Tissue Specific Accumulation of Hydroxyurea in Elasmobranchs

89 Pages·2013·1.7 MB·English

Tissue Specific Accumulation of Hydroxyurea in Elasmobranchs

by

David Iain Fraser

A Thesis
Presented to
The University of Guelph

In partial fulfilment of requirements
for the degree of
Master of Science
in
Integrative Biology

Guelph, Ontario, Canada

© David Iain Fraser, October, 2013
ABSTRACT

TISSUE SPECIFIC ACCUMULATION OF HYDROXYUREA IN ELASMOBRANCHS

David Iain Fraser Advisor:
University of Guelph, 2013 Professor J.S. Ballantyne

Hydroxyurea is an antibiotic and antiproliferative agent used clinically in the treatment of
select cancers, sickle cell anemia, HIV, and myeloproliferative diseases. Hydroxyurea has never
been detected in elasmobranch tissues and has only been detected in trace amounts in a single
eukaryote. Using the colorimetric assay for hydroxyurea determination originally described by
Fabricius and Rajewsky (1971), hydroxyurea was found at relatively high levels in the plasma
and tissues of marine, freshwater and euryhaline elasmobranchs and confirmed by gas
chromatography-mass spectrometry. The presence of marginal amounts of hydroxyurea in the
liver of a teleost (Oncorhynchus mykiss), and absence in a holostean (Amia calva), and a dipnoan
(Protopterus dolloi) suggest elasmobranchs may be unique in their ability to accumulate
hydroxyurea. In adult little skates (Leucoraja erinacea), hydroxyurea was found to accumulate
predominantly in the spiral valve, liver, plasma, rectal gland and stomach to levels ranging from
60-250 µM. Levels of hydroxyurea accumulated in L. erinacea are high enough to have
antineoplastic and antimicrobial effects. These findings provide evidence that hydroxyurea may
be an important component of the innate immune response of elasmobranchs.
ACKNOWLEDGEMENTS

I would first like to thank my advisor Dr. James Ballantyne for his support and extreme
patience during both my undergraduate and graduate research in his lab. Although, I am not in
the least bit sorry to say I now take it as a personal challenge to swim with and photograph whale
sharks before you! I am also grateful to my committee members Drs. Patricia Wright and
Nicholas Bernier for their invaluable expertise. On behalf on myself and past members of the
Ballantyne lab I need to thank the staff of the Hagen Aqualab. Robert Frank, Matt Cornish, and
Mike Davies as well as aqualab volunteers Kaitlyn Wagner, Dustin Kelch, and Zachary Millar
who were essential to ensuring the wellbeing of my skates and rays. For encouraging me to
pursue graduate studies I wish to thank my friends and family, marie Thérèse Rush, and OVC
graduate Tessa. I want to give a special shout out to my Mom, Alina Fraser, for proofreading and
providing feedback on nearly everything I have written in my 26 years of life. Although you still
have to read the chapter myself and Jim wrote on euryhaline elasmobranchs. I know it’s long and
technical but your efforts to avoid reading it are not amusing! I would be amiss if I didn’t also
thank the members of the Wright and Bernier labs, especially Andy Turko even if he did break
into my house that one time, for all the positive interactions and meaningful conversations over
the years. I’m sure the local pubs would like to thank us as well. Last, but certainly not least, I
need to thank the beautiful and intelligent Kristina Victoria Mikloska. I can’t even begin to
express the ways she has helped me through the last year of my graduate studies. Something I
will be eternally grateful for. Kristina, thank you.

iii
TABLE OF CONTENTS

ABSTRACT .................................................................................................................................... ii
ACKNOWLEDGEMENTS ........................................................................................................... iii
TABLE OF CONTENTS ............................................................................................................... iv
LIST OF FIGURES ........................................................................................................................ v
LIST OF TABLES ......................................................................................................................... vi
LIST OF ABBREVIATIONS ....................................................................................................... vii
CHAPTER 1. INTRODUCTION ................................................................................................... 1
1.1 Subclass: Elasmobranchii ..................................................................................................... 2
1.2 The ornithine urea cycle (OUC) and urea biosynthesis in elasmobranchs ........................... 2
1.3 Low incidence of disease in Elasmobranchii ........................................................................ 7
1.4 Hydroxyurea: introduction .................................................................................................. 10
1.5 Hydroxyurea: mechanism of action and metabolism in vertebrates ................................... 13
1.6 Methodological approach.................................................................................................... 16
1.7 Thesis goals and hypotheses ............................................................................................... 17
CHAPTER 2. MATERIALS AND METHODS .......................................................................... 20
2.1 Housing and sampling of adult experimental fish .............................................................. 21
2.2 Housing and staging of skate embryos ............................................................................... 23
2.3 Chemical suppliers .............................................................................................................. 26
2.4 Preparation of tissues, whole embryos and yolk sacs for measurement of urea and
hydroxyurea .............................................................................................................................. 26
2.5 Measurement of hydroxyurea in acidified plasma and tissue samples ............................... 27
2.6 Measurement of urea in acidified plasma and tissue samples ............................................ 29
2.7 In vitro incubations with urea precursors, OUC intermediates, and hydroxyarginine ....... 29
2.8 Gas Chromatography-Mass Spectrometry .......................................................................... 32
2.9 Using TargetP 1.1 to predict the presence of mTPs of nNOS and iNOS ........................... 32
2.10 Statistical analysis ............................................................................................................. 34
CHAPTER 3. RESULTS .............................................................................................................. 35
3.1 Hydroxyurea in elasmobranchs and tissue accumulation ................................................... 36
3.2 Comparing hydroxyurea of euryhaline elasmobranchs in marine, brackish and freshwater
conditions .................................................................................................................................. 42
3.3 Measurements of hydroxyurea in teleost fish ..................................................................... 42
3.4 In vitro incubations with urea precursors, OUC intermediates, and hydroxyarginine ....... 42
3.5 Predictions of the subcellular localization of nitric oxide synthetase (NOS) ..................... 45
CHAPTER 4. DISCUSSION ........................................................................................................ 49
4.1 Elasmobranchs display tissue specific accumulation of hydroxyurea ................................ 50
4.2 Potential role of hydroxyurea in elasmobranchs resistance to disease ............................... 51
4.3 Hydroxyurea in elasmobranchs is not a byproduct of urea synthesis ................................. 55
4.4 Distribution of hydroxyurea and urea in elasmobranch tissues .......................................... 56
4.5 Theoretical mechanism of hydroxyurea synthesis .............................................................. 60
4.6 Conclusions and perspective ............................................................................................... 65
REFERENCES ............................................................................................................................. 68
iv
LIST OF FIGURES

Figure 1. Schematic diagram of arginine metabolism, the ornithine urea cycle (OUC),
purine degradation and conversion of uric acid to urea in vertebrates, highlighting
pathways of urea biosynthesis…………………………………………………….6

Figure 2. The Lewis structure diagrams or urea and hydroxyurea…………….………..….12

Figure 3. Leucoraja erinacea embryos at different stages of development…………..........25

Figure 4. Visual representation of the assay performed to measure hydroxyurea and nitrite
following the procedure described by Fabricius and Rajewsky (1971)………….28

Figure 5. The Lewis structure diagrams of urea cycle intermediates (arginine, ornithine,
citrulline and argininosuccinate), alternative sources of urea (creatine and
allantoate), and N-hydroxyarginine………………………………………..…….31

Figure 6. Hydroxyurea (µM±SE) and urea concentrations (mM±SE) measured in the blood
and tissues of adult Leucoraja erinacea…………....……………………………39

Figure 7. Whole body hydroxyurea (µM±SE) concentrations measured in
Leucoraja erinacea embryos at stages 2 and 3 of development……........………40

Figure 8. Overlaid GC-MS chromatograms of a 10 µg/ml standard of hydroxyurea,
deproteinized liver sample from Leucoraja erinacea, and deproteinized plasma
sample from Leucoraja erinace......................…………………………………...41

Figure 9. Hydroxyurea (µM±SE) concentrations measured in deproteinized plasma samples
from euryhaline (Dasyatis sabina) and freshwater elasmobranchs (Potamotrygon
motoro, Himantura signifer) held in saltwater (SW, 35ppt), brackish (BW, 15-
18ppt) and freshwater (FW, 0-0.7ppt) conditions………………………………..43

Figure 10. (A) Hydrolysis of arginine catalyzed by arginase to produce ornithine and urea.
(B) Theoretical hydrolysis of hydroxyarginine catalyzed by arginase…………..63
Figure 11. Proposed mechanism and subcellular localization of hydroxyurea formation in
elasmobranchs tissue……………………………………………………………..64

v
LIST OF TABLES

Table 1. Summary of features of the metabolism and biochemistry of elasmobranch fishes
that may confer disease resistance through beneficial antineoplastic and/or
antimicrobial effects……………………………………………………………....9

Table 2. Measurement of hydroxyurea (µM±SE) concentrations in deproteinized plasma
samples of marine (Raja rhina, Leucoraja erinacea, Chiloscyllium indicum),
euryhaline (Dasyatis sabina) and freshwater (Potamotrygon motoro, Himantura
signifer) elasmobranchs………………………………………………………….38

Table 3. Measurement of hydroxyurea (µM±SE) concentrations in deproteinized liver
samples from an elasmobranchs (Leucoraja erinacea) and three teleost species,
the bowfin (Amia calva), rainbow trout (Oncorhynchus mykiss) and African
lungfish (Protopterus sp.)………………………………………………………..44

Table 4. Potential precursors of hydroxyurea synthesis incubated at 15 ºC with liver and
spiral valve tissue samples from Leucoraja erinacea (n=3)……………………..46

Table 5. Predicting the presence of mitochondrial targeting pepetides (mTP) from protein
sequences of elasmobranch and teleost neuronal and inducible nitric oxide
synthetase………………………………………………………………………...48

Table 6 Literature summary of effective dose levels of hydroxyurea at which 50% of a
cellular process (e.g. DNA synthesis) is inhibited (ED ) for cancer cell lines,
50
bacterial, viral, and protozoan systems…………………………………………..54

vi
LIST OF ABBREVIATIONS

ADC arginine decarboxylase
AFB aflatoxin B1
1
AGM agmatinase
ALLC allantoicase
ALLN allantoinase
ARG arginase
ASL argininosuccinate lyase
ASS argininosuccinate synthetase
ATP adenosine-5'-triphosphate
Bac1p arginine transporter of inner mitochondrial membrane
BSTFA N,O-Bis(trimethylsilyl)trifluoroacetamide
BW brackish water
CAD carbamoyl phosphate synthetase II-aspartate transcarbamoylase-dihydroorotase
complex
CPS carbamoyl phosphate synthetase
CPS I carbamoyl phosphate synthetase I
CPS III carbamoyl phosphate synthetase III
CRT creatinase
ddH O double distilled water
2
diTMS dimethylsilyl group
DNA deoxyribonucleic acid
ED effective dose at which 50% inhibition of a cellular process occurs
50
eNOS endothelial nitric oxide synthetase
FAU Florida Atlantic Unviersity
FDA food and drug administration
FW freshwater
GAMT guanidinoacetate methyltransferase
GAPDH Glyceraldehyde-3-phosphate
GAT glycine amidinotransferase
GCMS gas chromatography-mass spectrometry
GS glutamine synthetase
HIV Human Immunodeficiency Virus
HU hydroxyurea
iNOS inducible nitric oxide synthetase
LDH lactate dehydrogenase
LLC Lewis lung carcinoma
mTP mitochondrial targeting peptide
vii
NADH nicotinamide adenine dinucleotide
NADPH nicotinamide adenine dinucleotide phosphate
ND not detectable
NED N-(1-Naphthyl)ethylenediamine dihydrochloride
NM not measurable
nNOS neuronal nitric oxide synthetase
NOS nitric oxide synthetase
NOx nitric oxide
OTC ornithine transcarbamylase
OUC ornithine urea cycle
PCA perchloric acid
RBC red blood cell
RNA ribonucleic acid
RC reliability class (1 most reliable-5 less reliable)
SE standard error
SLC solute carrier transporters
SP secretory pathway signal
SW saltwater
TMCS trimethylchlorosilane
triTMS trimethylsilyl group
UGL ureidoglycolate lyase
UOD urate oxidase
UTA urea transporter A
UTB urea transporter B
XOD xanthine oxidase

viii
CHAPTER 1. INTRODUCTION
1
1.1 Subclass: Elasmobranchii

Elasmobranchs (sharks, skates, and rays), a subclass of Chondrichthyan fishes, are an
ancient group of primarily marine fishes distributed widely throughout the world’s oceans, and
occasionally in tropical rivers and lakes. Marine elasmobranchs are characterized by their
unusual solute system; most notably their ability to synthesize and accumulate urea to high
concentrations in their tissues (Smith 1935). Urea is a nitrogenous waste product and is used to
detoxify ammonia in many vertebrates. However, in marine species of elasmobranchs, urea
accumulation is part of a strategy to maintain an internal osmolarity close to that of seawater
with urea being the primary solute accumulated (Ballantyne 1997). This strategy enables marine
species to function as osmoconformers. A consequence is that the metabolism of elasmobranchs
has been greatly shaped by the high requirement to produce and retain urea (Ballantyne 1997). In
species inhabiting freshwater and brackish conditions the need to produce and retain urea is
reduced, although euryhaline species, such as the bull shark (Carcharhinus leucas Müller and
Henle 1839) and Atlantic stingray (Dasyatis sabina Lesueur 1824), do maintain an ability to
produce high concentrations of urea and can alter urea content by changing urea biosynthesis
and/or excretion during movement between variable salinity habitats (Ballantyne and Fraser
2012).
1.2 The ornithine urea cycle (OUC) and urea biosynthesis in elasmobranchs

The majority of urea accumulated by elasmobranchs is produced by the OUC with the
liver being the primary site of urea biosynthesis, although extrahepatic urea synthesis is also
known to occur, especially in the skeletal muscle (Ballantyne 1997; Steele et al. 2005; Kajimura
et al. 2006). The key enzymes involved in the OUC of elasmobranch are carbamoyl phosphate
2