1986. Other inducible forms metabolize lipophilic xenobiotics into water-soluble compounds



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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--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

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.




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.


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

Induced

Sequenced

Selected Species

 

 

 

 

Mammals

+

+

Mouse (Mus musculus), Rat (Rattus norvegicus), Human (Homo sapiens)

Birds

+

+

Chicken (Gallus gallus), Herring Gull (Larus argentatus)

Reptiles

+

-

Eastern Painted Turtle (Chrysemys picta picta)

Amphibians

+

-

Newt (Pleurodeles waltl), Tiger Salamander (Ambystoma tigrinum)

Bony Fish

+

+

Zebrafish (Danio rerio), Mummichog (Fundulus heteroclitus)

Cartilaginous Fish

+

-

Skate (Raja erinacea)

Jawless Fish

-

-

Atlantic Hagfish (Myxine glutinosa), Sea Lamprey (Petromyzon marinus)

Invertebrates

-

-

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

Trout 1A

Scup 1A

Frog 1A

Chicken 1A4

Rat 1A1

Pig 1A1

Human 1A2

Skate Fragment

 

 

 

 

 

 

 

 

 

 

Salmon 1A

1.00

0.88

0.74

0.54

0.51

0.54

0.51

0.51

0.74

Trout 1A




1.00

0.80

0.57

0.56

0.58

0.55

0.55

0.74

Scup 1A







1.00

0.59

0.56

0.58

0.56

0.56

0.74

Frog 1A










1.00

0.60

0.62

0.60

0.61

0.67

Chicken 1A4













1.00

0.59

0.59

0.58

0.65

Rat 1A1
















1.00

0.79

0.79

0.66

Pig 1A1



















1.00

0.77

0.67

Human 1A2






















1.00

0.64

Skate Fragment

























1.00

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.





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