alterations in the hepatic biotransformation of steroids as a potential

181 Pages·2005·1.58 MB·English

ALTERATIONS IN THE HEPATIC BIOTRANSFORMATION OF STEROIDS AS A
POTENTIAL MECHANISM FOR CONTAMINANT INDUCED CHANGES IN
PLASMA TESTOSTERONE CONCENTRATIONS IN JUVENILE ALLIGATORS
(Alligator mississippiensis)

By

MARK PAUL GUNDERSON

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2005
Copyright 2005

by

MARK PAUL GUNDERSON
This is dedicated to my family
ACKNOWLEDGMENTS
Completing this dissertation has been a long process, and there is an extensive list
of people that deserve acknowledgement for their contributions. I would first of all like
to thank my major advisor, Dr. Louis Guillette, for the support, encouragement, and
assistance he has provided as well as the research framework that has made these projects
possible. It has truly been a memorable experience that I will never forget. I also thank
Dr. David Evans who has been a supportive mentor. I will always value the time I spent
working in his laboratory at the Mount Desert Island Biological Laboratory. I credit him,
along with my undergraduate advisor Dr. Eugene Bakko, with introducing me to research
as an undergraduate and inspiring me to pursue my doctorate in zoology. I also
acknowledge the other members of my committee. I am grateful to Dr. Taisen Iguchi for
the helpful comments on manuscripts as well as his generous hospitality and provision of
research funds during my stay in Japan. I also thank Dr. David Julian for his perceptive
input throughout this process as well as Dr. William Buhi for his role as the outside
member on my committee.
Many of the projects included in this dissertation required extensive collaboration
and could not have been accomplished without the cooperation and manpower of the
people from the Guillette laboratory and the Florida Fish and Wildlife Conservation
Commission, as well as collaborators at other universities. Specifically, I thank Drs.
Gerald LeBlanc, Bruce Blumberg, Y. Katsu, and Satomi Kohno for their support and
comments on this work. I gratefully acknowledge James B. McLachlan, Cynthia Ryder,
iv
Matthew Milnes, Dieldrich Bermudez, Thea Edwards, Teresa Bryan, Brandon Moore,
Jenny Gates, Stefan Kools, Ruben Valdes, Edward F. Orlando and Gerald Binczik for
their contributions. The Florida Fish and Wildlife Conservation Commission played a
critical and much appreciated role in the collection of animals and I specifically thank
Allan Woodward, Dwayne Carbaneau, Lindsey and Troy Horde, John White, Christopher
Vischer, Chris Tucker and the Florida Fish and Wildlife Conservation Commission for
the help they offered in the collection of animals. I also thank the South Florida Water
Management District for the use of the Riverwoods Field Laboratory (100 Riverwoods
Circle, Lorida FL.33857). Funding for these projects came from the U.S. EPA, National
Science Foundation, JSPS, Crest, and JST.

v
TABLE OF CONTENTS

page
ACKNOWLEDGMENTS.................................................................................................iv
LIST OF TABLES...............................................................................................................x
LIST OF FIGURES..........................................................................................................xii
ABSTRACT.....................................................................................................................xiv
CHAPTER
1 ALTERATIONS IN THE HEPATIC BIOTRANSFORMATION OF STEROIDS
AS A POTENTIAL MECHANISM FOR CONTAMINANT INDUCED
CHANGES IN PLASMA TESTOSTERONE CONCENTRATIONS IN
JUVENILE AMERICAN ALLIGATORS...................................................................1
Introduction...................................................................................................................1
Types of Hepatic Biotransformation............................................................................2
Hormonal Regulation of Steroid Hydroxlase Enzymes................................................4
Induction of Steroid Biotransformation Enzymes by Xenobiotics...............................6
Cytochrome P-450 Nomenclature.........................................................................7
CYP1 Family in Alligators....................................................................................7
CYP2 Family in Alligators....................................................................................8
CYP3 Family in Alligators....................................................................................9
Regulation of CYP3A by Nuclear Receptors and Xenobiotics..................................10
Cytochrome P-450 Enzymes and Plasma Steroid Concentrations.............................12
Summary of Experiments...........................................................................................14
Chapter 2: Plasma Sex Steroid Concentrations and Phallus Sizes of Juvenile
Alligators Inhabiting 3 Sites in South Florida.................................................15
Chapter 3: Variation in T Biotransformation Enzyme Activities in Juvenile
Alligators Inhabiting 5 Sites in Florida............................................................15
Chapter 4: Regulation of Sexually Dimorphic Patterns of CYP3A Transcript
through Developmental Exposure to Ethanol and Pesticide Mixtures............16
Chapter 5: Up-Regulation of CYP3A by Toxaphene and Dexamethasone in
Female Juvenile Alligators and Its Effect on Plasma Testosterone
Concentrations.................................................................................................17
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2 VARIATION IN SEX STEROIDS AND PHALLUS SIZE IN JUVENILE
AMERICAN ALLIGATORS COLLECTED FROM THREE SITES WITHIN
THE KISSIMMEE-EVERGLADES DRAINAGE IN FLORIDA (USA).................25
Introduction.................................................................................................................25
Materials and Methods...............................................................................................27
Study Sites...........................................................................................................27
Animal Collection...............................................................................................28
Radioimmunoassay of Steroids...........................................................................28
Statistical Analysis..............................................................................................29
Results.........................................................................................................................30
Plasma Sex Steroids............................................................................................30
Plasma T.......................................................................................................31
Plasma E ......................................................................................................32
2
Evaluation of Spatial and Temporal Patterns of Phallus Size.............................32
S.E Associated with Phallus Size, Plasma T, and E Concentrations.................33
2
Discussion...................................................................................................................34
3 ALTERATIONS IN SEXUALLY DIMORPHIC BIOTRANSFORMATION OF
TESTOSTERONE IN JUVENILE AMERICAN ALLIGATORS FROM
CONTAMINATED LAKES......................................................................................46
Introduction.................................................................................................................46
Materials and Methods...............................................................................................50
Study Sites...........................................................................................................50
Animal and Tissue Collection.............................................................................52
Cytosol and Microsome Preparation From Alligator Livers...............................53
Testosterone Hydroxylase and Oxido-Reductase Activity..................................53
Testosterone UDP-Glucuronosyltransferase Activity.........................................55
Sulfotransferase Activity.....................................................................................56
Statistical Analysis..............................................................................................57
Results.........................................................................................................................57
Testosterone Hydroxylase Activities...................................................................58
Testosterone Oxido-Reductase Activities...........................................................59
Testosterone UDP-Glucuronosyltansferase Activity .........................................60
Testosterone Sulfotransferase Activity ...............................................................61
Discussion...................................................................................................................62
4 THE EFFECTS OF DEVELOPMENTAL EXPOSURE TO ETHANOL AND
ORGANOCHLORINE COMPOUNDS ON HEPATIC CYP3A AND NUCLEAR
RECEPTORS IN THE AMERICAN ALLIGATOR.................................................76
Introduction.................................................................................................................76
Materials and Methods...............................................................................................80
Statistical Analysis..............................................................................................81
Results.........................................................................................................................82
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Mean Relative CYP3A and Nuclear Receptor Gene Expression in Yearling
Alligators Exposed to Contaminant Mixtures during Development................82
CYP3A.........................................................................................................82
Nuclear Receptors........................................................................................83
Nuclear Receptors with Sexually Dimorphic Patterns of Expression in
Control Animals (AR, ERα, ERβ)...................................................................83
AR................................................................................................................83
CYP3A vs. AR.............................................................................................83
ERα..............................................................................................................84
CYP3A vs. ERα...........................................................................................86
ERβ...............................................................................................................87
CYP3A vs. ERβ...........................................................................................88
Nuclear Receptors that did not Exhibit Sexually Dimorphic Gene mRNA
Patterns in the Control Groups.........................................................................89
GR, PR, and SXR.........................................................................................89
CYP3A vs. GR.............................................................................................89
CYP3A vs. PR..............................................................................................90
CYP3A vs. SXR...........................................................................................90
Step Wise Regression Examining the Relationships between Mean Relative
CYP3A Gene Expression and Mean Relative AR, ERα, ERβ, GR, PR, and
SXR Gene Expressions....................................................................................90
Discussion...................................................................................................................91
5 UP-REGULATION OF AN ALLIGATOR CYP3A GENE BY TOXAPHENE
AND DEXAMETHASONE AND ITS EFFECT ON PLASMA
TESTOSTERONE CONCENTRATIONS...............................................................102
Introduction...............................................................................................................102
Methods....................................................................................................................107
CYP3A Up-Regulation Study...........................................................................107
Field Study.........................................................................................................109
RNA Isolation, RT-PCR, and qRT-PCR...........................................................110
Statistical Analysis............................................................................................112
CYP3A Up-Regulation Study....................................................................112
Field Study.................................................................................................113
Results.......................................................................................................................114
CYP3A and Nuclear Receptor Up-Regulation in Female Yearling
Alligators........................................................................................................114
Mean Relative Gene Expression among Treatments.................................114
Simple Linear Regression (CYP3A vs. 6 Nuclear Receptors)..........................115
CYP3A vs. AR...........................................................................................115
CYP3A vs. ERα.........................................................................................115
CYP3A vs. ERβ.........................................................................................116
CYP3A vs. GR...........................................................................................116
CYP3A vs. PR............................................................................................116
CYP3A vs. SXR.........................................................................................117
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Stepwise Regression...................................................................................117
Plasma T Concentrations...................................................................................118
Field Study.........................................................................................................119
Mean Relative CYP3A and SXR Gene Expression...................................119
Discussion.................................................................................................................120
Up-regulation of alligator CYP3A by known inducers.....................................121
Relationship of Nuclear Receptors to CYP3A Expression...............................121
CYP3A and Plasma T Concentrations..............................................................123
Stress and Plasma T Concentrations?................................................................125
CYP3A Expression in Wild Populations...........................................................126
Summary............................................................................................................128
6 CONCLUSIONS......................................................................................................139
Summary of Findings and Conclusions....................................................................139
Future Directions Based on the Findings of this Dissertation..................................145
The Development of Non-Invasive Techniques to Identify the Steroid
Metabolism Status of an Animal....................................................................145
Urinary Clearance of Steroids...........................................................................146
The Hypothalamus, Pituitary, Growth Hormone and CYP Enzymes...............147
Nuclear Receptor Response Elements...............................................................147
Relationship between Plasma T and Hepatic Enzyme Activity........................148
Implications of this Dissertation...............................................................................149
LIST OF REFERENCES.................................................................................................152
BIOGRAPHICAL SKETCH...........................................................................................165

ix
LIST OF TABLES
Table page

2-1 Sediment and water contaminant data for selected sites in the Kissimmee River
drainage. ..................................................................................................................41
2-2 Plasma testosterone (T) and estradiol-17β(E ) concentrations in juvenile alligators
2
collected from 3 sites in south Florida.....................................................................42
2-3 Relationship of plasma testosterone concentrations to snout-vent length in juvenile
alligators collected from 3 sites within the Everglades watershed...........................42
2-4 Mean phallus tip lengths (mm) and cuff diameters (mm) in male juvenile alligators
collected from 3 sites within the Everglades watershed..........................................43
2-5 Relationship of phallus tip length and cuff diameter to snout-vent length (SVL) in
juvenile alligators collected from 3 sites within the Everglades watershed.............43
2-6 Relationship of phallus tip length and cuff diameter to plasma testosterone (T)
concentrations in juvenile alligators collected from 3 sites within the Everglades
watershed..................................................................................................................44
2-7 Relationships of initial estradiol-17β (E ) concentrations to snout-vent length
2
(SVL) in female juvenile alligators collected from 3 sites within the Everglades
watershed..................................................................................................................44
3-1 Summary of total testosterone hydroxylase and oxido-reduction activities in lake
Woodruff, Lake Apopka, Lake Okeechobee, and Water Conservation Area 3A
juvenile alligators.....................................................................................................69
3-2 Summary of total testosterone UDP-glucuronosyltransferase and sulfotransferase
activities in Lake Woodruff, Lake Apopka, Lake Okeechobee, and Water
Conservation Area 3A (WCA 3A) juvenile alligators.............................................70
4-1 Simple linear regressions examining the relationship between mean relative
CYP3A and AR, ERα, ERβ, GR, PR, or SXR gene expression within each
treatment group........................................................................................................97
4-2 Forward step-wise regression analyses examining the relationship between mean`
relative CYP3A and AR, ERα, ERβ, GR, PR, and/or SXR gene expression within
each treatment group................................................................................................99
x