Introduction
The cytochrome P450 enzymes compose a well-studied superfamily of monooxygenase
hemoproteins involved in the hydroxylation of both endogenous and exogenous substrates. P450
isozymes are found in animals, plants, and bacteria, and in all mammalian cells except mature
red blood cells and skeletal muscle cells. Most P450s perform essential physiological functions
(e.g. steroid hormone biosynthesis, glucocortoid production, fatty acid hydroxlation) and are thus
expressed constitutively (Waterman et al., 1986). Other inducible forms metabolize lipophilic
xenobiotics into water-soluble compounds.
Induction can occur at either the transcriptional or posttranscriptional level; inducers
include anthropogenic pollutants (2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD),
phenylhydrazine, furans), barbiturates, and many other aliphatic and aromatic compounds. As
terminal oxidases in electron transport chains, P450 enzymes follow the general equation derived
by Mason (1957): H+ + XH2 + NADPH + O2  XHOH + NAPD+ + H2O, where X is the
xenobiotic substrate. These reactions can release highly reactive free radicals that lead to
membrane destruction and carcinogenesis. For this reason, P450 metabolism results in the toxic
effects of many common environmental pollutants.
Chlorinated dioxins, other planar halogenated aromatic hydrocarbons (HAHs),
polynuclear aromatic hydrocarbons (PAHs), and aromatic amines induce cytochrome P450 1A
(CYP1A) expression. Located predominantly in the hepatic and extrahepatic tissues, CYP1A is
induced through ligand activation of aryl hydrocarbon receptor (AHR). Upon activation in the
cytosol, AHR is translocated to the nucleus where it dimerizes with ARNT (AHR nuclear
translocator), creating an active transcription factor that initiates transcription of the CYP1A
gene. Stegeman (1993) proposes that CYP1A induction is a likely prerequisite for the
carcinogenesis that typically results from PAHs and HAHs. Some CYP1A metabolites (e.g.
benzo[a]pyrene:diol epoxide through CYP1A biotransformation) can damage DNA by
forming free radicals or adducts (Timbrell, 2000).
Although the CYP1A/AHR system efficiently metabolizes PAHs and HAHs, it is unclear
whether this is the primary function of CYP1A/AHR, as these toxins do not exist in nature. Song
et al. (2002) recently identified an endogenous AHR ligand that competes effectively with
TCDD for AHR forms in both mammals and fish. This endogenous ligand may induce CYP1A
for a physiological role. AHR-null mice have deleterious changes in the heart, ovary, liver, and
immune system, indicating that CYP1A induction is an adaptive function of AHR (Benedict et
al., 2000; Lahvis et al., 2000; Robles et al., 2000; Thurmond et al., 2000; Fernandez-Salguero et
al., 1997, 1995; Schmidt et al., 1996). Natural compounds that may act as inducers for CYP1A
through AHR or alternate pathways include caffeine, arachidonic acid, and some hormones.
Orthologous CYP1A genes have been sequenced and characterized in most vertebrates:
birds, mammals, amphibians, and bony fish. Mammals show two types: CYP1A1 and CYP1A2.
Hahn et al. (1998) successfully induced CYP1A in cartilaginous fish (little skate, Raja erinacea),
but found no evidence for CYP1A induction in jawless fish (sea lamprey, Petromyzon marinus,
and Atlantic hagfish, Myxine glutinosa). Photoaffinity labeling and velocity sedimentation
studies of more primitive animals (mollusks, annelids, echinoderms, and a urochordate) show no
specific binding to AHR proteins (Butler et al., 2001; Hahn et al., 1994). If CYP1A exists in
these phyla, it is a variant with a different induction pathway. This indicates that the ancestral
gene of CYP1A (as it is known) originated no earlier than 400 million years ago, in the lower
Devonian period when the jawed vertebrates diverged from the jawless fish and other primitive
vertebrates. A summary of known CYP1A genes is shown in Table 1.
<insert Table 1>
The cartilaginous and bony fish diverged from each other prior to the divergence of
tetrapods in the lower Devonian, 400 million years ago. The elasmobranchs (skates, sharks, and
rays) diverged from the chimaeroids in the upper Devonian. Rays and skates together made a
final divergence, from the shark lineage, in the Jurassic period, 150 million years ago. At this
point, most elasmobranch lineages had become extinct; by the beginning of the Cenozoic, the
extant Chondrichthys taxa were represented. On the contrary, bony fish evolution continued at an
expontential rate, resulting in more than 24,600 extant species (Nelson, 1994). CYP1A has been
cloned and sequenced from numerous teleosts.
This study aims to induce, clone, and sequence a novel CYP1A gene in little skate, Raja
erinacea, and to determine the phylogenetic relationship of skate CYP1A to that of bony fish and
tetrapods.
Materials and Methods
CYP1A Induction
A mature female little skate (Raja erinacea) was obtained from the Marine Biological
Laboratory, Woods Hole, MA, and kept in constant-flow sea water at 14C. It weighed 0.5kg,
had a total length of 45cm, and a disc width of 25cm. It was injected intraperitoneally with the
model CYP1A inducer -naphthoflavone (BNF, 50 mg kg-1 body weight) suspended in soybean
oil. The skate was anesthetized and euthanized 48 hours post-injection and the liver, pancreas,
spleen, spiral valve, stomach, rectal gland, shell gland, gill lamellae, kidney, heart, muscle, and
brain were dissected and preserved in RNAlater, a tissue storage reagent (Ambion Diagnostics,
Austin, TX), and flash frozen in liquid nitrogen and kept at –80C until processed.
cDNA Preparation
Total RNA was isolated from liver tissue with RNA STAT-60 (Tel-test, Friendswood, TX).
Liver mRNA (Poly(A)+) was isolated with Oligotex spin columns (Qiagen, Valencia, CA).
Complementary DNA was generated from 1 g poly(A)+ mRNA with oligo-d(T)-primed reverse
transcription using the Omniscript RT kit (Qiagen) and a Gene-AMP 2400 thermocycler.
Oligonucleotide Primers
Degenerate primers were kindly provided by Celine Godard (Woods Hole Oceanographic
Institution, Woods Hole, MA). Primer sequences were: 203F1A, 5-GTIGTIWSIGTIGCI
AAYGT-3; and 336R1A, 5-GTRTCRAAICCIGCICCRAAIARRTC-3; the primers were
synthesized by Life Technologies (Invitrogen, Carlsbad, California).
PCR Amplification
Amplification of 10 L cDNA was accomplished in 50 L reactions with 10X Gold Buffer,
2mM MgCl2, 0.8 mM of each of four dNTPs, 100 M of each primer, and 5 units of Amplitaq
Gold polymerase (Perkin Elmer, Foster City, CA). PCR conditions were: an initial denaturation
step of 10 min at 95C, 35 cycles of 15 sec denaturation at 95C and 90 sec annealing/extension
at 50C, and a final extension at 50C for 10 min.
Cloning and DNA Sequencing
A PCR product of the predicted length (400 bp) was visualized on 1% ethidium bromide stained
agarose gel, purified with the GeneClean kit (Qbiogene, Carlsbad, CA), cloned into pGEM-T
Easy vector (Promega, Madison, WI) and transformed into JM109 competent cells (Promega).
Plasmid DNA was checked for insert with EcoRI restriction enzyme digestion; clones containing
the 400 bp insert were further purified with the QIAprep Spin Miniprep kit (Qiagen) and sent to
the University of Maine-Orono DNA Sequencing Facility for sequencing.
Sequence Analysis
The NCBI BLASTX search program (www.ncbi.nlm.nih.gov) was used to compare the resulting
nucleotide sequence with known genes. Related CYP1A sequences were aligned with ClustalX
(Higgins and Sharp, 1988). The PAUP program was used to conduct phylogenetic analyses
(Sinauer Assocs., Sunderland, MA; Swofford, 1993).
Results
Identification of a CYP1A-like Gene in Raja erinacea
A CYP1A homolog from little skate liver tissue was amplified using RT-PCR with degenerate
primers CYP1A-203F/CYP1A-336R. The primary PCR product was approximately 400bp as
expected. The product was cloned with the pGEM-T Easy vector and JM109 competent cells
(Promega). Insert size was confirmed with digestion by EcoRI; two 400bp clones were selected
for sequencing. The consensus partial nucleotide sequence was compared to known sequences
with a standard nucleotide-nucleotide BLAST search.
The highest scoring BLAST results indicate that the fragment is a highly conserved
region of a CYP1A gene. The partial cDNA (393 bp) is most similar to the CYP1A gene from
atlantic salmon, Salmo salar. There was high amino acid identity, 51% to 88%, among the skate,
salmon, trout, scup, frog, chicken, rat, pig and human CYP1A polypeptides. The skate partial
nucleotide and corresponding amino acid sequence is shown in Figure 1. Amino acid identities
between the fragment and various CYP1A genes are presented in Table 2. <insert Figure 1,
Table 2>
Phylogenetic Analysis
The skate CYP1A fragment was aligned with fourteen various CYP1 sequences (Table 3) using
ClustalX software. Phylogenetic analyses were made with PAUP. An unrooted cladogram,
presented in Figure 2-1, shows the CYP1 sequences to cluster in four distinct monophyletic
groups, or clades: CYP1Bs, mammals CYP1As, bony fish CYP1As, and the skate CYP1A,
which stands as its own group. A phylogenetic tree (Figure 2-2) was created with monkey
CYP2B as an outgroup. The skate CYP1A appears between the mammal and bony fish CYP1As;
as in the unrooted cladogram, the plaice gene (PleplA) does not cluster with the other bony fish
and has apparently been misidentified. Finally, a maximum parsimony tree, Figure 2-3, was
constructed with the plaice CYP1A as the outgroup. Skate CYP1A appears in the same lineage
as the bird and mammal CYP1As, in a group divergent from the bony fish. A more complete
phylogenetic analysis will be performed once the full-length amino acid sequence is obtained.
<insert Table 3, Figures 2-1, 2-2, 2-3>
Discussion
This study confirms the presence of an inducible CYP1A homolog in little skate, Raja erinacea.
This data provides the first molecular evidence for CYP1A in cartilaginous fish and substantiates
the findings of Hahn, et al., (1998) that CYP1A and its regulation by AHR is a well-conserved
characteristic of jawed fish and their descendants.
The CYP1A homolog in R. erinacea is most similar to the CYP1A gene of atlantic
salmon, Salmo salar, with 74% shared amino acid identity. The skate CYP1A also shares
significant identity with bird, amphibian, and mammalian forms of CYP1A. Interestingly,
phylogenetic analysis shows the skate CYP1A to fall between the bony fish and mammalian
CYP1As. Further research, particularly the cloning of the full-length CYP1A sequence, will
clarify this result. It is expected that the full-length gene will cluster within the bony fish clade.
Further research will also show if elasmobranch CYP1A regulation is equivalent to the
regulation observed in bony fish. This study and work by Hahn, et al. (1998) suggest that the
coupled AHR/CYP1A signal transduction pathway is similar, if not equal, to that found in more
diverged taxa.
The question of the fundamental function of AHR remains unanswered. Although the
presence of an AHR/CYP1A pathway in the earliest jawed vertebrates appears to indicate that
induction of CYP1A may be the primary function of AHR, the discovery of AHR homologs in
lamprey (Hahn, et al., 1998), soft-shell clam (Butler, et al, 2001), Caenorhabditis elegans
(Powell-Coffman, et al, 1998), and Drosophila melanogaster (Duncan, et al, 1998) show that
AHR likely serves an important ancestral role. Studies of invertebrate AHR forms may elucidate
the original function of AHR and the regulation of its expression.
Table 1
Summary of Known CYP1A Genes
Taxa
Mammals
Birds
Reptiles
Amphibians
Bony Fish
Cartilaginous Fish
Jawless Fish
Invertebrates
Induced
+
+
+
+
+
+
-
Sequenced Selected Species
+
+
+
-
Mouse (Mus musculus), Rat (Rattus norvegicus), Human (Homo sapiens)
Chicken (Gallus gallus), Herring Gull (Larus argentatus)
Eastern Painted Turtle (Chrysemys picta picta)
Newt (Pleurodeles waltl), Tiger Salamander (Ambystoma tigrinum)
Zebrafish (Danio rerio), Mummichog (Fundulus heteroclitus)
Skate (Raja erinacea)
Atlantic Hagfish (Myxine glutinosa), Sea Lamprey (Petromyzon marinus)
Soft-Shell Clam (Mya arenaria)
Figure 1
393bp Consensus Partial Nucleotide and Translated Amino Acid Sequence for ReCYP1A cDNA
GTG GTG TCG GTG GCG AAT GTC ATT TGC GCC CTG TGC TTC GGG AAG CGC TAC AGT CAC GAA GAC CAA GAG CTC
V
V
S
V
A
N
V
I
C
A
L
C
F
G
K
R
Y
S
H
E
D
Q
E
L
CTC AAC ATC GTC AAT GTC AGC GAT GAG TTC GGC AAA ATC GTA GCC GCT GGC AAC CCC GCC GAT TTC ATC CCG
L
N
I
V
N
V
S
D
E
F
G
K
I
V
A
A
G
N
P
A
D
F
I
P
ATC CTG AGA TTC CTC CCG AAC CAC TCG ATG GAC AAG TTC ATC GCT ATC AAC AAG AGA TTC GCC ACT TTC GTT
I
L
R
F
L
P
N
H
S
M
D
K
F
I
A
I
N
K
R
F
A
T
F
V
GAG AAC ATT GTC ATG GAG CAT TAC CGC ACA TTT GAC AAG GAT AAC ATT CGG GAT ATA ACC GAT TCG CTG ATC
E
N
I
V
M
E
H
Y
R
T
F
D
K
D
N
I
R
D
I
T
D
S
L
I
GGT CAC TGC CAG GAT AAA AAA GTG GAC GAG AAT GCC AAT ATC CAA ATA TCC GAT GAA AAG ATT GTC GGC ATC
G
H
C
Q
D
K
K
V
D
D
N
A
N
I
Q
I
S
D
E
K
I
V
G
I
GTG AAT GAC CTC TTC GGC GCC GGC TTC GAC ACA
V
N
D
L
F
G
A
G
F
D
T
Table 2
Amino Acid Identities between Aligned CYP1A Sequences
Salmon 1A
Salmon 1A
Trout 1A Scup 1A Frog 1A Chicken 1A4 Rat 1A1
1.00
Trout 1A
Pig 1A1 Human 1A2 Skate Fragment
0.88
0.74
0.54
0.51
0.54
0.51
0.51
0.74
1.00
0.80
0.57
0.56
0.58
0.55
0.55
0.74
1.00
0.59
0.56
0.58
0.56
0.56
0.74
1.00
0.60
0.62
0.60
0.61
0.67
1.00
0.59
0.59
0.58
0.65
1.00
0.79
0.79
0.66
1.00
0.77
0.67
1.00
0.64
Scup 1A
Frog 1A
Chicken 1A4
Rat 1A1
Pig 1A1
Human 1A2
1.00
Skate Fragment
Table 3
GenBank Accession Numbers for CYP1 Genes Used in Phylogenetic Analysis
Gene
Label
Organism
Species
Accession Number
CYP1A
Funhe1A
Mummichog
Fundulus heteroclitus
AAD01809
CYP1A
Ammma1A
Sand lance
Ammodytes marinus
CAC342
CYP1A
Lizau1A
Mullet
Liza aurata
AAB70307
CYP1A
Limli1A
Dab
Limanda limanda
CAA0495
CYP1A
Angja1A
Japanese eel
Anguilla japonica
BAA8824
CYP1A
Plepl1A
Plaice
Pleuronectes platessa
CAB5136
CYP1A1
Oncmy1A1
Rainbow trout
Oncorhynchus mykiss
A28789
CYP1A1
Micto1A1
Tomcod
Microgadus tomcod
IS6646
CYP1A1
Ratno1A1
Rat
Rattus norvegicus
IO4RTMC
CYP1A1
Oviar1A1
Sheep
Ovis aries
AAD1431
CYP1A1
Homsa1A1
Human
Homo sapiens
NP_00075
CYP1A4
Galdo1A4
Chicken
Gallus domesticus
X99453
CYP1B1
Musmu1B1
Mouse
Mus musculus
NP_034
CYP1B1
Homsa1B1
Human
Homo sapiens
Q16678
Figure 2-1
Unrooted Cladogram with Fish, Bird, and Mammal CYP1 Genes
Four distinct clades: CYP1Bs, bird and mammal CYP1s (with plaice CYP1A, “Plepl1A”, as
irregular result), skate CYP1A, and bony fish CYP1As. The plaice gene has likely been
misidentified or has an incorrect GenBank entry.
Figure 2-2
Phylogenetic Tree Showing Divergence of CYP1 Genes
The bony fish CYP1A genes clade together, with the exception of the plaice gene (see Figure 4-1
and Results). The skate CYP1A gene appears between the bony fish clade and the chicken and
human CYP1As.
Figure 2-3
Phylogenetic Analysis of CYP1 Genes using Maximum Parsimony
Tree constructed using maximum parsimony with bootstrapping. Most bony fish CYP1A genes
are clustered together, as are the bird and mammal CYP1As. The skate CYP1A fragment is most
closely associated with the bird and mammal clade.
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