggested that some
efflux pathways on the plasma membrane were intact. In vivo work suggested that
chronic dieldrin exposure altered hepatic proteins involved in intracellular trafficking.
In the current study, dieldrin pretreatment increased BaP accumulation by cells in the
presence of retinoic acid. Although more substrate was made available for metabolism,
production of polar metabolites by cells and their elimination into the medium was not
increased. These results suggested that delivery of material to sites of metabolism in the
cell (the MFO system) was not rate limiting. One possibility is that the trafficking
compartment observed in the whole animal is not intact in the RTL-Wl cell-line.
Additional work is necessary to confirm this response and therefore warrants further
investigation.
56
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