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. >
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
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 14C. 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 –80C until processed.
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.
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).
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 95C, 35 cycles of 15 sec denaturation at 95C and 90 sec annealing/extension at 50C, and a final extension at 50C 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.
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).
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.
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.
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.
Summary of Known CYP1A Genes
Mouse (Mus musculus), Rat (Rattus norvegicus), Human (Homo sapiens)
GenBank Accession Numbers for CYP1 Genes Used in Phylogenetic Analysis
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.