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thesis entitled

Cloning and Expression Analysis of
Atlantic Salmon (Salmo salar) CYPlA

 

presented by

Christopher Benjamin Rees

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6/01 cJCIFiC/DateOmpSS—ots

 

CLONING AND EXPRESSION ANALYSIS OF
ATLANTIC SALMON (SALMO SALAR) CYPlA

By

Christopher Benjamin Rees

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

DEPARTMENT OF FISHERIES AND WILDLIFE

2001

ABSTRACT

CLONING AND EXPRESSION ANALYSIS OF
ATLANTIC SALMON (SALMO SALAR) CYPlA

By

CHRISTOPHER BENJAMIN REES

Environmental pollution, such as polyaromatic hydrocarbons (PAH’s), polychlorinated
biphenyls (PCB’s), and dioxins, is suspected of causing recent declines in Atlantic
salmon (Salmo salar) populations. I hypothesize that cytochrome P4501A (CYPlA) is
inducible by B-Naphthoflavone (BNF), a PAH, in Atlantic salmon. To characterize the
fimction and expression of this gene, I first determined the cDNA sequence of Atlantic
salmon CYPlA through molecular cloning, the first CYPlA gene cloned for any salmon
species. In addition, phylogenetic analysis of the CYPlA coding region as well as the
deduced amino acid sequence placed this gene most closely related to rainbow trout
CYPlA genes. I then demonstrated the inducibility of this gene by treating salmon with
B-Naphthoflavone and visualizing through Northern blotting procedures. Furthermore,
based on the Atlantic salmon CYPlA sequence, a highly specific and sensitive
competitive reverse transcription-polymerase chain reaction was developed and used to
determine the levels of CYPlA expression. This method was utilized to quantify the
levels of inducibility of CYPlA in liver, gill, kidney and brain tissue as well as to
demonstrate induction of CYPlA in wild Atlantic salmon from streams known to contain
PCB’s, PAH’s, and dioxins. These studies provide a foundation for studying CYPlA in
salmon as well as other species, for quantitatively assessing biomarker responses in

Atlantic salmon, and for providing valuable tools to manage dwindling salmon stocks.

DEDICATION

To my family and friends for all of their love and support.

iii

ACKNOWLEDGMENTS

I would like to first thank my committee, Dr. Robert Batie, Dr. Richard W. Hill, and Dr.
Weiming Li for their constant enthusiasm and support throughout the duration and
completion of my research. I would also like to thank Dr. Donald Garling for sitting in
place of Dr. Hill during my oral examinations. My advisor, Dr. Weiming Li, deserves a
special thank you for one, giving me the opportunity to pursue an advanced degree, and
two, for his incredible patience and understanding. I would also like to thank Dr. Steve
McCormick of the USGS Conte Anadromous Fish Laboratory, Turner’s Falls,
Massachusetts for assistance and collaboration during collection of wild salmon.
Gratitude is extended to Roger Greil and Trent Sutton of Lake Superior State University
Fish Hatchery as well as Phil Hulbert and the staff at Adirondack Fish Hatchery for
donation of hatchery salmon for this project. Sequencing expertise was provided by Tom
Newman and the personnel at the Michigan State University (MSU) DNA Sequencing
Facility. An acknowledgment goes to Marla Sanbom for her help with RACE and
molecular cloning. Processing of RNA samples during quantitative PCR analysis would
not have been possible without the diligent work of Linda F erkey. A great thanks goes to
Hong Wu for her assistance in Northern blotting experiments and Bradley Young for
assistance in statistical analysis. Also, this project would not have been possible without
the cooperation of Dr. Kim Scribner, Scot Libants, and the rest of the Molecular Ecology
Lab at MSU. Funding for this project was provided by the J aqua Foundation and

Michigan State University.

iv

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................ vii
LIST OF FIGURES ......................................................................................................... viii
CHAPTER ONE: Cloning and induction of CYPlA in Atlantic salmon (Salmo salar)... 1

ABSTRACT .................................................................................................................. 1
INTRODUCTION ........................................................................................................ 2
MATERIALS AND METHODS .................................................................................. 4
Fish ....................................................................................................................... 4
Tissue collection .................................................................................................... 4
RNA extraction ...................................................................................................... 4
First strand cDNA synthesis .................................................................................. 5
Polymerase chain reaction (PCR) ......................................................................... 5
Rapid amplification of cDNA ends (RACE) .......................................................... 6
Molecular cloning ................................................................................................. 7
Sequencing ............................................................................................................. 7
Phylogenetic analysis ............................................................................................ 8
Induction ................................................................................................................ 8
Northern blotting ................................................................................................... 8
RESULTS ................................................................................................................... 10
Atlantic salmon C YPIA ....................................................................................... 10
Phylogenetic analysis .......................................................................................... 10
Induction of C YPIA ............................................................................................. 11
DISCUSSION ............................................................................................................. 13
CHAPTER TWO: Quantitative PCR analysis of CYPlA induction in Atlantic salmon
(Salmo salar) ............................................................................................................... 17
ABSTRACT ................................................................................................................ 17
INTRODUCTION ...................................................................................................... 18
MATERIALS AND METHODS ................................................................................ 21
Fish handling and sampling ................................................................................ 21
Total RNA isolation and storage ......................................................................... 22
Internal standard synthesis ................................................................................. 22
Competitive RT -PCR ........................................................................................... 24
PCRfragment visualization and data generation ............................................... 26
Statistical analysis ............................................................................................... 26

RESULTS ................................................................................................................... 27

Internal standard and standard curves ............................................................... 27
Induction of C YPIA in brain, gill, liver, and kidney in hatchery-raised salmon 27

C YPIA expression in wild salmon ....................................................................... 28
DISCUSSION ............................................................................................................. 29
REFERENCES ................................................................................................................. 49

vi

Table 1:

Table 2:

Table 3:

Table 4:

Table 5:

Table 6:

LIST OF TABLES

Primers used in PCR and RACE amplifications of CYPIA cDNA.
Phylogenetic analysis of cytochrome P450 genes.

Phylogenetic analysis of cytochrome P450 proteins.

CYP genes and accession numbers used in the phylogenetic analysis.
Quantitative RT-PCR analysis of CYPIA levels in tissues of hatchery salmon.

Quantitative RT—PCR analysis of CYPIA expression in tissues of wild salmon.

vii

Figure 1:
Figure 2:
Figure 3:
Figure 4:
Figure 5:
Figure 6:

Figure 7:

Figure 8:

Figure 9:

LIST OF FIGURES

Electrophoretic analysis of amplified CYPlA cDNA’s.

cDNA and deduced amino acid residue sequence of Atlantic salmon CYPIA.
Alignment of heme-binding region (amino acid residues 456-465).

Northern blot analysis of CYPIA total RNA from Atlantic salmon liver.
Construction of internal standard.

Generation of standard curve.

Representative gel pictures for gill, liver, kidney, and liver samples acclimated
at 17°C.

Representative gel pictures for gill, liver, kidney, and liver samples acclimated
at 11°C . ‘ ,

Representative gel picture of actin normalization.

Figure 10: Quantitative PCR of gill and liver tissue CYPIA for samples collected from

wild salmon from two Massachusetts rivers.

viii

CHAPTER ONE: Cloning and induction of CYPIA in Atlantic salmon (Salmo salar)

ABSTRACT
Environmental contaminants are implicated for recent declines in Atlantic salmon stocks
across the eastern coast of the United States and Canada (F airchild et a1. 1999). To
understand the function of cytochrome P450 enzymes in the metabolism of these
toxicants, I have cloned a CYPIA gene in Atlantic salmon using reverse transcription-
polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE).
The cloned cDNA possesses all characteristic motifs of teleost CYPIA genes including
the start codon, heme-binding region, stop codon, and putative poly-adenylation signal.
This gene is also characterized by a long 3’ untranslated region (UTR; 1025bp)
containing three AUUUA sequences, which are a target for its rapid degradation. The
Atlantic salmon CYPIA shares 97.4% and 96.7% sequence identity with the
corresponding rainbow trout CYP1A1 and CYP1A3 genes, respectively. In addition, the
deduced amino acid sequence of salmon CYPIA shows 96.9% and 96.2% identity with
rainbow trout P4501A1 and P4501A3 enzymes, respectively. Phylogenetic analysis of a
sample of P450 genes established that this Atlantic salmon CYPIA gene is closely
related to rainbow trout CYPIA genes as well. Northern analysis showed that CYPIA
mRNA is significantly higher in the liver of Atlantic salmon injected intraperitoncally
with B-Naphthoflavone (BNF) suggesting that the cloned gene is inducible (Rees 2001;
this paper). The sequence of the CYPIA cDNA is useful in development of biomarkers

for exposure of Atlantic salmon to a broad range of xenobiotics.

INTRODUCTION

Cytochrome P450 constitutes a ubiquitous enzyme system noted for its
inducibility and diversity (Andersson and Forlin 1992). These enzymes have been found
in all species studied to date including plants, animals, bacteria, and other organisms
(Nelson et al. 1996). P450’s are involved with the detoxification of organic compounds
such as xenobiotics (Mehmood et a1. 1996, Goksoyr and Husoy 1998), inhaled odorants
(Dahl and Hadley 1991, Nef et al. 1989, Lazard et al. 1991), steroids (Brittebo and Rafter
1984, Ding and Coon 1994), drugs (Nef et al. 1989, Schlenk et al. 1993), and carcinogens
(Hrelia et al. 1996, Kawajiri and Fujii-Kuriyama 1991). Historically, mammals have
been the primary targets of P450 examinations. More recently, fish species have become
models of P450 research.

In fish, the most intensively studied P450 genes are CYPlA’s (Nelson et al.
1996). Their sequences have been determined in rainbow trout (Heilmann et al. 1988,
Berndtson and Chen 1994), plaice (Leaver et al. 1993), Atlantic tomcod (Roy et al.
1995), toadfish and scup (Morrison et al. 1995), killifish (Monison et a1. 1998), red sea
bream (Mizukami et a1. 1994), and sea bass (Stien et a1. 1998). These experiments have
provided tools to understand the structure and function of cytochrome P450 proteins in
general, and the role of CYPIA in metabolism of a variety of toxicants in particular.

I attempted to clone a CYPIA gene from Atlantic salmon to determine its
structure and evolutionary relationship to other teleost CYPIA cDNA’s. This
information will be useful to understand the steep decline of Atlantic salmon populations
in response to environmental pollutants across the eastern seaboard, including both the

United States and Canada (Tufts 2000). These pollutants have long been suspected of

negatively impacting Atlantic salmon populations (W aldichuk 1979). Recent research
has focused on the possibility that pesticides may be affecting the return rates of Atlantic
salmon in Canada (Fairchild et al. 1999). The problems associated with salmon exposure
to environmental pollution are two fold. Firstly, one of the most sensitive stages of
stream life, smoltification (Moyle and Cech 2000), coincides seasonally with the peak
agricultural application of pesticides (Albanis et a1. 1998). Secondly, many studies
suggest that pesticides may have profound effects (decreased reproductive success; lower
recruitment, etc.) on teleosts at sublethal concentrations (Little et al. 1990, Jones et al.
1998, Gagne et al. 1999), which over time, may lead to population decline. Therefore,
the acute effects pesticides have on Atlantic salmon development may be subtle,
emphasizing the need for molecular markets. In order to understand these effects at the
molecular level, I examined the cDNA sequence, deduced amino acid sequence, and

inducibility of CYPIA in Atlantic salmon liver.

MATERIALS AND METHODS

Fish

Thirty Atlantic sahnon (Salmo salar) approximately 20cm in length and 50 grams
in weight were acquired from a fish hatchery (Lake Superior State University, Sault Ste.
Marie, Michigan) and acclimated for 2 weeks at the Michigan State University Lower
River Laboratory in an 8001 flow through tank (1 fish 20 1") at 11.5°C. A 12 hour light-
dark cycle was maintained during the acclimation period. Salmon were fed Purina
AquaMax© Grower 400 (lot A-5D04) daily at a level of 3.0% body weight. Two days

prior to tissue collection, salmon were taken off of feed.

Tissue collection
Fish were given an overdose of MS-222. The liver was removed, sectioned 3

times, immediately stored in at least a 10x volume of RNALater© (Ambion; Austin,

Texas), and subsequently stored at -80°C.

RNA extraction

Total RNA was extracted using TRIzol® Reagent according to the
manufacturer’s protocol (Life Technologies; Rockville, MD), resuspended in 50g]
diethylpyrocarbonate-treated water, and quantified on a UV spectrophotometer (Beckman
DUO 7400; Fullerton, CA). RNA solution was mixed with 3 volumes of 70% ethanol and
3 M sodium acetate to a final concentration of 0.3 M and stored at -80°C until further

analysis. Storage of RNA in this condition is stable (Sambrook et al. 1989).

First strand cDNA synthesis

Total RNA (1-5 pg) was mixed with 10pM p1" Oligo am” and diluted with
deionized water to a final volume of 12p]. This mixture was heated at 70°C for 10
minutes and quick chilled on ice. Next, 4yl 5x first strand buffer, 2,111 0.1 M
dithiothreitol, and 1,111 10mM dNTP were also added. After incubation of this mixture at
42°C for 2 minutes, 200 units of MMLV-RT (Life Technologies) were added making the
final reaction volume 20,111. The reaction mixture was incubated at 42°C for 50 minutes
and inactivated at 70°C for 15 minutes. Finally, 10 units of RNAseH (Life Technologies)
were added, incubated at 37°C for 20 minutes, and inactivated at 94°C for 5 minutes.

The synthesized cDNA was stored at -20°C until. used for PCR.

Polymerase chain reaction (PCR)

To isolate a CYPIA gene in Atlantic sahnon liver, I first aligned three rainbow
trout CYPIA sequences (Heilmann et al. 1988; Bemdtson and Chen 1994) using the
CLUSTAL W algorithm. Then, using a region spanning the heme-binding domain and
the helical region, I selected degenerate primers that flanked a region of approximately
580bp in known teleost genes (Table 1). Degenerate primers were synthesized
(Macromolecular Structure, Synthesis, and Sequencing Facility, Michigan State
University).

PCR master mix consisted of the following constituents (final reaction
concentrations in parentheses): 2a] of cDNA, dNT P’s (200uM), 5x PCR Buffer (1x),
MgClz (4mM), WML38 sense primer (500nM), and WML39 antisense primer (500nM).

The final 50p] reaction was amplified in a FTC-200 MJ Research Thennocycler (1 cycle

94°C 4 min; 30 cycles 94°C 1 min, 56°C 45s, 72°C 1 min; 1 cycle 72°C 5 min; 1 cycle
4°C forever). The product was subsequently cloned and sequenced as described in the

Materials and Methods section.

Rapid amplification of cDNA ends (RA CE)

Gene specific primers (GSP) for 5’ and 3’ RACE were designed by using the
sequence of the previous PCR product. RACE was carried out utilizing the Advantage 11
RACE system (Clontech; Palo Alto, CA) according to the manufacturer’s protocol. One
pg of total RNA from Atlantic salmon liver was used as template for synthesis of 5’ and
3’ RACE Ready cDNA. GSP’s were used individually with the universal primer mix
supplied in the kit for both 3’ and 5’ RACE. Nested GSP’s were used to enhance the
efficiency of RACE amplification (Table 1).

To clone a hill length CYPlA cDNA, a two-step long distance RACE-PCR was
utilized. Briefly, a 3’ RACE GSP (WMLS 1) was designed to extend the cDNA to the 3’
end (poly A tail) while a 5’ RACE GSP (WML52) was used to extend to the 5’ end. The
two RACE products produced by this method were expected to have a region of overlap
of ~200bp. Both RACE products were cloned and sequenced as previously described and
analyzed using BLAST on the world-wide-web. To eliminate the possibility of
producing a hybrid sequence of a full length CYPIA gene, I conducted a second RACE
with a GSP designed upstream from the start codon. The expected product from this

RACE would include all functional domains of a single, full-length CYPIA gene.

Molecular cloning

All PCR products were size-fractionated on a 1% agarose gel containing 0.1,ug/ml
ethidium bromide (Sambrook et a1. 1989). PCR products were cleaned using the Wizard
DNA clean-up system (Promega Corporation; Madison, WI), ligated into a pGEM - T
Easy vector (Promega Corporation), and subsequently electroporated into DH5a
competent cells (Clontech ). Plasmid DNA minipreps were extracted by Wizard DNA

Miniprep Kit (Promega Corporation).

Sequencing

Preliminary sequencing reactions were performed using the Sequitherrn
EXCELTM 11 DNA Sequencing Kit (Epicentre Technologies; Madison, WI). Hex-labeled
T7 (forward sequencing primer) and SP6 (reverse sequencing primer) primers were
purchased from Integrated DNA Technologies (Coralville, IA). Once CYPIA positive
inserts were identified using dideoxy sequencing and BLAST, extended sequencing was
carried out by the Plant Biology DNA Sequencing Facility, Michigan State University.
Primer walking was used for shorter inserts, between 1-1.5kb. In vitro transposon
insertion was used (Genome Priming System, New England Biolabs) for sequencing of
templates greater than 1.7kb. DNA sequence comparison and analysis was performed
either with BLAST (l_1t_tp://www.ncbi.nhn.nih.gov/BLAST), Sequencher 3.1 (Gene Codes
Corporation; Ann Arbor, Nfl), MacVector 6 (Oxford Molecular Group PLC; Huntsville,

MD), or DNASTAR (DNASTAR, Inc.; Madison, WI).

Phylogenetic analysis

The coding region of Atlantic salmon CYPIA was aligned to the coding region of
a sample of P450 genes using the CLUSTAL W algorithm. Genetic relationships and
distances were generated using the Neighbor-joining Method. This same analysis was
also performed on CYPlA amino acid sequences as well. Genes selected for this
analysis comprised mostly teleost CYPIA genes, several mammalian CYPIA genes, and
two rainbow trout P450 genes of other subfamilies (CYP2K1 and CYP3A27). Initially,
teleost genes were aligned after which subsequent mammalian genes were aligned.

Finally, rainbow trout CYP2K1 and CYP3A27 were added to the alignment.

Induction

To demonstrate the inducibility of the CYPIA gene in Atlantic sahnon, randomly
selected individuals (n=4) were sampled and given either an intraperitoneal injection of
B-Naphthoflavone (BNF, a potent inducer of CYPIA; 50mg kg'1 body weight) dissolved
in corn oil (10mg BNF ml’1 corn oil) or corn oil alone. Individual sahnon were then
placed for 48 hrs in a 40 1 flow-through aquarium (20 1 h"; 0.5R), sacrificed with an
overdose of MS-222, and dissected for liver tissues which were immediately stored in

RNALater© (Ambion).

Northern blotting
Total RNA (7,ug) from control and induced samples were separated on 1%
agarose-formaldehyde gels and transferred to positively charged nylon membranes

(Ambion). P450 full sequence clones were used as probes and were labeled using the

DIG Random Primed DNA Kit (Roche Diagnostics Corp.; Indianapolis, IN). Blots were
hybridized according to the manufacturer’s protocol and washed (68°C, 0.1x SSC, 0.1%
SDS) followed by color detection reactions (Roche Diagnostics Corp.). Probes for actin
mRNA were produced by the same labeling technique as above from an Atlantic sahnon

fl-actin cDNA (Rogers et al. 1999, GenBank Accession AF 012125) fragment confirmed

by sequencing.

RESULTS

Atlantic salmon C YPIA

Initial PCR amplification of CYPIA from Atlantic salmon liver cDNA produced
a fragment approximately 580bp long (Figure 1A). Molecular cloning and sequencing of
this PCR product established that it was homologous to rainbow trout CYPIA and
exactly 576bp long. GSP’s obtained fiom this preliminary sequence extended to the 3 ’
and 5’ end by RACE reactions. The resulting CYPIA fragments were 1,669bp and
1,394bp respectively with a region of overlap of 205bp (Figure 1B). Both 3’ and 5’
RACE fragments were roughly 95% homologous to both rainbow trout CYP1A1 and
CYP1A3 cDNA molecules.

To verify that the two overlapping products encode a single CYPIA gene, a long
distance 3’ RACE fragment of 2626bp was obtained (Figure 1C). Sequence analysis
indicated this product included 32bp of the 5’ untranslated region (UT R), a 1569bp
coding region, and a 1025bp 3’ UTR containing 3 AUUUA sequences. It encodes a
protein of 523 amino acid residues. The obtained sequence also possesses all major
firnctional domains and characteristics of previously discovered CYPIA molecules
including the heme-binding cysteine (position 463), arginine codon (position 246)
integral to enzymatic function, stop codon (position 523), and putative polyadenylation
signal (Figure 2).

Phylogenetic analysis
This gene shares 97.4% sequence identity with the corresponding rainbow trout

CYP1A1 gene and 96.7% sequence identity with rainbow trout CYP1A3. In addition,

10

this gene shares between 70-76% nucleotide homology with other teleost CYPIA genes.
The deduced amino acid sequence shows 96.9% amino acid identity with rainbow trout
p4501A1 and 96.2% amino acid similarity with rainbow trout P4501A3. Likewise, the
salmon CYPlA protein demonstrates between 68-83% amino acid homology with other
teleost CYPIA proteins. The heme-binding region encompassing the amino acid
sequence F GMGKRRCIG (positions 456-465) is highly conserved over all teleosts
including Atlantic salmon (Figure 3).

Multiple sequence alignment using the CLUSTAL W algorithm followed by
construction of phylogenetic tree using the Neighbor-j oining method suggested that
Salmo salar CYPIA is most highly related to Oncorhyncus mykiss CYPIA genes of the
genes compared (Table 2). The phylogenetic tree also showed that Atlantic salmon
CYPIA is related to representative teleost CYPIA genes, followed by mammalian
CYPIA cDNA’s, and finally members of Oncorhyncus mykiss CYP subfamilies 2 and 3.
The same analysis based on predicted amino acid sequences demonstrated the same
relationship (Table 3). The phylogenetic methods carried out here showed that Atlantic

sahnon CYPIA is a close relative of rainbow trout CYPIA genes.

Induction of C YPIA

In the Northern analysis, the CYPIA probe hybridized with mRN A approximately
2.7kb in length from salmon treated with B-Naphthoflavone (Figure 4). Total RNA
extracted from control salmon did not show a visible hybridization band. In addition, a
probe designed fiom an amplified PCR product of the Atlantic salmon B-actin gene

hybridized with mRNA of approximately 1.2kb from both control and induced samples

11

with both bands showing roughly the same density. This suggests the difference in
CYPIA mRNA levels between control and induced sahnon is not due to different loading

of total RNA (data not shown).

12

DISCUSSION

There are several lines of evidence that suggest the cDNA I have cloned is a full-
length CYPIA molecule from Atlantic sahnon. It contains all of the positional
characteristics of a full-length coding cDNA including the start codon and a stop codon
followed by the poly A tail. This cDNA also carries qualities characteristic of many
teleost CYPIA genes such as the heme-binding domain, arginine codon integral to
enzymatic function, and a rather large 3’ UTR (1025bp). The coding region (1569bp),
which encodes a protein of 523 amino acid residues, is the same size as the rainbow trout
P4501A protein. This gene shows 97.4% and 96.7% sequence identity with rainbow
trout CYP1A1 and CYP1A3, respectively. The deduced amino acid sequence of this
salmon gene is highly homologous to rainbow trout CYP1A1 and CYP1A3 proteins
(96.9% and 96.2% respectively). Furthermore, this gene was demonstrated to be
inducible by BNF, a strong characteristic of CYPIA genes. This is the first sequenced
cytochrome P450 gene in Atlantic salmon.

It is especially interesting that this gene has a 3’ UTR as long as 1025bp. The
RNA sequence AUUUA recurs frequently in the 3’ UTR of many CYPIA genes. In
mammalian species, where CYP1A1 genes generally have 1 or 2 AUUUA (Sagarni et al.
1991), this sequence has been postulated to be involved with RNA degradation (Fukuhara
et al. 1989; Shaw and Karnen 1986; Binder et al. 1989). This property of 3’ UTR’s in
teleost CYPIA molecules has not been reported or discussed previously. Atlantic salmon
CYPIA has 3 AUUUA sequences in the 3’ UTR suggesting this RNA is likely to be

targeted for rapid removal in cells.

13

Whether Atlantic salmon has two CYPIA genes needs to be explored. It has been
demonstrated that rainbow trout have two separately functioning CYPlA genes, CYP1A1
and CYP1A3 (Bemdtson and Chen 1994). Salmo, which is the sister genus of
Oncorhyncus (Allendorf and Waples 1996), is likely to possess two CYPIA molecules in
all species of the genus. This raises two issues. The first issue dealt with isolation of one
single CYPIA gene. During initial PCR amplification, the primer sequences utilized in
these experiments showed 100% homology to both rainbow trout CYPIA genes. In
addition, the product amplified also showed less than a 1% difference in sequence
identity between both CYPlA genes in rainbow trout. Therefore, the RACE primers
initially utilized also were “non-distinguishing” between the possibility of two CYPIA
genes in Atlantic sahnon liver. The first CYPIA gene published in rainbow trout was
named CYP1A1 (Heihnann et a1. 1988). Recently, it has been reported that this gene
actually was a chimeric sequence incorporating both CYP1A1 and CYP1A3 character
(Rabergh et al. 2000). For that reason, even though overlapping 5’ and 3’ RACE CYPIA
products were produced, I was not sure whether this was a chimeric sequence. The
CYPIA genes in rainbow trout difi‘er mostly in their 5’ UTR. Therefore, I designed
another RACE primer in a nonhomologous 5 ’ region upstream from the start codon. The
long distance RACE product obtained using this GSP contained 32bp of the 5’ UTR as
well as the entire coding region and 3’ UTR. Therefore, it is likely this cDNA is a single
CYPIA gene from Atlantic sahnon liver.

The second issue dealt with the naming of this gene. As discussed in previous
teleost CYPIA cloning papers (Morrison et al. 1998; Nelson et al. 1996), careful

considerations are essential to correctly assign nomenclature to each new CYPIA gene

14

published. In recent years, functional properties as well as extended sequence analyses
have become necessities in distinguishing families and subfamilies of CYPIA genes.
One of the major ways to distinguish between CYP1A1 and CYP1A3 in rainbow trout is
by looking for presence or absence of xenobiotic regulatory elements (XRE) in the 5’
flanking region of each gene. CYP1A1 contains no XRE’s while CYP1A3 contains two
XRE’s (Bemdtson and Chen 1994). Unfortunately, the 5’ UTR of this salmon gene isn’t
large enough to verify whether or not it contains XRE’s. Another possible way to assign
a name to the CYPIA gene from Atlantic salmon is by comparison to rainbow trout
CYPlAland CYP1A3. However, rainbow trout CYPlA genes show 96% homology
suggesting a more recent gene duplication event, and thus making comparisons in this
manner problematic. Conservatively, I have asSigned this Atlantic salmon P450 gene as
CYPIA. However, based on strict sequence comparison, it is likely this cDNA is a
CYP1A1 molecule.

The nucleotide sequence of CYPIA is useful as a tool for continued research on
Atlantic salmon. Highly accurate and expedient techniques now exist which use
sequence information to quantitatively assess expression levels of particular genes. One
of these techniques, quantitative PCR, has been used previously to study expression of
CYPIA genes (V anden Heuvel et al. 1994; Miller et al. 1999). Using CYPIA as a
biomarker in this manner will open a new avenue of pesticide research and hopefully will
shed light on some of the causes of Atlantic salmon decline over the last few decades.
For instance, this biomarker would make it possible to characterize the impact of
environmental exposure across many sahnon streams or even across entire regions, thus

making it easier for scientists to focus their efforts for salmon restoration.

15

Further studies are needed to characterize the family of cytochrome P450 genes in
Atlantic sahnon and to understand their complex diversity and evolutionary relationships
to the same genes of other teleosts and mammals. It is apparent that P450 genes have
been genetically modified in many ways to account for and metabolize the many
compounds with which fish interact throughout their life history. P450 genes from
subfamily 2 and 3 have been identified in rainbow trout. Based on the genetic similarity
of rainbow trout and Atlantic salmon, these gene subfamilies would be expected to exist
in Atlantic sahnon as well. Likewise, sequence and evolutionary comparisons are needed
from the Pacific salmon species to grasp knowledge of how salmonid species in general
have adapted to their changing environments.

In conclusion, I have cloned a single, hill-length CYPIA cDNA fiom Atlantic
salmon. This cDNA contains all functional domains of teleost CYPIA genes, is most
closely related to rainbow trout CYP1A1 through sequence homology comparisons and
phylogenetic analysis, and is BNF inducible. I also believe this gene is targeted for rapid

degradation due to the presence of AUUUA sequences in its 3’ UTR.

l6

CHAPTER TWO: Quantitative PCR analysis of CYPIA induction in Atlantic salmon

(Salmo salar)

ABSTRACT
CYPIA genes are highly inducible by a broad range of environmental pollutants
(Goksoyr and Husoy 1998, Croce et al. 1995) and are suspected of causing declines in
populations of Atlantic salmon (Salmo salar; Fairchild et al. 1999). I developed a highly
sensitive technique, quantitative reverse transcription-polymerase chain reaction (RT-
PCR), for measuring the levels of induction of salmon CYPIA gene expression. Two
groups of 100 salmon maintained at 11°C and 17°C received an intraperitoneal injection
(50mg kg'l) of either B-Naphthoflavone (BNF) in corn oil (10mg BNF ml'l corn oil) or
corn oil alone. After a 48 h exposure sahnon gill, kidney, liver, and brain were collected
for RNA isolation and analysis. The highest base levels of CYPIA expression (2.56
X109 molecules/ 100ng RNA) were found in gill tissue. In addition, all tissues show
induction of CYPIA by BNF. Kidney had the highest mean induction at 5 orders of
magnitude while gill tissue showed the lowest mean induction at 2 orders of magnitude.
This technique was also applied to salmon sampled from two Massachusetts streams.
Salmon liver and gill tissue sampled from Miller’s River (South Royalston, Worcester
County), known to contain polychlorinated biphenyls (PCB), showed on average a 2
order of magnitude induction over those collected from a stream with limited
contamination, Fourmile Brook (N orthfield, Franklin County). These results suggest that
quantitative PCR analysis of CYPIA expression is advantageous in studying ecotoxicity

on populations of wild Atlantic salmon.

17

INTRODUCTION

Atlantic salmon populations across Eastern Canada and the United States have
been suffering a steady decline for the past 30 years (Anderson et al. 2000). This decline
has resulted in the listing of Atlantic salmon as an endangered species in the state of
Maine as of November, 2000 (U .S. Department of Interior 2000). Fairchild et al. (1999)
suggested that endocrine disrupting chemicals inhibit salmon development during stream
life, home stream imprinting, or possibly smoltification. Other compounds such as
polyaromatic hydrocarbons, polychlorinated biphenyls, dioxins, and furans can also have
physiological and pathological effects on fish populations at sublethal concentrations
(Goksoyr and Husoy 1998). These compounds; and other pesticides are known to
stimulate expression of various members of the cytochrome P450 family of genes (Beyer
et al. 1996; Hodgson et al. 1995).

The cytochrome P450 detoxification system is involved in the metabolism of
compounds such as steroids, prostaglandins, eicosanoids, drugs, and xenobiotics (Nelson
et al. 1996; Larsen et al. 1992). It is an extensively studied enzyme system and has been
found in bacteria, plants, and animals. Cytochrome P450 genes are highly diverse
(Buhler et al. 1998; Sarasquete and Segner 2000). Approximately 120 different
subfamilies of cytochrome P450 (CYP) genes have been identified (Nelson et al. 1996)
and characterized by a wide range of xenobiotic-metabolizing functions.

The most intensively studied cytochrome P450 molecule is arguably P4501A1.
Its gene (CYP1A1) is highly inducible by polyaromatic hydrocarbons (PAH’s),
polychlorinated biphenyls (PCB’s), furans, and dioxins. The mechanism of this induction

has been examined closely in rainbow trout (Buhler et a1 1998; Cao et al. 2000; Porter

18

and Coon 1991). There are many factors other than environmental pollutants that also
affect the expression of this gene such as stage of development, age, temperature, season,
species, and genetic strain differences (Grosvik et al. 1997; Vignier et al. 1996; Goksoyr
and Larsen 1991). Because this gene is inducible by a wide variety of compounds, it has
been utilized as a biomarker for detecting environmental contamination and stress in fish
populations (Anderson et al. 1995).

Studies of CYPIA inducibility and expression in fish have previously relied on
techniques such as Northern blotting, Western blotting, ELISA, or 7-ethoxyresorufin O-
deethlyase (EROD) enzyme kinetics (Anderson and Goksoyr 1994; Croce et al. 1995;
Schlezinger and Stegeman 2001; Grosvik et al. 1997). These methods, although
informative, require much fish tissue, time, and are often qualitative rather than
quantitative in nature. In the last 10 years, a new and innovative technique for measuring
gene expression, quantitative reverse transcription-polymerase chain reaction (RT-PCR),
has been developed (Cousinou et al. 2000; Miller et al. 1999). As reported by Vanden
Heuvel et al. (1993), RT-PCR is at least 10 fold more sensitive in detecting CYPIA
induction over EROD activity and radioirnmunoassay and at least 100 fold more sensitive
than Northern or slot blotting in measuring CYPIA RNA. Also, because PCR is an
amplification process, only a very small amount of tissue is required for analysis. A
requirement in studying P450 levels with quantitative PCR is one must know the
nucleotide sequence of the CYPIA cDNA in order to design highly specific primers for
use in the procedure. The sequence of CYPIA in Atlantic salmon has been determined

(see Chapter 1, this thesis).

l9

The immediate goal of this study is to develop a sensitive and expedient assay to
study toxicity responses of Atlantic sahnon to persistent organic contaminants known to
induce the CYPIA gene. This paper will show that quantitative PCR is a highly
advantageous technique in studying P450 expression in Atlantic salmon in both lab-based
experiments and specifically biomarker response studies with wild populations. There
were two parts in developing this study. The first part attempted to develop the
quantitative PCR method to study CYPIA induction using Atlantic salmon as a subject in
the laboratory. Secondly, I applied the PCR method to assess toxic responses of wild
Atlantic salmon. The ultimate goal of my study is to develop a highly sensitive
biomarker to study the toxicity effects of PCB’s, dioxins, and polyaromatic hydrocarbons

on wild Atlantic salmon populations.

20

MATERIALS AND METHODS

Fish handling and sampling

For the laboratory induction study, 200 juvenile Atlantic salmon weighing 35g i
7g and 15cm 1 2cm in length were acquired from Adirondack Fish Hatchery, Saranac
Lake, NY and transported to Michigan State University where they were acclimated for 2
weeks at 11°C. The fish were then divided into two equal groups for another two-week
acclimation period, one group at 11°C, the other at 17°C. Randomly selected individuals
were sampled through unbiased netting procedures and given either an intraperitoneal
injection of B-Naphthoflavone (BNF, Sigma Chemical Corp.; St. Louis, MO; 50mg kg'1
body weight) dissolved in corn oil (10mg ml") or corn oil alone. Individual salmon were
then placed for 48 h in an appropriate temperature 40 l flow-through aquarium (20 l h'l).
A 48 h exposure results in the maximum expression of CYPIA (Grosvik et al. 1997).
Injected sahnon were then sacrificed with an overdose of MS-222 (Sigma Chemical
Corp.) and tissues (gill, liver, brain, and kidney) were collected and immediately stored in
RNALater© at -20°C (Ambion; Austin, TX).

To sample wild salmon, 10 juvenile Atlantic sahnon were collected by electro-
shocking from two Massachusetts streams 25.8km apart, Miller’s River (South
Royalston, Worcester County) and F ourmile Brook (Northfield, Franklin County).
Fourmile Brook samples were collected on October 17th (2000) and Miller’s River was
sampled on November 8th (2000). Miller’s River, known to contain fish with tissue
concentrations of PCB’s between 0.8 — 5.5 ,ug/g (US Army Corps of Engineers 1995),

was expected to have salmon with higher levels of CYPIA expression. All tissues

21

collected were immediately stored in RNALater© and shipped to Michigan State

University for further analysis.

Total RNA isolation and storage

Tissue samples that had been stored in RNALater© (1 sample of liver, gill, brain,
and kidney from each injected salmon) were homogenized and total RNA was extracted
using Trizol Reagent (Life Technologies; Rockville, MD) according to the
manufacturer’s protocol. Total RNA was resuspended in 50 ,ul of diethylpyrocarbonate-
treated water (DEPC-HZO) and quantified (Sambrook et al. 1989) using a Beckrnan DU
7400 spectrophotometer (Fullerton, CA). Long-term storage of RNA samples was
canied out by adding 3 volumes of 95% ethanol, 1/10 volume of 3 M sodium acetate, and
placing at -80°C. Storage of RNA in this manner maintains stability of RNA for greater

than 6 months (Sambrook et al. 1989).

Internal standard synthesis

An internal standard (IS) that contained a T7 promoter, both CYPIA forward and
reverse primer sequences, and a poly-deoxythymidilic acid tail was synthesized by the
method of Vanden Heuvel et al. (1993) and is outlined in Figure 5. Briefly, using human
genomic DNA as a template for PCR, the WML53 5’-TAA TAC GAC TCA CT A TAG
GCT GTC TTG GGC CGT TGT GTA CCT TGT GCA ACT TCA TCC ACG TTC
ACC-3’ and WML54 5’-TTT TTT TTT TTT TTT TTT TAT CCT TGA TCG TGC AGT
GTG GGA TGG GAA GAG CCA AGG ACA GGT AC-3’ internal standard primers

(Macromolecular Structure, Sequencing, and Synthesis Facility, Michigan State

22

University) amplified a B-globin product of approximately 360bp under the following
conditions: 3 mM MgC12, 0.4 mM dNTP’s, 0.6nM forward internal standard primer, 0.6
nM reverse internal standard primer, a 1x concentration of PCR buffer, and 2.5 units of
Taq DNA Polymerase (all reagents were from Life Technologies). This reaction was

performed with 1 cycle at 94°C for 4 min, 30 cycles at 94°C for 20 sec, 59°C for 30 sec,

and 72°C for 30 sec, and 1 cycle at 72°C for 5 min. The size of the product was verified
on a 1% TAE agarose gel with a 100bp DNA ladder loaded (Life Technologies). I then
diluted this product 1/ 100 with DI H20 and reamplified the IS PCR product with the
same reaction conditions. Unincorporated primers and primer-dimers were cleaned from
the concentrated PCR product using the Wizard DNA Clean-Up System (Promega Corp.;
Madison, WI). Once a purified PCR product was obtained, in vitro transcription was
carried out using the Riboprobe In Vitro Transcription System (Promega Corp.)
according to standard protocol. The rcRNA was then treated with RNase-fiee Dnase
(Promega Corp.) to remove excess DNA template and subsequently extracted with water-
saturated (pH 4.9) phenol/chloroform (24:1). The aqueous phase was isolated and
extracted with chloroform/isoamyl alcohol (24:1) followed by an overnight ethanol

precipitation at —20°C. To remove free nucleotides, the precipitated sample was spun

down for 10min at 12,000g, resuspended in 20 pl DEPC-Hzo, and filtered through a G-
50 Sephadex column (Amersharn Pharrnacia Biotech; Piscataway, NJ) pre-equilibrated in
0.1% SDS. The filtered sampled was precipitated overnight. After spinning the sample
for 10min at 12, 000g the rcRNA pellet was washed with 70% ethanol, resuspended in

DEPC-HZO, and quantified with a UV spectrophotometer.

23

Competitive RT -PCR

It is necessary to gain an estimate of the levels of P450 mRNA in each tissue prior
to spiking each RNA sample with internal standard. A standard curve was generated for
each tissue and treatment to be analyzed (data not shown for all tissues) by co-reverse
transcription and co-amplification of a constant amount of total RNA (100ng) against a
dilution series of internal standard (1010 molecules —— 103 molecules). These “range
finding” experiments allow one to determine the relative levels of a particular gene of
interest between several tissues and/or treatments (V anden Heuvel 1998). These
preliminary reactions help to determine the exact amount of internal standard to spike
into each sample. Once initial range-finding experiments were concluded for each tissue,
it was determined that it would be possible to use only one of the standard curves
generated, thus reducing errors introduced through the use of several curves. Next,
reverse transcription (all reagents were fiom Life Technologies) was performed on all
samples in a final volume of 20 pl containing a 1x concentration of First Strand Buffer,
0.01 M dithiothreitol, lmM of each deoxynucleotide triphosphate, 2.5pM oligo(dT)18, 5
units of MMLV reverse transcriptase, 1 unit RNAsin (Promega Corp.), 100ng of total
RNA, and varying amounts of internal standard predetermined from initial range-finding
experiments. The reaction mix was incubated at 42°C for 50 min and inactivated at
70°C for 15 min. Immediately, 1 unit of RNase H (Life Technologies) was added and
then each reaction was incubated at 37 °C for 20 min, inactivated at 94°C for 5 min, after
which 2 pl were taken and spiked into a PCR master mix. The 50 pl PCR mix contained
3 mM MgC12, 2.5 units Taq Polymerase, 30 pmol of each hex-labeled (Integrated DNA

Technologies; Coralville, IA) forward and reverse primer (WML51 5’- CTG TCT TGG

24

GCC GTT GTG TAC CTT GTG-3’ and WML52 5’- TAT CCT TGA TCG TGC AGT
GTG GGA TGG-3’), and 0.4 mM dNTP’s. A “hot start” was utilized where each
reaction was heated to 94°C for 2 nrin afier which Taq was added. Then, the reactions
were heated to 94°C for 4 min, followed by 30 cycles of a 94°C denaturation for 20 sec, a
70°C annealing step for 30 sec, and a 72°C extension step for 30 sec. An additional 5
min extension step was included at the conclusion of the 30 cycle main reaction.

Because the efficiency of reverse transcription and PCR varies from tube to tube
(V anden Heuvel et al. 1994), four controls were used in my reactions. First, the internal
standard controls for variability of reverse transcription and PCR amplification. The IS is
roughly the same size as the target gene product and contains the same primer recognition
sequence, thus it should amplify at the same efficiency as the target gene. Secondly, to
assure IS was spiked at the expected concentration, a blank IS RT-PCR reaction was also
included and visualized. Thirdly, the laser scanner used to visualize PCR products was
calibrated across all gels by loading an absorbance standard (AS), which was simply a
1/10th dilution of a single P450 PCR product. Finally, to assure the total RNA was
loaded for each RT-PCR reaction at expected levels, I adapted the standard procedure
used in many quantitative PCR studies (V anden Heuvel et al. 1994; Loitsch et al. 1999).
I sampled several cDNA samples that corresponded to lower or higher levels of P450
compared to other samples in a group of Atlantic salmon. Using hex-labeled primers
ACT 1N1 and ACTIN2 designed fi'om an Atlantic salmon B-actin cDNA (Rogers et al.
1999, GenBank Accession AF 012125) fragment confirmed by sequencing, I amplified a
fi'agrnent of the actin gene using 25 cycles of the same conditions as was used for P450.

If actin fragment amplification was equal across all samples I then concluded that any

25

difference in P450 levels in the corresponding samples was due to individual variation

and not experimental error introduced by RNA loading.

PCR fragment visualization and data generation

PCR products were electrophoresed on a 4% non-denaturing polyacrylamide
(BioRad; Hercules, CA) gel at 20 V cm'l. The size of the products was verified using a
hex-labeled MAPMARKERT" molecular size standard (Bioventures Inc.; Murfreesboro,
TN). Densitometric readings were calculated using an FMBIO H Laser Scanner (Hitachi
Genetic Systems; Alameda, CA) and software (ReadImage version 1.5, Analysis v8.0).

Target RNA was computed as described by Vanden Heuvel (1998).

Statistical analysis

The data (estimated copies of RNA) were transformed logarithmically to bring
data to normality. The main effects and possible interactions of treatments in laboratory
induction experiments were analyzed with a 2-way AN OVA. For samples collected from
streams, student t-tests were utilized for detecting differences between streams. All

analyses were carried out using Statistical Analyses System (SAS Institute; Cary, NC).

26

RESULTS

Internal standard and standard curves

A PCR reaction using internal standard primers WML53 and WML54 amplified a
fiagment from human genomic DNA approximately 360bp long. Transcription of this
PCR product yielded the rcRNA molecule of approximately 360bp. Reverse transcription
of this rcRNA molecule and subsequent PCR amplification using primers WML51 and
WML52 resulted in a cDNA of approximately 320bp. The observations of these IS
products are in accordance with expected results.

Standard curves for all tissues and treatment groups were estimated and showed a
correlation coefficient (r) of 0.85 or higher. The standard curve used for this study
obtained from liver tissue is shown in Figure 6. The point at which log [mRNA/IS] = 0
tells the amount of internal standard to spike into the RT reactions. It was determined
that a range of internal standard concentrations (1x107-1x109 molecules per reaction)
could be used for subsequent analysis. After these initial range-finding experiments, final
computation of P450 RNA in all tissues was estimated through the use of one

representative standard curve (Figure 6).

Induction of C YPIA in brain, gill, liver, and kidney in hatchery-raised salmon

Mean levels of CYPIA mRNA are reported for all treatment groups in Table 5.
Representative gel pictures of these results are given in Figures 7 and 8. In a controlled
laboratory setting, CYPIA mRNA was affected by treatment with BNF in all four tissues
(gill, liver, kidney, and brain; ANOVA, p < 0.05). Gill tissue demonstrated the highest

overall base level of P450 expression at 2.56 x 109 molecules per 100ng total RNA. The

27

lowest base level, 6.52 x 105 molecules per 100ng total RNA of P450 expression, was
seen in brain tissue. Kidney tissue showed the greatest induction potential from base
levels with a mean induction of ~4 orders of magnitude. The lowest mean induction for
the tissues studied was in gill at approximately 2 orders of magnitude. In all cases, base
levels of CYPIA mRNA were lower in salmon maintained at 17°C than salmon
maintained at 11°C (ANOVA p < 0.05). Overall, ANOVA indicated that no interactions
existed between BNF treatment and temperature (p > 0.05).

In cases with highly variable results, total RNA samples were analyzed with actin
amplification to make sure initial RNA concentrations were accurately quantified and
diluted. A representative gel picture for actin visualization is given in Figure 9. These

results showed that initial total RNA dilutions Were accurate.

C YPIA expression in wild salmon

The quantitative PCR analysis showed that Miller’s River sahnon CYPlA levels
were on average approximately 100 times greater in both gill and liver tissue than salmon
sampled from Fourmile Brook. Representative gel pictures of these results are given in

Figure 10 and a summary of these results are given in Table 6.

28

DISCUSSION

I have demonstrated that CYPIA is highly inducible in Atlantic salmon gill, liver,
kidney, and brain tissues. Each tissue showed at least 2 orders of magnitude induction
over control levels. Interestingly, gill tissue samples had the highest base levels of P450
expression. Generally, it has been reported that liver tissue demonstrates the highest
levels of CYPIA (Goksoyr and Husoy 1998). It is likely that high concentrations of
CYPIA do exist in the gill lamellae for the following reasons. The gill constitutes less
than 1% of the body weight of a fish but more than 90% of the surface area. It is a
primary route of exposure for water borne contaminants, and an important secondary
route for ingested compounds since it directly receives all of the blood flow fi‘om the
heart. In addition, it was discovered that an induction limit exists across all tissues. All
inductive responses seen in the laboratory study crested at approximately 1x10ll
molecules per 100ng total RNA 1 order of magnitude except for brain tissue. This
result could possibly be due to the presence of AUUUA sequences, which is believed to
be involved with degradation of mRNA molecules, in the 3’ untranslated region of
CYPIA (in Chapter 1, this thesis). Even in kidney tissue, which showed the second
lowest (brain tissue had the lowest) base levels of CYPIA expression, induced samples
had a mean mRNA level of ~ 1x10ll molecules per 100ng total RNA. Therefore, I
conclude that kidney tissue has the highest induction potential of all tissues. Brain
mRNA levels appeared to have a delayed response to BNF induction, perhaps simply due
to the fact that liver, kidney, and gill CYPIA metabolized the majority of the injected

sample before it reached the brain. Brain CYPIA levels also could have been less

29

pronounced due to a blood-brain-barrier although this possibility has not been
investigated.

I also discovered that there was an overall mean decrease in CYPIA expression in
all tissues during acclimation at higher temperatures (AN OVA p < 0.05). Previous
studies on P450 levels have also found significant effects of temperature (Grosvik et al.
1997; Andersson and F orlin 1992). There are several possibilities that may explain this
finding. The first is the solubility of BNF increases with increasing temperatures thus
making it easier physiologically for salmon to rid the body of the contaminant. However,
this seems unlikely since we are only dealing with an increase in temperature of 6°C.
Another possibility is increased temperatures suppress the Ah-receptor complex which
mediates production of CYPIA mRNA transcripts (Goksoyr and Husoy 1998). Thirdly,
there may be a decreased activation of heat shock protein complexes that are involved
with stimulation of the Ah-receptor complex (Goksoyr and Husoy 1998). This last
possibility could also be related to suppression of the Ah-receptor complex.

I developed a standard curve-based quantitative PCR method to assess expression
levels of CYPIA in Atlantic salmon gill, kidney, liver, and brain tissue. This method was
utilized in both a controlled lab induction study as well as on samples from natural
streams. I believe developing quantitative PCR assays such as this will be useful in the
following years in monitoring the current status of Atlantic salmon populations, helping
to discover cause and effect relationships for the reasons behind Atlantic salmon decline,
and providing solutions to establish sound management plans for restoration of wild

salmon.

30

Recently, a quantitative PCR study was carried out using standard curves with the
Antarctic fish Trematomus bernacchi and the effects environmental pollution has on
P450 levels in this species (Miller et al. 1999). The current study and the Miller paper
contradict traditional quantitative PCR experiments where single samples are quantified
using a dilution series of internal standard for each sample (V anden Heuvel et al. 1993).
Using standard curves in quantitative PCR studies allow for more samples to be analyzed
in a shorter amount of time while still producing results that correspond to results seen in
traditional quantitative PCR experiments (Tsai and Wiltbank 1996). In fact, the time
between sampling to production of results can be reduced to just three days. Overall, the
quantitative PCR experiment becomes more economical without reducing accuracy and
sensitivity. Finally, quantitative PCR is both quantitative and qualitative in nature, a
contradiction to traditional RNA-based biomarker analysis where qualitative data is the
result. This characteristic can enhance understanding of biomarker responses seen in
Atlantic salmon and other species.

Finally, further research is needed in studying the overall effects PCB’s and
similar compounds are having on the health of Atlantic salmon. I have demonstrated that
it is possible to assess CYPIA levels in wild Atlantic salmon using quantitative PCR.
Using this method, it would be possible to quantify CYPIA levels in Atlantic salmon
through gill biopsy sampling, a definite advantage from the viewpoint of restoration and
conservation. This type of sampling could be performed on either adult or juvenile fish.
However, knowing how CYPIA is altered in response to PCB’s and other compounds is
not enough. There is a need to acquire better physiological knowledge of how increased

levels of CYPIA correlate with levels of sex steroids or hormones, activity of Na+,K+-

31

ATPase, or other metabolic processes. In addition, further research is needed to establish
a distribution of PCB related contamination in New England rivers and streams. Using
quantitative PCR as a tool, it would be possible to quantify the CYPIA levels of different
Atlantic sahnon runs across the east coast, thus making it easier to focus research projects
and restoration efforts. Moreover, the possibility exists to adapt quantitative PCR to study
other gene expression in fish, such as olfactory receptor expression during different life
stages. Some pesticides, namely carbofuran at a concentration of 1-6ppb, can abolish
olfactory sensitivity in fish (Waring and Moore 1997). Olfactory function is essential for
juvenile imprinting and the ability for adult sahnon to find their natal stream for
spawning. Thus olfaction is integral for survival of this species. It is still unknown why
Atlantic salmon stocks are decreasing across the Eastern portion of North America. Is it
a case of endocrine disruption? What is causing increased precocity in sahnon
populations? Could this effect be due to abolishment of olfactory sensitivity during
exposure to low levels of pesticides? I feel with the usefulness of a biomarker such as
CYPIA expression as well as the analytical power of quantitative PCR these questions

can be answered.

32

Table 1: Primers used in PCR and RACE amplifications of CYPIA cDNA.

 

 

Application Nucleotide sequence Primer Name

PCR 5’-CCT TGK KRA TGA ART AGC CMT TGA GG-3’ WML38
5’-CCA ACR TMA TCT SWG GMA TGT GC-3’ WML39

RACE 5’-CTG TCT TGG GCC GTT GTG TAC CTT GTG-3’ WML51
5’-TAT CCT TGA TCG TGC AGT GTG GGA TGG-3’ WML52
5’-GCT CAG TTC CTG ATG CAG TCT TTC CTG-3’ WML55
5’-CGG CTC ATT TGG CT C ATA ACG GAA GAT-3’ WML56

 

33

Table 2: Phylogenetic analysis of cytochrome P450 genes.
Gene accession numbers and references for the sequences used in this analysis are
given in table 4. Multiple sequence alignment was carried out using the Clustal
W algorithm (1515 nucleotides of the coding region). The phylogenetic tree and
genetic distances were determined using the Neighbor-Joining Method.

Cytochrome P450 nomenclature followed that of Morrison et al. 1998.

 

Diccntrarchus labrax CYPIA
Chactodon capistratus CYPIA

i ““" Stcnotomus chrysops CYPIA
r—{': Limanda limanda CYPIA

 

Fundulus heteroclitus CYPIA
Liza salicns CYPIA

Opsanus tau CYPIA

Micro gadus tomcod CYPIA

It Oncorhyncus mykiss CYPlAlVI

 

 

 

 

 

 

Oncorhyncus mykiss CYP1A1V3
Oncorhyncus mykiss CYP1A1V2
L Oncorhyncus mykiss CYP1A3
Salmo salar CYPlA
" Anguilla japonica CYPIA
l: Homo sapicns CYP1A1
Macaca iris CYP1A1
—— Mus musculus CYP1A1
l—‘—- Homo sapicns CYPlBl
L——— Rattus norvegicus CYPlBl
Oncorhyncus mykiss CYP2K1
65 5 Oncorhyncus mykiss CYP3A27

I I l 1 I I I

60 50 40 30 20 10 0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

34

Table 3: Phylogenetic analysis of cytochrome P450 proteins.

Analysis was carried out as described in Table 2 utilizing 505 amino acid residues

of each enzyme. Accession numbers for the genes encoding these enzymes are

given in table 4.

120.5

 

— Fundulus heteroclitus CYPIA
—- Liza salicns CYPIA

--—— Diccntrarchus labrax CYPIA
E Limanda limanda CYPIA

 

Stenotornus chrysops CYPIA
L. Chactodon capistrahrs CYPIA

. Opsanus tau CYPIA

Micro gadus tomcod CYPIA
Oncorhyncus mykiss CYP1A1V1
Oncorhyncus mykiss CYP1A1V3
Oncorhyncus mykiss CYP1A1V2

 

:—--=----- Oncorhyncus mykiss CYP1A3

Salmo salar CYPIA

 

 

 

Anguilla japonica CYPIA
Homo sapicns CYP1A1
Macaca iris CYP1A1

 

 

 

 

 

 

._._ Mus musculus CYP1A1
r—— Homo sapicns CYPlBl
L— Rattus norvegicus CYPlBl

 

Oncorhyncus mykiss CYP2K1

 

Oncorhyncus mykiss CYP3A27

 

120

100

35

1

Table 4: CYP genes and accession numbers used in the phylogenetic analysis.

 

 

Name in analysis (General Name) Reference GenBank Accession Number
Fundulus heteroclr'tus CYPlA (Killifish) Morrison etal., 1998 AF026800
Liza saliens CYPlA (Mullet) Sen et al., 1999 AF072899
Dicentrarchus labrax CYPlA (Seabass) Stien et al., 1998 U783l6
Limanda limanda CYPIA (Sand dab) Robertson, 1997 M001724
Stenotomus chrysops CYPIA (Scup) Morrison et al., 1995 Ul4l62
Chaetodon capistratus CYPIA (Butterflyfish) Vrolijk et al., 1995 Ul9855
Opsanus tau CYPlA (Toadfish) Monison et al., 1995 Ul4161
Microgadus tomcod CYPIA (T omcod) Roy et al., 1995 1.41886
Oncorhyncus mykiss CYPlAlVl (Rainbow trout) Bemdtson and Chen, 1994 869278
Oncorhyncus mitts: CYP1A1V3 Bailey et al., 1997 U62797
Oncorhyncus mykiss CYPlAlVZ Bailey et al., 1997 U62796
Oncarhyncus nay/tics CYP1A3 Bemdtson and Chen, 1994 869277
Salmo salar CYPlA (Atlantic salmon) Rees 2001 (this paper) AF 361643
Anguilla japonica CYPIA (Japanese eel) Mitsuo et al., 1999 AB020414
Homo sapiens CYP1A1 (Hurmn) Jaiswal et al., 1985 K03l9l
Macaca iris CYP1A1 (Crab eating monkey) Ohrnachi et al., 1993 Dl7575
Mus musculus CYP1A1 (Mouse) Kimura et al., 1984 Y00071
Homo saplens CYPlBl Sutter et al., 1994 U03688
Rattus norvegicus CYPlBl (Rat) Battacharyya et al., 1995 X83867
Oncorhyncus mykiss CYP2K1 Buhler et al., 1994 L11528
Oncorhynau mykiss CYP3A27 Lee et al., 1998 U96077

 

36

Table 5: Quantitative RT-PCR analysis of CYPIA levels in tissues of hatchery salmon.
Each value represents the mean number of CYPlA mRNA transcripts per 100ng
total RNA for each treatment group (n = 6-9). Induced animals were treated with
50mg kg'l BNF (see Methods section). In all four tissues, the control P450
mRNA level was significantly lower than the induced P450 mRN A level (p <
0.05). In addition, for each tissue, there was a significant decrease in P450
mRNA during increased temperatures (p < 0.05). No interaction was seen
between temperature and BNF treatment (p > 0.05).

Low Temp = 11°C; High Temp = 17°C

 

 

 

 

 

 

 

 

 

 

 

Control , Induced
Tissue High Temp Low Temp High Temp Low Temp
Gill 2.56E+09 2.03E+11 3.94E+11 2.29E+12
Liver 1.25E+07 9.00E+08 1.07E+11 5.76E+11
Kidney 1.21E+06 2.79E+06 1.00E+10 2.49E+11
Brain 6.52E+05 2.86E+06 1.39E+09 3.17E+09

 

37

 

Table 6: Quantitative RT-PCR analysis of CYPIA expression in tissues of wild sahnon.
Each value represents the mean number of CYPIA mRNA transcripts per 100ng
total RNA for each tissue sampled (n = 7). Miller’s River P450 mRNA levels
were significantly higher than Fourmile Brook in both liver and gill tissue

samples (p < 0.05). See Methods section for description of rivers.

 

 

 

 

 

 

 

mRN A Levels
Tissue Fourmile Brook Miller's River
Gill 6.39E+07 1.43E+10
Liver 1.29E+09 1.97E+11

 

 

38

   

A. B. C.
Figure 1: Electrophoretic analysis of amplified CYPIA cDNA’s. All of these 1%
agarose gels were stained with ethidium bromide (0.1pg/ml).
A) RT-PCR products using primers WML38 and WML39. Representative lanes are:
(l) CYPIA PCR product using Salmo salar liver cDNA as the template; (2)
negative control with no template loaded; (3) 100bp DNA ladder (Gibco BRL).
B) 5’ and 3’ RACE-PCR products.
Gene specific primers (GSP) were WML51 for 3’ RACE and WML52 for 5’
RACE. Representative lanes are: (l) 5’ RACE; (2) GSPl + GSP2 positive
control; (3) GSPl negative control; (4) 3’ RACE; (5) GSPl + GSP2 positive
control; (6) GSP2 negative control (7) 100bp DNA ladder.
C) 3’ long distance RACE product using GSP’s WML52 and WML56.
Representative lanes are: (1) 3’ RACE showing 2.7kb RACE product and (2)

100bp DNA ladder.

39

TGTWMAMMMTMATAMMTGGTTCTCATGATAC TACOCATTATTGU: TCABTCTC TGTGTCTGAGGGGC TGGTGGOCATN 88

T V L H I L P I I G S V 8 V S E G L V A I 21
TAACACTATWC TGGTGTMATGTTCATMTWMMATCCCWTGAMCGGCTCCCAGGACCAAAGOCCC TGCCOATOA 1 82
VTLCLVVUFHKYKHTEIPEGLKRLPGPKPLPI 53
TCGGMATGTGOTGGABGTGCMAACAACCCTCAOCTOAGCCTGACTGCCATGAGTGAGCGCTACGGCTCAGTCTTCCABATCCAGATWTGC 288
IGNVLEVHNNPHLSLTAISERYGSVFOIOIGI 85
MTGTGGTTGTTCTGABTGGOAGCGAGACAGTOCGCCAGGCTCTTATCMGOMGGGGMGACTTCGCCGGGAGGCCCGATCTATAOABCTTCA 384
RPVVVLSGSETVROALIKOGEDFAGRPDLYSF 117
ABTTCATCAACGACNCAABAOCTTGGCCTTCAGCACCWTGGGGTATGGCGCGCCCGCCGCMGCTAGCTATGAGCGICCTCCGCTCTT 480
KFINDGKSLAFSTDKAGVWRARRKLAHSALRS 148
TWTWTOGACCCCAGAGTACTOCTGTGOCCTWGTCTGCWGTACCTGGTMAACAOCTGACCTOCG 578
FATLEGSTPEYSCALEEHVCKEGEYLVKOLTS 181
TCATWTGTOAGTMCABCTTTGACCCCTTCOGOCATATTGTOGTATONTGGCCAACGTCATCTGTGGAATGTGCTTCGGCOGGCGCTW 872
VUDVSGSFDPFRHIVVSVANVICGHCFGRRYs 213
ATMTWTGTTWTTGGTGAACTTGMTGATGAGTTTWTGGTGMCABCWCCTGCABACTTCATTCOGATCCTTC 788
HDDOELLSLVNLSDEFGOVVGSGNPADFIPIL- 245
GTTMCTACCAMCCGCACCATWTTTATGGATATOMTGACCGTTTOAACBCCTTTGTGCAGMGATTGTGAGTGAGCACTATWT 884
rYLPNRTHKRFIDINDRFNAFVOKIVSEHYES 277
ATWMOATCOGTGACATCACTGACT000TCATTGACCACTGTGAGGADAWTAGATGAGAATGCCAACATCCAGGTTTCTGATG 880
YDKDNIRDITDSLIDHOEDRKLDENANIOVSD 308
MWGTMATTGTCMTGATCTGTTTWAGGTTTTGACACTATCAWACAGCTCTGTC TTGGGCTGTTGTGTACCTTGTMCTTMC 1 058
EKIVGIVNDLFGAGFDTISTALSWAVVYLVAY 341
CMTCCAGGMAGAC TGCATCWTGACWTGMATTGAATCGCACTGCCCGTCTC TCAGACMAACCAACTTACCTC TGCTGG 1 152
PEIOERLHOELTEKVGLNRTPRLSDKTNLPLL 373
AABCCTTCATCCTGGMATCTTCCWACTOTTCGTTCC TGCCGTTCACCATCCCACAC TGCACGATCMGGATACATOCCTCMTGGCTACTTCA 1 248
EAFILEIFRHSSFLPFTIPHCTIKDTSLNGYF 405
"WCTBTGTCTTCATCMCCMTWAGGTCMOCATMCCCGGAGC TGTGGAAGGAGCC TTC TTTATTCAACCCTGADCGTTTOC 1 344
IPKDTCVFINOWOVNHDPELWKEPSLFNPDRF 437
TGAGTNTGATGGCACAGAM:TCAACAAGOTWTGCTOGTATTCGGCATGGGCMGCGCCGCTGCATCGGTGAGGCCATCM 1 440
LSADGTELNKLEGEKVLVFGIGKRRTIGEAIG 488
WW“:TACCTCTTCTTGGCCATCCTWTOCAMNCTGOWTTOCWC TWACOCGCTGGACATGACCOCAGABTW 1 538
RNEVYLFLAILLOBLRFOEKPGHPLDHTPEVG 501
TCACTATMACAAMGCTGTCAGC TmTAGCCTGCGGOCATGMGGCAGGAGGAGTGAAGGACATGGTCACATTTATGATTC TOWAT 1 832
LTMKHKRCOLKASLRPWG'OEE— 522

OACTATAAC TGATTTATAGTTAGCCCTACATABTTGATGGCATGAGATGAGTTCAGATTTMAAGCCAGAGGGTACATTTTCTO TOTAW 1 728
WTTWCTTMTTCATWTTMCMTWTGAGWI’GTABACMTTTGMATCGATAMATTATMTCTG 1 824
MATMTATGTMGGGGATTACMACMGTGTTGTATTGGATMAGGACCTTCATGACCAGCATACCACAGGGCC TAC TAGAGTTTGTCAGGTGTT 1 820
NTTCAGAAGATATCAGGAGAGACTATTGCTGTTTAGC TGCCTTTTTGCCTACATCATCGTGTTTTAC TGC TCCTCTTGCCTTGACCACABC TCAT 2018
ATAC TGTATCAGCTCGCTTTMWTCMTC TATAAMCATACACATMACCCATAGTGGGGTTTTMAGAGCATTGTTTTTTAGTTCCATGT 21 1 2
TTGGGTATMTTTTTTGC TMAGMTTTTTTGAGGCAMMTGTTACMCC TACATGATMAGTGCCTGTTC TCGTAC TTTTGAGTTATTGTTCAG 2208
GTGGTCACAGACAGAGTTGMTGGATGTATCCTCCCMTTC TMGCTAGATATTTTTATTCCACTGMTMATCAGTCMATGTGGACACMCACA 2304
CTGAABC TATGTTTGTATCCCACCMCATGTGATTGAT TGTTTGAGTACATTTTTMGCCTAGATGTATTTTGAAMGTCCTTTTTTGMATGTGC 2400
CTAMATGTGTATATATTGTGGTGGCTTTGTATGACTTTATCAGGATGCCAMATACGTATGTATTAC TGAC TGTGTTATMATGTCAACATTTTA 2488
TMTGTAADWTCTTGTMACTAAMOCCCAACTGTATACACTATGCATTGTGTTGTTMTAGCTATCTAGGTAGC TAC TGAMTW 2582

TWAWWMMAMAM 2828

Figure 2: cDNA and deduced amino acid residue sequence of Atlantic salmon CYPIA.
The start codon, arginine residue critical to enzymatic function (position 246),
heme-binding cysteine codon (position 463), stop codon (positon 523), ATTTA

(AUUUA) sequences, and putative poly-adenylation signal are all underlined.

40

FGMGKRRCI G ButterflyfishlA
FGIGERRCI G KillifishlA
FGMGKRRCI G MulletlA
FGMGKRRCI G SalmonlA
FGMGKRRCI G SanddablA
FGMGKRRCI G ScuplA
FGLGKRRCI G SeabasslA
FGIGKRRCI G ToadfishlA
FGMGKRRCI G TomcodlA
FOB/@KRRCI G TroutlAlVl
FGMGKRRCI G TroutlAlVZ
FGMGKRRCI G TroutlAlV3
FGMGKRRCI G TroutlA3

Vrolijk et al. 1995
Morrison et al. 1998

Sen et al. 1999

Rees (this study)
Robertson 1997
Morrison et al. 1995
Stien et al. 1998
Morrison et al. 1995

Roy et al. 1995
Bemdtson and Chen 1994
Bailey et al. 1997

Bailey et al. 1997
Bemdtson and Chen 1994

Figure 3: Alignment of heme-binding region,(amino acid residues 456-465).

Comparison of heme-binding regions fi'om representative teleost P4501A

enzymes. Boxed areas show amino acid residue differences from sahnon

P4501A.

4l

 

“in.

 

 

 

Control Induced

Figure 4: Northern blot analysis of CYPIA total RNA from Atlantic salmon liver.
Lane 1: untreated salmon liver; Lane 2: salmon liver treated with B-
Naphthoflavone. The 2.7kb CYPIA mRNA was hybridized with a digoxigenin-

labeled CYPlA firll-length cDNA probe.

42

WML53

A L

‘ 267b B-Globin Gene Human Genomic DNA 7
a? P ( )

 

11'

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

r7 WML 51
86 F Spacer Gene B GF
30 cycles PCR ‘QGF WML52 dm
1 WML 54
T7 WML 5‘ 30 F B-Globin Gene nor wm. 52 am

 

 

 

 

 

 

 

 

 

358bp Internal Standard (ds-DNA)

in vitro transcription

1

r7 WMLSl BGF B-Globin Gene BGF WMLSZ d(’1')

 

 

 

 

 

 

 

 

 

 

A
V

358bp Internal Standard (re-RNA)

Figure 5: Construction of internal standard.
A schematic flow diagram showing the steps for synthesis of the rcRNA internal
standard used in this quantitative PCR study (modified from Vanden Heuvel et

al,1993)

43

L19 Standard Curve

 

log [RNA/IS]

 

 

 

 

Figure 6: Generation of standard curve.
The standard curve used for quantifying the number of CYPIA transcripts in
100ng of total RNA. A constant amount of total RNA (100ng) was co-amplified
against a dilution series of internal standard (1010-103 molecules IS). Data points
used in the curve are generated by taking log absorbance [RNA/IS]/log
absorbance [IS].

Abbreviations: IS, internal standard; CYPIA, cytochrome P4501A

Samples Acclimated to 17° C

 

Brain Samples Gill Samples
W M u 321bp " r - ~ ..
AS NC . ls '3 43 NC» a .. .. .. .
5' -~ w 208bp .. w w
Control (n = 9) Induced (n = 9) Control (n = 7) Induced (n - 8)
Kidney Samples Liver Samples
M W "" 321bp W , he . _,
Netti e rs AS NCn. .. 1.. MASIS
-.._._ ---- -- --- w.- -' 208bp W - . ... -
Control (n = 8) Induced (n = 8) Control (n - 9) Induced (n =- 9)

Figure 7: Representative gel pictures for gill, liver, kidney, and liver samples acclimated
at 17°C. RNA (100ng) was co-amplified against a known concentration of
internal standard (IS). Induced sahnon received an intraperitoneal injection of B-
Naphthoflavone (50mg kg’1 body weight) while control salmon received an
injection of corn oil alone. Sample size is indicated in parentheses for each
treatment group. The bands near the top of each gel are the 321bp IS. The bands
at the bottom of each gel represent the 208bp CYPIA fragment. CYPIA mRNA

levels are determined by taking the density ratio of IS/CYPl A.

Abbreviations: NC, negative control (water control); AS, absorbance standard; IS,

internal standard positive control.

45

Samples Acclimated to 11 ° C

Brain Samples Gill Samples
W’ W 32191’ “ ..
NC ' lSAS NC «nnuAsrs
.-.... .. W " 208bp w- w .-
Control (11 = 9) Induced (n = 8) Control (n = 9) Induced (n a g)
Kidney Samples Liver Samples
W a. a...- h... .- 321bp ~' W a -
AS 1 l 3‘1-1 IS IS AS an enut- i
" ----s...a .~--..N6W ' 20891) v- n... W 11C '
Control (n = 8) induced (n -= 6) Control (11 ___ 9) in duced (n _ 8)

Figure 8: Representative gel pictures for gill, liver, kidney, and liver samples acclimated
at 11°C. RNA (100ng) was co-amplified against a known concentration of
internal standard (IS). Sample size is indicated in parentheses for each treatment
group. Induced salmon received an intraperitoneal injection of B-Naphthoflavone
(50mg kg'l body weight) while control salmon received an injection of corn oil
alone. The bands near the top of each gel are the 321bp IS. The bands at the
bottom of each gel represent the 208bp CYPIA fragment. CYPIA mRNA levels

are determined by taking the density ratio of IS/CYPlA.

Abbreviations: NC, negative control (water control); AS, absorbance standard; IS,

internal standard positive control.

46

Kidney Samples

321bp —> U W has in new. "Ms M

 

250bp —->

rs NC ,. ‘ AS
208bp —> , w W u 1......

Control Induced

Figure 9: Representative gel picture of actin normalization. RNA (100ng) was co-
amplified against a known concentration of internal standard (IS). Induced
salmon received an intraperitoneal injection of B-Naphthoflavone (5 0mg kg'1
body weight) while control salmon received an injection of corn oil alone. The
bands near the top of the gel are the 321bp IS. The bands at the bottom of the gel
represent the 208bp CYPIA fragment. The bands in the middle are the 250bp

actin fragments.
Abbreviations: NC, negative control (water control); AS, absorbance standard; IS,

internal standard positive control.

n = 8 for both control and induced groups.

47

Gill Samples Liver Samples
NW “up.” {—321bp—+-M MM

1

3 NC ' AS 18 NC " ‘ ‘ as
*-W ~“No—zoshp—p W.“ W

Fourmile Brook Miller’s River Fourmile Brook Miller’s River

Figure 10: Quantitative PCR of gill and liver tissue CYPIA for samples collected fi'om
wild salmon from two Massachusetts rivers. RNA (100ng) was co-amplified
against a known concentration of internal standard (IS). The bands near the top of
each gel are the 321bp IS. The bands at the bottom of each gel represent the
208bp CYPIA fragment. mRNA quantities are determined by taking the density

ratio of IS/CYPlA.
Abbreviations: NC, negative control (water control); AS, absorbance standard; IS,

internal standard positive control.

n = 7 for all groups.

48

REFERENCES

Albanis, T.A., Hela, D.G., Sakellarides, T.M., and Konstantinou, I.K., 1998. Monitoring
of pesticide residues and their metabolites in surface and underground

waters of Irnathia (N. Greece) by means of solid-phase extraction disks and
gas chromatography. J. Chromatogr. A. 823, 59-71.

Allendorf, F.W., Waples, RS, 1996. Conservation and Genetics of Salrnonid Fishes. In:
Avise, J .C., Hamrick, J.L., (Eds), Conservation Genetics: Case Histories
from Nature. Chapman and Hall, New York, pp. 269-294.

Anderson, J., Rossi, 8., Tukey, R., Vu, T., Quattrochi, L., 1995. A biomarker, P450
RGS, for assessing the induction potential of environmental-samples.
Environ. Toxicol. Chem. 14, 1159-1169.

Anderson, J.M., Whoriskey, F.G., Goode, A., 2000. Atlantic salmon on the brink. Marine
Matters: Endangered Species Update. 17, 15-21.

Andersson, T., F orlin, L., 1992. Regulation of the cytochrome P450 enzyme-system in
fish. Aquat. Toxicol. 24, 1-19.

Andersson, T., Goksoyr, A., 1994. Distribution and induction of cytochrome P450 1A1
in the rainbow-trout brain. Fish Physiol. Biochem. 13, 335-342.Andersson,
T., Forlin, L., 1992. Regulation of the cytochrome P450 enzyme-system in
fish. Aquat. Toxicol. 24, 1-19.

Bailey, G.,You, L., Harttig, U., 1997. Cloning, sequencing, and functional expression of
two trout CYPIA cDNAs in yeast. Unpublished. GenBank Accession
Numbers U62796 and U62797.

Bemdtson, A., Chen, T., 1994. Two unique CYPl genes are expressed in response to 3-
methylcholanthrene treatment in rainbow-trout. Arch. Biochem. Biophys.
310, 187-195.

Beyer, J ., Sandvik, M., Hylland, K., Fjeld, E., Egaas, E., Aas, E., Skare, J., Goksoyr, A.,
1996. Contaminant accumulation and biomarker responses in flounder
(Platichthysflesus L) and Atlantic cod (Gadus morhua L) exposed by caging
to polluted sediments in Sorljorden, Norway. Aquat. Toxicol. 36, 75-98.

Bhattacharyya, K.K., Brake, P.B., Eltom, S.E., Otto, S.A., Jefcoate, CR, 1995.
Identification of a rat adrenal cytochrome P450 active in polycychc
hydrocarbon metabolism as rat CYPlB 1. Demonstration of a unique tissue-
specific pattern of hormonal and aryl hydrocarbon receptor-linked
regulation. J. Biol. Chem. 270, 11595-11602.

Binder, R., Hwang, S.P., Ratnasabapathy, R., Williams, D.L., 1989. Degradation of
apolipoprotein H mRNA occurs via endonucleolytic cleavage at 5'-AAU-

49

3'/5'-UAA—3' elements in single-stranded loop domains of the 3'-noncoding
region. J. Biol. Chem. 264, 16910-16918.

Brittebo, E.B., Rafter, J .J ., 1984. Steroid metabolism by rat nasal mucosa: Studies on
progesterone and testosterone. J. Steroid Biochem. 20, 1147-1151.

Buhler, D., Wang-Buhler, J ., 1998. Rainbow trout cytochrome P450s: purification,
molecular aspects, metabolic activity, induction and role in environmental

monitoring. Comp. Biochem. Physiol. C. Pharmacol. Toxicol. Endocrinol.
121, 107-137.

Buhler, D., Yang, Y., Dreher, T., Miranda, C., Wang, J., 1994. Cloning and sequencing
of the major rainbow-trout constitutive cytochrome-p450 (CYP2K1) -
Identification of a new cytochrome-P450 gene subfamily and its expression
in mature rainbow-trout liver and trunk kidney. Arch. Biochem. Biophys.
312, 45-5 1 .

Cao, 2., Hong, J ., Peterson, R.E., Aiken, J.M., 2000. Characterization of CYP1A1 and
CYP1A3 gene expression in rainbow trout (Oncorhynchus mykiss). Aquat.
Toxicol. 49, 101-109.

Cousinou, M., Nilsen, 13., Lopez-Barea, J., Dorado, G., 2000. New methods to use fish
cytochrome P4501A to assess marine organic pollutants. Sci. Total
Environ. 247, 213-225.

Croce, B., Stagg, R., Smith, M., 1995. Effects of exposure to a combination of beta-
naphthoflavone and resin acids on biotransformation enzymes in Atlantic
sahnon parr (Salmo salar). Aquat. Toxicol. 32, 161 -1 76.

Dahl, A., Hadley, W., 1991. Nasal cavity enzymes involved in xenobiotic metabolism -
Effects on the toxicity of inhalants. Crit. Rev. T oxicol. 21, 345-372.

Ding, X., Coon, M.J., 1994. Steroid metabolism by rabbit olfactory-specific P450 ZGl.
Arch. Biochem. Biophys. 315, 454-459.

Fairchild, W.L., Swansburg, E.O., Arsenault, J .T., Brown, SB, 1999. Does an
association between pesticide use and subsequent declines in catch of

Atlantic salmon (Salmo salar) represent a case of endocrine disruption?
Environ. Health Perspect. 107, 349-358.

Fukuhara, M., Nagata, K., Mizokarrri, K., Yamazoe, Y., Takanaka, A., Kato, R., 1989.
Complete cDNA sequence of a major 3-methylcholanthrene-inducible
cytochrome P-450 isozyme (P-450AF B) of Syrian hamsters with high
activity toward aflatoxin B1. Biochem. Biophys. Res. Commun. 162, 265-
272.

Gagne, F., Pardos, M., Blaise, C., Turcotte, P., Quemerais, B., Fouquet, A., 1999.
Toxicity evaluation of organic sediment extracts resolved by size exclusion

50

chromatography using rainbow trout hepatocytes. Chemosphere 39, 1545-
1 570.

Goksoyr, A., Husoy, A.M., 1998. Irnmunochemical approaches to studies of CYPIA
localization and induction by xenobiotics in fish. Fish Ecotoxicology 86,
165-202.

Goksoyr, A., Larsen, H., 1991. The cytochrome-P450 system of Atlantic salmon (Salmo
salar). 1. Basal properties and induction of P450-1A1 in liver of immature
and mature fish. Fish Physiol. Biochem. 9, 339-349.

Grosvik, B., Larsen, H., Goksoyr, A., 1997. Effects of piperonyl butoxide and beta-
naphthoflavone on cytochrome P4501A expression and activity in Atlantic
salmon (Salmo salar L). Environ. Toxicol. Chem. 16, 415-423.

Heilrnann, L., Sheen, Y., Bigelow, S., Nebert, D., 1988. Trout-P4501A1 - cDNA and
deduced protein-sequence, expression in liver, and evolutionary
significance. DNA 7, 379-387.

Hodgson, E., Rose, R., Ryu, D., Falls, G., Blake, B., Levi, P., 1995. Pesticide-
metabolizing enzymes. Toxicol. Lett. 82-83, 73-81.

Hrelia, P., Fimognari, C., Maffei, F., Vigagni, F., Mesirca, R., Pozzetti, L., Paolini, M.,
Forti, G., 1996. The genetic and non-genetic toxicity of the fungicide
vinclozolin. Mutagenesis 11, 445-453.

J aiswal, A.K., Gonzalez, F .J ., Nebert, D.W., 1985. Human dioxin-inducible cytochrome
Pl-450: complementary DNA and amino acid sequence. Science 228, 80-
83.

Jones, S.B., King, L.B., Sappington, L.C., Dwyer, F .J ., Ellersieck, M., Buckler, DR,
1998. Effects of carbaryl, permethrin, 4-nonylphenol, and copper on
muscarinic cholinergic receptors in brain of surrogate and listed fish species.
Comp. Biochem. Physiol. C. Pharmacol. Toxicol. Endocrinol. 120, 405-
414.

Kawajiri, K., Fujii-Kuriyama, Y., 1991. P450 and human cancer. Jpn. J. Cancer. Res.
82, 1325-1335.

Kimura, S., Gonzalez, F.J., Nebert, D.W., 1984. Mouse cytochrome P3-450: complete
cDNA and amino acid sequence. Nucleic Acids Res. 12, 2917-2928.

Larsen, H., Celander, M., Goksoyr, A., 1992. The cytochrome-P450 system of Atlantic
salmon (Salmo-salar) .2. Variations in hepatic catalytic activities and
isozyme patterns during an annual reproductive-cycle. Fish Physiol.
Biochem. 10, 291-301.

51

Lazard, D., Zupko, K., Poria, Y., Nef, P., Lazarovits, J., Horn, 8., Khen, M., Lancet, D.,
1991. Odorant signal termination by olfactory UDP-glucuronosyl
transferase. Nature 349, 790-793.

Leaver, M.J., Pirrit, L., George, S.G., 1993. Cytochrome P450 1A1 cDNA from plaice
(Pleuronectes platessa) and induction of P450 1A1 mRNA in various
tissues by 3-methylcholanthrene and isosafrole. Mol. Mar. Biol. Biotech. 2,
338-345.

Lee, S., Wang-Buhler, J., Cok, 1., Yu, T., Yang, Y., Miranda, C., Lech, J., Buhler, D.,
1998. Cloning, sequencing, and tissue expression of CYP3A27, a new
member of the CYP3A subfamily fi'om embryonic and adult rainbow trout
livers. Arch. Biochem. Biophys. 360, 53-61.

Little, E.E., Archeski, R.D., Flerov, B.A., Kozlovskaya, V.I., 1990. Behavioral indicators
of sublethal toxicity in rainbow trout. Arch. Environ. Contarn. Toxicol. 19,
380-385.

Loitsch, S.M., Kippenberger, S., Dauletbaev, N., Wagner, T.O., Bargon, J ., 1999.
Reverse transcription-competitive multiplex PCR improves quantification of
mRNA in clinical samples-application to the low abundance CFTR mRNA.
Clin. Chem. 45, 619-624.

Mehmood, Z., Williamson, M., Kelly, D., Kelly, S., 1996. Metabolism of organochlorine
pesticides: The role of human cytochrome P450 3A4. Chemosphere 33,
759-769.

Miller, H., Bembo, D.G., Macdonald, J .A.,Evans, C.W., 1999. Induction of cytochrome
P4501A(CYP1A) in T rematomus bernacchii as an indicator of

environmental pollution in Antarctica: assessment by quantitative RT-PCR.
Aquat. Toxicol. 44, 183-193.

Mitsuo, R., Sato, M., Itakura, T., 1999. Cloning, sequencing, and phylogenetic analysis
of complementary DNA of novel cytochrome P4501A in Japanese eel
(Anguilla japonica). Unpublished. GenBank Accession AB020414.

Mizukami, Y., Okauchi, M., Arizono, K., Ariyosh, I.T., Kito, H., 1994. Isolation and
sequence of cDNA-encoding a 3-methylcholanthrene-inducible cytochrome-
P450 fi'om wild red-sea brearn, Pagrus major. Mar. Biol. 120, 343-349.

Morrison, H., Weil, E., Karchner, S., Sogin, M., Stegeman, J., 1998. Molecular cloning
of CYPIA from the estuarine fish F undulus heteroclitus and phylogenetic
analysis of CYPl genes: update with new sequences. Comp. Biochem.
Physiol. C. Pharmacol. Toxicol. Endocrinol. 121, 231-240.

52

Morrison, H.G., Oleksiak, M.F., Comell, N.W., Sogin, M.L., Stegeman, J .J ., 1995.
Identification of cytochrome P-450 1A(CYP1A) genes from two teleost
fish, toadfish (Opsanus tau) and scup (Stenotomus chrysops), and
phylogenetic analysis of CYPIA genes. J. Biochem. 308, 97-104.

Moyle, P.B., Cech, J .J ., 2000. Fishes: An Introduction to Ichthyology. 4th edition.
Prentice Hall, New Jersey, pp. 267-275.

Nef, P., Heldman, J., Lazard, D., Margalit, T., Jaye, M., Hanukog, L.I., Lancet, D., 1989.
Olfactory-specific cytochrome-P450 - cDNA cloning of a novel

neuroepithelial enzyme possibly involved in chemoreception. J. Biol.
Chem. 264, 6780-6785.

Nelson, D., Koymans, L., Kamataki, T., Stegeman, J ., Feyereisen, R., Waxman, D.,
Waterman, M., Gotoh, 0., Coon, M., Estabrook, R., Gunsalus, I., Nebert,
D., 1996. P450 superfamily: Update on new sequences, gene mapping,
accession numbers and nomenclature. Pharrnacogenetics 6, 1-42.

Ohmachi, T. 1993. Molecular cloning and sequence analysis of cDNA encoding crab-
eating monkey (Macaca iris) cytochrome P-450. Unpublished. GenBank
Accession D17575.

Porter, T., Coon, M., 1991. Cytochrome P450 - Multiplicity of isoforrns, substrates, and
catalytic and regulatory mechanisms. J. Biol. Chem. 266, 13469-13472.

Rabergh, C.M.I., Vrolijk, N.H., Lipsky, M.M., Chen, T.T., 2000. Differential expression
of two CYPIA genes in rainbow trout (Oncorhyncus mykiss). Toxicol. App.
Pharmacol. 165, 195-205.

Robertson, RE. 1997. Limanda limanda mRNA for CYPIA protein. Unpublished.
GenBank Accession AJ 001724.

Rogers, S.A., Llewellyn, L., Sweeney, G.E., Wigham, T., 1997. Sequence of a full length
Atlantic salmon (Salmo salar) beta actin cDNA. Unpublished. GenBank
Accession AF 012125.

Roy, N.K., Kreamer, G.L., Konkle, B., Grunwald, C., Wirgin, I., 1995. Characterization
and prevalence of a polymorphism in the 3' untranslated region of
cytochrome P4501Al in cancer-prone Atlantic tomcod. Arch. Biochem.
Biophys. 322, 204-213.

Sagami, 1., Ohmachi, T., Fujii, H., Kikuchi, H., Watanabe, M., 1991. Hamster
cytochrome P-450 IA gene family, P-4501A1 and P-4501A2 in lung and
liver: cDNA cloning and sequence analysis. J. Biochem. (Tokyo) 110, 641-
647.

Sarnbrook, J., Fritsch, E.F., Maniatas, T., 1989. Molecular Cloning: A Laboratory
Manual. 2"d edition. Cold Spring Harbor Laboratory Press, New York.

53

Sarasquete, C., Segner, H., 2000. Cytochrome P4501A(CYP1A) in teleostean fishes. A
review of immunohistochemical studies. Sci. Total Environ. 247, 313-332.

Schlenk, D., Ronis, M., Miranda, C., Buhler, D., 1993. Channel catfish liver
monooxygenases - Immunological characterization of constitutive
cytochromes-P450 and the absence of active flavin-containing
monooxygenases. Biochem. Pharmacol. 45, 217-221.

Schlezinger, J .J ., Stegeman, J .J ., 2001. Induction and suppression of cytochrome P450
1A by 3,3',4,4',5- pentachlorobiphenyl and its relationship to oxidative stress
in the marine fish soup (Stenotomus chrysops). Aquat. Toxicol. 52, 101-
1 15.

Sen, A., Buhler, D.R., Wang-Buhler, J .L., 1999. Cloning and sequencing mullet (Liza
saliens) liver cytochrome P4501A1. Unpublished. GenBank Accession
AF 072899.

Shaw, G., Kamen, R., 1986. A conserved AU sequence from the 3' untranslated region
of GM-CSF mRNA mediates selective mRNA degradation. Cell 46, 659-
667.

Stien, X., Amichot, M., Berge, J ., Lafaurie, M., 1998. Molecular cloning of a CYPIA
cDNA from the teleost fish Dicentrarchus labrax. Comp. Biochem.
Physiol. C. Pharmacol. Toxicol. Endocrinol. 121, 241-248.

Sutter, T.R., Tang, Y.M., Hayes, C.L., Wo, Y.Y., Jabs, E.W., Li, X., Yin, H., Cody,
C.W., Greenlee, W.F., 1994. Complete cDNA sequence of a human dioxin-
inducible mRNA identifies a new gene subfamily of cytochrome P450 that
maps to chromosome 2. J. Biol. Chem. 269, 13092-13099.

Tsai, S.J., Wiltbank, M.C., 1996. Quantification of mRNA using competitive RT-PCR
with standard-curve methodology. Biotechniques 21, 862-866.

Tufts, B. 2000. Science may be the key to salmon restoration. Atlantic Salmon Journal
49

US Army Corp of Engineers, 1995, Birch Hill Reservoir PCB Investigation -
Phase 2: Waltham, MA Hydraulics and Water Quality Branch, Water
Control Division, Engineering Directorate, Department of the Army, New
England Division Corps of Engineers.

US Department of Interior. 2000. Endangered and Threatened Species; Final Endangered
Status for a Distinct Population Segment of Anadromous Atlantic salmon
(Salmo salar) in the Gulf of Maine. Federal Register 65, 69459-69483.

Vanden Heuvel, JP, 1998. PCR Protocols in Molecular Toxicology. CRC Press, New
York, pp. 41 -99.

54

Vanden Heuvel, J .P., Clark, G.C., Kohn, M.C., Tritscher, A.M., Greenlee, W.F., Lucier,
G.W., Bell, D.A., 1994. Dioxin-responsive genes: Examination of dose-
response relationships using quantitative reverse transcriptase-polymerase
chain reaction. Cancer Res. 54, 62-68.

Vanden Heuvel, J .P., Tyson, F .L., Bell, D.A., 1993. Construction of recombinant RNA
templates for use as internal standards in quantitative RT -PCR.
Biotechniques. 14, 395-398.

Vignier, V., Vanderrneulen, J., Mossman, D., 1996. Assay conditions and basal activity
of CYPIA-dependent mixed function oxidase in parr and smolt of Atlantic
salmon (Salmo salar). Comp. Biochem. Physiol. C. Pharmacol. Toxicol.
Endocrinol. 113, 325-330.

Vrolijk, N.H., Lin, C., Chen, T.T., 1995. Characterization and expression of a CYPIA
gene fiom a tropical teleost, Chaetodon capistratus. Unpublished. GenBank
Acession Ul9855.

Waldichuk, M. 1979. The assessment of sublethal effects of pollutants in the sea Review
of the problems. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 286, 399-424.

Waring, C., Moore, A., 1997. Sublethal effects of a carbarnate pesticide on pheromonal
mediated endocrine function in mature male Atlantic salmon (Salmo salar
L) parr. Fish Physiol. Biochem. 17, 203-211.

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