Marine invertebrate cytochrome P450: Emerging

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147–157. Boyle, S.M., James, M.O., 1996. ... 46, 21–24. Boyle, S.M., Popp, M.P., Smith, C.W., Greenberg, R.M., James, M.O., 1998b. ... Physiol. 121C, 351–360.
Comparative Biochemistry and Physiology, Part C 143 (2006) 363 – 381 www.elsevier.com/locate/cbpc

Review

Marine invertebrate cytochrome P450: Emerging insights from vertebrate and insect analogies Kim F. Rewitz ⁎, Bjarne Styrishave, Anders Løbner-Olesen, Ole Andersen Department of Life Sciences and Chemistry, Roskilde University, P.O. Box 260, 4000 Roskilde, Denmark Received 5 January 2006; received in revised form 11 April 2006; accepted 12 April 2006 Available online 29 April 2006

Abstract Cytochrome P450 enzymes (P450s) are responsible for the oxidative metabolism of a plethora of endogenous and exogenous substrates. P450s and associated activities have been demonstrated in numerous marine invertebrates belonging to the phyla Cnidaria, Annelida (Polychaeta), Mollusca, Arthropoda (Crustacea) and Echinodermata. P450s of marine invertebrates and vertebrates show considerable sequence divergence and the few orthologs reveal the selective constraint on physiologically significant enzymes. P450s are present in virtually all tissues of marine invertebrates, although high levels usually are found in hepatic-like organs and steroidogenic tissues. High-throughput technologies result in the rapid acquisition of new marine invertebrate P450 sequences; however, the understanding of their function is poor. Based on analogy to vertebrates and insects, it is likely that P450s play a pivotal role in the physiology of marine invertebrates by catalyzing the biosynthesis of signal molecules including steroids such as 20-hydroxyecdysone (the molting hormone of crustaceans). The metabolism of many exogenous compounds including benzo(a)pyrene (BaP), pyrene, ethoxyresorufin, ethoxycoumarin and aniline is mediated by P450 enzymes in tissues of marine invertebrates. P450 gene expression, protein levels and P450 mediated metabolism of xenobiotics are induced by PAHs in some marine invertebrate species. Thus, regulation of P450 enzyme activity may play a central role in the adaptation of animals to environmental pollutants. Emphasis should be put on the elucidation of the function and regulation of the ever-increasing number of marine invertebrate P450s. © 2006 Elsevier Inc. All rights reserved. Keywords: Benzo(a)pyrene; CYP; CYP1A; Ecdysteroids; Evolution; Hydroxylase; Mixed function oxidases; Monooxygenases; PAH; Steroids; Xenobiotics

Contents 1. 2. 3. 4. 5. 6. 7.

8.

Introduction . . . . . . . . . . . . . . . . . . . . . . . The nature of the P450 enzyme system . . . . . . . . Marine invertebrate P450 genes and their expression . Phylogeny of marine invertebrate P450s . . . . . . . . Immuno- and DNA probe detection . . . . . . . . . . Tissue distribution . . . . . . . . . . . . . . . . . . . P450 mediated metabolism of endogenous compounds 7.1. Ecdysteroids . . . . . . . . . . . . . . . . . . . 7.2. Sex steroids . . . . . . . . . . . . . . . . . . . 7.3. Non-steroidal endogenous substrates . . . . . . P450 mediated metabolism of xenobiotics . . . . . . .

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Abbreviations: AHR, Aryl hydrocarbon receptor; 20E, 20-hydroxyecdysone; BaP, Benzo(a)pyrene; P450, Cytochrome P450; E, Ecdysone; E20MO, Ecdysone 20monooxygenase; EST, Expressed sequence tag; PAHs, Polycyclic aromatic hydrocarbons; CYP1A IPP, CYP1A immunopositive protein; Phm: CYP306A1, Phantom; Dib: CYP302A1, Disembodied; Sad: CYP315A1, Shadow; Shd: CYP314A1, Shade. ⁎ Corresponding author. Tel.: +45 4674 2785; fax: +45 4674 3011. E-mail address: [email protected] (K.F. Rewitz). 1532-0456/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2006.04.001

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9. Regulation of P450s . . . . . 10. Development, sex and season 11. In summary and the future . Acknowledgements . . . . . . . . References . . . . . . . . . . . . .

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1. Introduction

incorporation of one atom of dioxygen into the substrates. The overall stoichiometric reaction is:

The cytochromes P450 comprise one of the largest and most versatile protein families. P450 enzymes are particularly known for the Phase 1 metabolism of a variety of lipophilic xenobiotics e.g. drugs, pesticides, polycyclic aromatic hydrocarbons (PAHs) and plant allochemicals (see Nelson et al., 1996). Many P450s have, however, endogenous functions, being specialized in the metabolism of signal molecules, such as steroid hormones, eicosanoids and pheromones (see Werck-Reichhart and Feyereisen, 2000; Feyereisen, 2005). The great diversity of this gene family is a result of consecutive gene duplications and the subsequent divergence of genes (Nelson and Strobel, 1987). Ancient P450s first evolved for important physiological functions e.g. sterol synthesis (Nelson and Strobel, 1987; Nelson, 1998). Changing environments and life strategies probably drove the functional diversification leading to the broad activity towards exogenous compounds. As plants developed protective phytoalexins, new P450s were recruited for the detoxification of these compounds (Nelson and Strobel, 1987; Gonzalez and Nebert, 1990). Mutations disrupting the function of P450 enzymes participating in physiologically important reactions often lead to embryonic lethality (Chávez et al., 2000; Warren et al., 2002, 2004; Petryk et al., 2003). These P450s usually have narrow substrate specificity and are often conserved between species and sometimes phyla to preserve important biosynthetic pathways. In contrast, many P450s that metabolize exogenous compounds have broader substrate specificities and no obvious phenotype associated with the knockout of their gene products (Gonzales and Kimura, 2003). In marine invertebrates, P450 enzymes with physiological functions have received little attention as the majority of studies have focused on the role of P450s in the metabolism of environmental pollutants, mainly PAHs. Several metazoan genomes are now sequenced, revealing the presence of approximately 50–150 different P450 genes, including some pseudogenes, in species so distinct as nematodes, arthropods and mammals. Although the primary sequence identity between P450 proteins often is below 20%, highly conserved residues of the canonical P450 motifs ensure that the structural fold of the protein remains conserved (see Graham and Peterson, 1999; Werck-Reichhart and Feyereisen, 2000). P450 enzymes catalyze a variety of reactions, including hydroxylations, epoxidations, oxidative deaminations, N-, O-, S-dealkylations and dehalogenations (see Mansuy, 1998). In general, these heme-thiolate enzymes are known for their monooxygenase activity, catalyzing the

SH þ O2 þ NADPH þ Hþ →SOH þ H2 O þ NADPþ P450 genes are assigned to families and subfamilies based on amino acid sequence similarities. The root symbol CYP is derived from cytochrome P450 and is followed by an Arabic number indicating the family, a letter indicating the subfamily and finally an Arabic number to designate individual members of subfamilies. Usually P450 enzymes with N 40% primary sequence identity are assigned to the same family. Enzymes that share N55% amino acid identity are in the same subfamily (Nelson et al., 1996). Since the number of new sequences has exploded over the last few years, related P450 families are now structured into higher order groups called Clans (Nelson, 1998, 1999). The Clan names inherently indicate family relationship. That is, the 2 Clan comprises CYP2 and other related enzymes such as CYP1 enzymes; the 3 Clan includes CYP3 and enzymes most closely related to this family e.g. CYP6 and CYP9; the 4 Clan is comprised of CYP4 and other closely related enzymes; the Mito Clan comprises mitochondrial P450s. In marine invertebrates, P450 genes and biochemical evidence of P450 enzyme activity have been demonstrated in Cnidaria, Annelida (Polychaeta), Mollusca, Arthropoda (Crustacea) and Echinodermata (for excellent reviews on earlier literature within this field see: Lee, 1981, 1998; James, 1989; Livingstone et al., 1989; Livingstone, 1990, 1991; den Besten, 1998; James and Boyle, 1998; Snyder, 2000). The present article aims at reviewing recent advances in marine invertebrate P450 research with critical appraisals of our current state of knowledge. We hope to draw attention to this largely unexploited field with important implications in developmental biology, endocrinology, physiology and ecotoxicology. The perspectives will rely to some extent on advances made in insect and vertebrate P450 research, as this is necessary to outline the prospects of future research. 2. The nature of the P450 enzyme system The approximately 500 amino acids in the primary sequences of P450s have several characteristic signature regions allowing for the easy identification of enzymes belonging to this family (Fig. 1). P450 enzymes may reside in the mitochondria (Class I) and/or in the membrane of the endoplasmic reticulum (ER) (microsomal P450s; Class II) depending on their N-terminal target sequence. It has sometimes been implied that microsomal P450s are involved in the biotransformation of exogenous compounds (detoxification), whereas endogenous compounds often are the substrates of

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Fig. 1. Schematic presentation of P450 primary structure features, including the characteristics of the N-terminal target sequence of microsomal P450s located in the endoplasmic reticulum (Class II) and mitochondrial P450s (Class I), conserved structural core motifs and more variable regions. Numbers indicate the approximate localization.

mitochondrial P450s. However, some mitochondrial P450s show substantial activity towards exogenous compounds e.g. the mitochondrial CYP12A1 from the housefly Musca domestica, is capable of metabolizing xenobiotics including various insecticides (Guzov et al., 1998). In microsomal P450s, the N-terminal signal sequence consists of approximately 20 hydrophobic amino acids usually preceding one or two charged residues, acting as a halt-transfer signal and a proline/glycine rich region necessary for proper folding (Fig. 1) (Yamazaki et al., 1993; Chen et al., 1998; Kusano et al., 2001). In addition, a hydrophobic surface of the globular domain is believed to be associated with the ER membrane, placing the substrate channel in the hydrophobic environment of the membrane which allows lipophilic substrates to enter (Williams et al., 2000a). In contrast, mitochondrial P450s generally have several positive Nterminal residues which are proteolytically cleaved during import. Several other charged residues, two of which are in the helix-L, are important for redox partner interactions (see Werck-Reichhart and Feyereisen, 2000). However, in some cases the N-terminal sequence carries a dual signal resulting in both ER and mitochondrial localization e.g. the mammalian CYP1A1 and CYP2E1 are located in the ER but are translocated to the mitochondria when cleaved at an N-terminal position by a cytosolic endoprotease or when phosphorylated, respectively (Addya et al., 1997; Robin et al., 2002). Microsomal and mitochondrial P450s have different electron donor systems. Microsomal P450s accept electrons from the P450 reductase, a diflavin protein, containing both a FAD and a FMN cofactor. A mitochondrial specific adrenodoxin (an iron– sulfur (Fe–S) protein) and an adrenodoxin reductase are required for mitochondrial P450s (Degtyarenko and Archakov, 1993). Anandatheerthavarada et al. (1998) demonstrated that interactions of CYP1A1 with the mitochondrial redox partner change the substrate specificity of the enzyme. In vitro assays using subcellular fractions have demonstrated that the P450 mediated hydroxylation of ecdysone (E; an arthropod molting hormone) at position C-20 and C-26 occurs in both mitochondrial and microsomal fractions of arthropod tissues (Weirich et al., 1984, 1985; see Smith, 1985; Williams et al., 2000b; see Lafont et al.,

2005). The physiological significance of this subcellular distribution is currently unknown. However, it is interesting that the subcellular distribution may affect the catalytic competence of the enzyme. In the shore crab, Carcinus maenas, pyrene is converted to 1-hydroxypyrene in both mitochondrial and microsomal fractions prepared from the hepatopancreas (K.F. Rewitz, unpublished data) indicating the presence of mitochondrial P450s capable of metabolizing pyrene. The question remains as to whether different enzymes carry out the same enzymatic reactions or whether the same enzyme is present in both mitochondria and ER. 3. Marine invertebrate P450 genes and their expression The genomes of several invertebrates, including the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, are now sequenced, revealing the great diversity of invertebrate P450s. This is substantiated by the presence of 85 functional genes and 5 apparent pseudogenes in Drosophila (Feyereisen, 2005) and more than 80 genes in C. elegans (Menzel et al., 2005). Although numerous partial P450 sequences are now available from the genome of the sea urchin Strongylocentrotus purpuratus and through a number of expressed sequence tags (ESTs) from different species of marine invertebrates, it is beyond the scope here to review the complete number of these ESTs and genome sequences. Table 1 shows a list of P450s cloned from marine invertebrates (excluding ESTs and sequences annotated from genomes) with details of their regulation by external stimuli. Animals belonging to the phylum Cnidaria have received little attention with regard to their P450 enzyme systems; however, a number of ESTs representing P450 members of 2, 3 and 4 Clan have now been obtained from freshwater cnidarians, Hydra sp. In annelid polychaetes, two P450 sequences with highest homology to CYP4 enzymes were recently obtained from Nereis virens (Rewitz et al., 2004). Expression of one of these genes, CYP342A1 (GenBank accession no. AY453408), was induced by benz(a)anthracene, crude oil and the typical CYP4 inducer clofibrate. The other gene, CYP4BB1 (GenBank accession no. AY453407), was not induced significantly by any

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Table 1 P450 genes cloned from tissues of marine invertebrates Species

P450

Tissue

Regulation Regulators Endogenous

Phylum Annelida Nereis virens (worm) Nereis virens (worm) Capitella capitata (worm) Capitella capitata (worm) Phylum Mollusca Haliotis rufescens (abalone) Mytilus galloprovincialis (mussel) Mercenaria mercenaria (clam) Phylum Arthropoda (Subphylum: Crustacea) Carcinus maenas (crab) Carcinus maenas (crab) Panilirus argus (spiny lobster) Panilirus argus (spiny lobster) Penaeus setiferus (shrimp) Homarus americanus (lobster) Homarus americanus (lobster) Phylum Echinodermata Lytechinus anamesis (sea urchin) Lytechinus anamesis (sea urchin)

CYP4BB1 CYP342A1 CYP4AT1 CYP331A1

Intestine Intestine Whole animal Whole animal

CYP4C17⁎ CYP4Y1⁎ CYP30

Digestive gland Digestive gland Gonads

CYP330A1 CYP4C39 CYP2L1 CYP2L2 CYP4C16⁎ CYP4C18⁎ CYP45

Hepatopancreas Hepatopancreas Hepatopancreas Hepatopancreas Hepatopancreas Hepatopancreas Hepatopancreas

CYP4C19⁎ CYP4C20⁎

Pyloric caeca Pyloric caeca

References Effects

Exogenous

CO, BA, CF 3-MC BaP, FLU/3-MC

↑ ↑ ↑/↓

BNF



E, PoA

PB, BaP



20E

PB, H



Rewitz et al. (2004) Rewitz et al. (2004) Li et al. (2004) Li et al. (2004) Snyder (1998a) Snyder (1998a) Brown et al. (1998) Rewitz et al. (2003) Rewitz et al. (2003) James et al. (1996) Boyle et al. (1998a) Snyder (1998a) Snyder (1998a) Snyder (1998b) Snyder (1998a) Snyder (1998a)

E; ecdysone, PoA; ponasterone A, 20E; 20-hydroxyecdysone, CO; crude oil, BA; benz(a)anthracene, CF; clofibrate, 3-MC; 3-methylcholanthrene, BaP; benzo(a) pyrene, PB; phenobarbital, H; heptachlor, FLU; fluoranthene. ⁎ Indicate sequences that are only partially completed. Note that partial and full-length sequences resulting from genome and EST projects are not presented here.

of the compounds tested. Both CYP342A1 and CYP4BB1 metabolized pyrene to 1-hydroxypyrene, although CYP342A1 at a higher rate (Jorgensen et al., 2005a). Expression of CYP4AT1 and CYP331A1 in the polychaete Capitella capitata sp. I, an opportunistic invader and dominant inhabitant of heavily contaminated sediments, was also induced by PAHs (Li et al., 2004). In contrast, expression of CYP4Y1, a partially sequenced P450 from the mussel Mytilus galloprovencialis, was inhibited by crude oil (Snyder, 1998a; Snyder et al., 2001). In mammals, members of the CYP4 family are involved in fatty acid hydroxylation (Simpson, 1997). Compared to mammals, the insect CYP4 family is very diversified (Scott et al., 1994). That is, insects have more CYP4 genes than mammals, some of which are believed to be involved in the metabolism of xenobiotics (Scott et al., 1994; Danielson et al., 1998). The CYP342A1 (a member of the 4 Clan i.e. most closely related to CYP4 enzymes: Fig. 2) and CYP4BB1 mediated metabolism of pyrene (Jorgensen et al., 2005a) support the supposition that xenobiotics may be substrates of invertebrate CYP4 enzymes. In molluscs, CYP30 was obtained from gonadal tissue of the clam Mercenaria mercenaria. This protein shows highest sequence identity to mammalian CYP3 and subsequently to insect CYP6 enzymes (Brown et al., 1998). Due to its gonadal expression, it has been suggested that CYP30 may be involved in the metabolism of steroid hormones (Brown et al., 1998).

The first evidence of crustacean P450 genes came from the identification of CYP2L1 from the hepatopancreas of the spiny lobster Panulirus argus (James et al., 1996). Even though this protein is less than 40% identical to other CYP2 enzymes it was “forced” into this family based on the substantial sequence homology which makes it clear that it belongs in the 2 Clan (Fig. 2). Subsequently, several P450 sequences closely related to CYP2L1 were identified from the hepatopancreas of P. argus (Boyle et al., 1998a). In the shore crab C. maenas, CYP330A1, also a member of the 2 Clan, was isolated from the hepatopancreas (Rewitz et al., 2003). Sequencing of different clones indicates the presence of more than one closely related P450s in this tissue (K.F. Rewitz, unpublished data). This could indicate that the 2 Clan of P450 enzymes is diversified in crustaceans. In mammals, the 2 Clan and in particular the CYP2 family is the most expansive group of extremely versatile enzymes e.g. human CYP2D6 metabolizes more than 75 different drugs (Nebert and Russell, 2002). In Drosophila, and other insects, expansion of the 3 and 4 Clans has occurred and these are more diversified than the 2 Clan. Expression of CYP330A1 in the hepatopancreas of C. maenas was induced by the molting hormone E but also by phenobarbital and BaP. This indicates some degree of similarity to vertebrates where phenobarbital is a typical CYP2 enzyme inducer (Bresnick, 1993). The only crustacean P450 for which functions have been established is the CYP2L1. When expressed in yeast, this enzymes

Fig. 2. A phylogenetic Neighbor-Joining tree inferring the relationship of marine invertebrate P450s (boxed) to the full complement of Drosophila P450s (excluding P450 pseudogenes). This tree was generated using a ClustalX multiple alignment. Taxonomic names of marine invertebrates are abbreviated, with the two letters indicating the genus and species i.e. Carcinus maenas (Cm); Capitella capitata (Cc); Haliotis rufescens (Hr); Homarus americanus (Ha); Lytechinus anamesis (La); Mercenaria mercenaria (Mm); Mytilus galloprovencialis (Mg); Nereis virens (Nv); Panulirus argus (Pa); Penaeus setiferus (Ps).

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catalyzes the NADPH dependent 16-α-hydroxylation of testosterone and progesterone (Boyle et al., 1998b), which are substrates of vertebrate CYP2 enzymes (Gonzalez, 1989). In addition to these substrates, the partially purified P450 enzyme from the hepatopancreas microsomes, metabolizes the monooxygenation of benzphetamine, aminopyrine, 7-ethoxycoumarin and BaP (James, 1990). The P450 mediated biosynthesis and degradation of ecdysteroids has promoted the search for the specific P450 enzymes involved. CYP4C15 was isolated from the Y-organ of the freshwater crayfish Orconectes limosus (Dauphin-Villemant et al., 1999). Even though its expression is specific to the Y-organ, the major site of ecdysteroid production, there is no evidence indicating a role in the biosynthesis of ecdysteroids. Changes in expression of another P450, CYP45, in the hepatopancreas of the lobster Homarus americanus, coincides with changes in the hemolymph ecdysteroid levels (Snyder, 1998b). CYP45 is most closely related to members of the 3 Clan (Fig. 2), such as CYP3 and CYP6 enzymes, and is induced by 20-hydroxyecdysone (20E: the principal arthropod molting hormone), phenobarbital and the insecticide heptachlor (Snyder, 1998b; Snyder and Mulder, 2001). Numerous P450 genes, most of which are only partially sequenced, have been obtained from the ongoing genome sequencing project of the sea urchin S. purpuratus (see Dr. Nelson's P450 website: http://drnelson.utmem.edu/CytochromeP450.html). It is interesting to note the discovery of a CYP51 ortholog. This enzyme, the sterol 14α-demethylase, is required for sterol metabolism and believed to be one of the most ancient P450s due to its presence in bacteria, plants, fungi and animals (Yoshida et al., 2000). This finding is consistent with echinoderms being capable of de novo sterol biosynthesis; whereas this gene was lost in insects which like crustaceans are sterol heterotrophs (Kanazawa, 2001). It must be assumed that this gene is also present in polychaetes and in those molluscs capable of synthesizing sterols de novo. 4. Phylogeny of marine invertebrate P450s Fig. 2 shows the inferred phylogenetic relationship of those marine invertebrate P450s listed in Table 1 with Drosophila P450 proteins. Drosophila was chosen because it is one of the best characterized invertebrates and the species phylogeny makes it more suitable than e.g. C. elegans. None of the marine invertebrate P450s, presented in the tree, is clearly orthologous to Drosophila proteins, which reflects the enormous gap in our knowledge of P450s in these marine species. Obviously, crustaceans should posses orthologs of the Drosophila Halloween P450s involved in ecdysteroidogenesis (see Gilbert, 2004). In insects, the Halloween genes are among the few truly orthologous P450s. An EST (GenBank accession no. CN952331) from lobster H. americanus show significant homology to the Halloween P450, Shade (Shd; CYP314A1: Petryk et al., 2003) and another EST (GenBank accession no. DV111478) from the crab C. maenas indicates close relation to the Halloween P450, Disembodied (Dib; CYP302A1: Warren et al., 2002). The marine invertebrate P450 sequences presented in Fig. 2 are all grouping within the 2, 3 and 4 Clans. In marine invertebrates, the only apparent mitochondrial P450s are the above mentioned lobster and crab ESTs with homology to Halloween P450s and a

genomic annotated sequence from the sea urchin S. purpuratus with homology to vertebrate CYP27A. Furthermore, CYP10 identified from the dorsal bodies of the freshwater mollusc Lymnaea stagnalis has the signature of mitochondrial P450s. In a phylogenetic analysis, both CYP10 and the sea urchin CYP27A were grouped in the Mito Clan (data not shown). Although mitochondrial P450s are well represented in insect genomes, CYP44A1 is the only apparent mitochondrial P450 in C. elegans. The freshwater cnidarian Hydra magnipapillata EST project has identified a number of partial P450 sequences but no apparent mitochondrial P450s. It is unknown if annelids contain mitochondrial P450s; however, P450 activity towards PAHs was not observed in mitochondrial fractions from annelids (see Lee, 1998). Several marine invertebrate sequences appear to be members of the invertebrate specific CYP4C subfamily (Fig. 2). With the exception of CYP4C7, the function of members of this subfamily is unknown. CYP4C7 is selectively expressed in the corpora allata (CA) of the cockroach Diploptera punctata where it mediates the hydroxylation of sesquiterpenoids. It is believed that this P450 is part of a catabolic pathway to reduce the juvenile hormone level within the CA (Sutherland et al., 1998). It is most likely that the CYP1A family, which has received considerable attention due to its significance in the metabolism of PAHs in vertebrates, is vertebrate specific. CYP1A enzymes are not present in the genomes of fully sequenced invertebrates such as C. elegans and Drosophila (or any other insect sequenced). Although an EST (GenBank accession no. CA036213) from the earthworm Lumbricus rubellus shows 44% amino acid sequence identity to mouse CYP1A1, this partial sequence corresponds to a conserved part of P450s and it shares almost similar identity to CYP2 enzymes. Therefore the complete sequence is probably less than 40% identical to CYP1A family members and will likely be assigned to a new family within the 2 Clan. A partial P450 sequence differentially expressed in Pacific oyster Crassostrea gigas exposed to hydrocarbons has been reported as CYP1A-like (Boutet et al., 2004). However, based on sequence similarity is it unlikely that it represents a member of the CYP1 family. The marine invertebrate P450s grouping in the 2 Clan are the closest relatives to vertebrate CYP1A enzymes and they could be responsible for the metabolism of typical substrates of CYP1A enzymes such as BaP and ethoxycoumarin, reactions which have been amply reported to occur in marine invertebrates. However, caution should be taken when portending functional competence from sequence similarities because even single amino acid substitutions of important residues can change the catalytic competence of the enzyme (Chen et al., 2002). Yet when it comes to orthologous genes, sequence identity often indicates functional equivalence. The Halloween P450 enzymes (involved in the biosynthesis of E), Phantom (Phm: CYP306A1), Dib and Shadow (Sad: CYP315A1) (Warren et al., 2002, 2004), of the lepidopteran Manduca sexta are only 33–40% identical to dipteran orthologs but their functions are conserved (Rewitz et al., 2006a). RT-PCR facilitated techniques with degenerated oligo-nucleotide primers matching consensus sequences of vertebrate and insect P450s have resulted in the identification of a number of new marine invertebrate P450 sequences. Currently, the genome sequencing projects of sea urchin S. purpuratus together with the

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increased use of high-throughput methods, generating data such as ESTs, accelerate the identification of marine invertebrate P450s. As genomic and transcriptomic information on marine invertebrates continues to emerge, it will allow for the use of advanced molecular techniques such as thematic DNA array analysis. These growing resources will reveal the diversity of P450s in different phyla of marine invertebrates and provide important information about ancestral and truly orthologous P450 genes. For instance, CYP20 orthologs have been identified in cnidarians (H. magnipapillata, GenBank accession no. BP508840), annelids (leech Haementeria depressa, GenBank accession no. CN807321) and echinoderms (S. purpuratus, GenBank accession no. XP798308). Orthologs of CYP20 are also present in fungi and vertebrates making its origin ancient. Special attention should be given to orthologous genes of orphan P450s (P450s with unknown functions), particularly those evolutionarily conserved for the greatest period of time, since they are likely to catalyze fundamental physiological reactions. 5. Immuno- and DNA probe detection Cross-reaction with antibodies or DNA probes derived from vertebrate P450s has been used as preliminary evidence for the presence of marine invertebrate genes or proteins with sequence similarities. Multiple epitopes recognized by polyclonal antibodies and DNA probes to five major P450 families (CYP1A, CYP2, CYP3A, CYP4A and CYP11A) have been detected in different phyla of marine invertebrates, including Cnidaria, Mollusca, Arthropoda (Crustacea) and Echinodermata (den Besten et al., 1993; Boyle and James, 1996; Heffernan et al., 1996; Wootton et al., 1996; Oberdörster et al., 1998; Peters et al., 1998, 1999; see Snyder, 2000). Due to the low sequence identity between vertebrate and invertebrate P450 proteins, interpretation of data from such studies is a difficult and risky task. This is demonstrated by the fact that antibodies raised against vertebrate CYP1A proteins continually have been used to detect proteins in invertebrates (Peters et al., 1998; Danis et al., 2004; Shaw et al., 2000, 2004), even though the CYP1 family appears to be vertebrate specific. Thus, vertebrate CYP1A antibodies may crossreact with distantly related P450s (b40% identical primary structure) due to recognition of evolutionary broadly retained motifs or even with other proteins in these invertebrates. To exemplify the uncertainty using this approach, CYP30 from the clam, M. mercenaria, was not recognized by antibodies against its closest vertebrate homolog, CYP3A, despite ≈ 38% amino acid identity (Brown et al., 1998). Molluscs have received considerable attention due to their central role in ecotoxicology and applications in biomonitoring of environmental pollutants. A number of immunological studies have indicated cross-reactivity of digestive gland proteins with antibodies against vertebrate xenobiotic metabolizing P450 enzymes (CYP1A, CYP2B, CYP2E, CYP3A and CYP4A) (Peters et al., 1998; Wootton et al., 1995, 1996; Shaw et al., 2000, 2004). In addition, DNA probes synthesized from vertebrate CYP1A, CYP4 and CYP11A (a mitochondrial P450 involved in side chain cleavage of cholesterol during pregnenolone biosynthesis) sequences hybridized with digestive gland mRNA from Mytilus

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edulis and M. galloprovencialis (Wootton et al., 1995, 1996). In the pyloric caeca of the sea star, Asterias rubens, PCB induced 73fold increase in the level of CYP1A immunopositive proteins (CYP1A IPP) recognized by vertebrate CYP1A antibodies (Danis et al., 2004). In hepatopancreas microsomes from the blue crab Callinectes sapidus, scup CYP3A antibodies not only crossreacted with a protein of the expected size but also inhibited testosterone 6β-hydroxylation (a reaction catalyzed by CYP3A in vertebrates). This might indicate that a protein(s) with structural and functional similarities to vertebrate CYP3A is responsible for this activity in the blue crab (Oberdörster et al., 1998). The specificity of the antibodies used in the above mentioned experiments has not been determined since members of most of these families have not been found in invertebrates. Although such approaches may turn out to be useful for monitoring exposure to environmental contaminants, results obtained using these methods need critical interpretation and are preliminary, and thus, the biological information from these experiments is very limited. The rapid emergence of P450 sequences from marine invertebrates should be used to raise specific antibodies for future studies. Specific antibodies may prove invaluable in elucidating the regulation and function of marine invertebrate P450s. Antibodies raised against specific marine invertebrate P450s include spiny lobster P. argus anti-CYP2L and American lobster H. americanus anti-CYP45. Antibodies against CYP45 were used to detect xenobiotically induced variations in CYP45 protein levels (Snyder and Mulder, 2001), in agreement with the previously observed induction of CYP45 transcript levels (Snyder, 1998b). 6. Tissue distribution P450 mediated activity and expression are found in various tissues and organs of marine invertebrates. The highest levels and activities towards xenobiotics are usually observed in tissues processing food i.e. intestine of polychaetes, digestive gland of molluscs, hepatopancreas of crustaceans and pyloric caeca of asteroid echinoderms (Solé and Livingstone, 2005). In the polychaete N. virens, BaP-hydroxylase activity was concentrated in the microsomal fraction of the lower portion of the intestine, with lower activity in the upper pharynx, esophagus and upper part of the intestine (Fries and Lee, 1984). In crustaceans, P450 levels and activity towards xenobiotics are usually much higher in hepatopancreatic microsomes than e.g. in microsomes from the stomach, antennal gland and testes (James, 1989, 1990). Surprisingly, P450 reductase activity has been reported to be disproportionately low in the hepatopancreas of crustaceans, although this might be an artifact caused by proteolytic degradation (see James, 1989). In the echinoderms, A. rubens and Mathasterias glacialis, BaP-hydroxylase and P450 reductase activities are highest in the pyloric caeca. Lower activity is observed in the stomach and gonadal tissue (den Besten et al., 1990). Depending on the biological function, P450s can be very selectively expressed in specific tissues or, though at different levels, in various tissues. In mammals, members of the CYP4 family catalyzing fatty acid and prostaglandin ω-

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hydroxylations are expressed in most tissues. Less widely abundant are CYP2A1 and CYP2A2 enzymes being present only in the liver (Gonzalez, 1989). Similarly, in the shore crab C. maenas, CYP4C39 expression was detected in several tissues including the gills, hepatopancreas, heart, eye-stalk and epidermis whereas CYP330A1 (most closely related to CYP2 enzymes) was selectively expressed in the hepatopancreas (Fig. 3). The tissue-specific expression of six non-redundant C. maenas ESTs encoding P450s of the 2, 3 and 4 Clans is currently being investigated in our laboratory. The data indicate that the hepatopancreas is a major site of P450 gene expression (E. Dam, K.F. Rewitz, B. Styrishave, O. Andersen, unpublished data). This is in agreement with previous biochemical studies establishing the hepatopancreas as the major tissue of P450 activity in crustaceans. Unlike many mammalian hepatic P450s that metabolize xenobiotics (CYP1–4 families), steroidogenic P450s are almost exclusively expressed in endocrine tissues such as testes and ovaries (Gonzalez, 1989; Nebert and Russell, 2002). For the aromatase (CYP19), a strict spatio-temporal expression is achieved through distinct promoters and signal molecules (Bulun et al., 2003). Similarly, in insects the ecdysteroidogenic Halloween P450s, phm, dib and sad, are predominantly ex-

pressed in the prothoracic gland cells, the primary site of ecdysteroid production (Warren et al., 2002, 2004; Rewitz et al., 2006a). Although there are no rules, tissue-specific expression of the corresponding genes may provide clues to the catalytic competence of a vast number of orphan P450s. In general, P450s present in hepatic tissues often have specificity for a wide array of xenobiotics whereas P450s with physiologically important substrates often are selectively expressed in principal endocrine tissues. 7. P450 mediated metabolism of endogenous compounds P450s participate in the biosynthesis of signal molecules that coordinate developmental processes. The P450 mediated biosynthesis of steroidal precursors to active hormones occurs mainly in endocrine tissues (Warren et al., 2002, 2004; Niwa et al., 2005; Rewitz et al., 2006a). The inactivation of steroid hormones, prior to their excretion, usually occurs in hepatic tissues. In mammals, inactivation includes several hydroxylations catalyzed by members of the CYP2 and CYP3 family (Zimniak and Waxman, 1993). However, in insects, the catalytic activation of E to the active molting hormone, 20E, by Shd occurs in the fat body, which is analogous to the vertebrate liver,

Fig. 3. Northern analysis showing molt cycle changes in the tissue-specific mRNA abundance (20 μg total RNA/lane) of shore crab Carcinus maenas CYP330A1 and CYP4C39 (K.F. Rewitz, unpublished data). Experimental conditions as described previously (Rewitz et al., 2003). Bars represent samples of tissues pooled from three individuals. B, late postmolt (stage B); C, late intermolt (stage C3–C4); D, premolt (stage D1–D2). Samples were normalized to actin. He, hepatopancreas; Gi, Gill; Ep, epidermis; Te, testes, Vd, vas deferens; Es, eye stalk; Mu, muscle; Yo, Y-organ; Hrt, heart; In, intestine.

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but also in the midgut and Malpighian tubules (see Lafont et al., 2005). The expression and activity of shd is subject to a precise developmental regulation (Smith et al., 1983; Petryk et al., 2003; Rewitz et al., 2006b). 7.1. Ecdysteroids Ecdysteroids are arthropod molting hormones that control the execution of the developmental schedule during embryogenesis, post-embryonic development and reproduction (Fig. 4). P450s play a pivotal role in the biosynthesis of these steroid hormones by catalyzing the terminal hydroxylations converting ecdysteroid precursor into 20E (see Chang, 1993; Gilbert et al., 2002; Gilbert, 2004; Lafont et al., 2005). Dependent on insect species, varying ratios of E and 3-dehydroecdysone (3DE) are synthesized and secreted by the prothoracic glands from dietary cholesterol or phytosterols (Warren et al., 1988; Kiriishi et al., 1990; see Gilbert et al., 2002). 3DE is immediately converted to E by a hemolymph 3-ketoreductase (see Lafont et al., 2005). Recently, the Drosophila Halloween genes mediating the final four sequential hydroxylations leading to 20E (Fig. 4), that code for Phm (the 25-hydroxylase), Dib (the 22-hydroxylase), Sad (the 2-hydroxylase) and Shd (the 20-hydroxylase), were characterized as being P450 genes (Chávez et al., 2000; Warren et al., 2002, 2004; Petryk et al., 2003). These genes were characterized using molecular genetics and their biochemical functions established in cellular homogenates of Drosophila S2 cells transiently transfected with vectors expressing the genes (Warren et al., 2002, 2004; Petryk et al., 2003). In crustaceans, the Y-organ is the equivalent of the insect prothoracic gland. The Y-organ produces and secretes E and 3DE (Lachaise et al., 1986, 1989; Spaziani et al., 1989; Dauphin-Villemant et al.,

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1997; Wang et al., 2000) which are converted to 20E in peripheral tissues. Since the biosynthetic pathways are similar in crustaceans and insects (Lachaise et al., 1993; Gilbert et al., 2002), it is reasonable to assume that crustaceans may possess orthologs of the insect Halloween genes. In insects, the transformation of E to 20E is catalyzed by Shd also known as the ecdysone 20-monooxygenase (E20MO) (Petryk et al., 2003; Rewitz et al., 2006b). Biochemical evidence for the occurrence of this reaction in crustacean species has been amply reported (Chang et al., 1976; Chang and O'Connor, 1978; James and Shiverick, 1984; Soumoff and Skinner, 1988; Wang et al., 2000; see Grieneisen, 1994). For instance, E is hydroxylated to 20E in extracts of crab testes and hepatopancreas (Chang et al., 1976; Chang and O'Connor, 1978). Furthermore, injected [3H]-E was converted into 20E in the female lobster H. americanus (Snyder and Chang, 1991). In insects, the fat body, midgut and Malpighian tubules are the primary sites of Shd activity and expression (Smith et al., 1983; see Smith, 1985; Petryk et al., 2003; see Lafont et al., 2005; Rewitz et al., 2006b). In crustaceans, conversion of E to 20E occurs mainly in the green gland, ovaries, testes, hepatopancreas and epidermis (Chang et al., 1976; Chang and O'Connor, 1978; James and Shiverick, 1984; Soumoff and Skinner, 1988; Böcking et al., 1995; Spaziani et al., 1997). Whereas Phm is microsomal and Dib and Sad are mitochondrial, the localization of the E20MO activity is associated with both mitochondrial and microsomal fractions depending on species and maybe developmental stage (Bollenbacher et al., 1977; Feyereisen and Durst, 1978; Mitchell and Smith, 1986; see Weirich et al., 1984, 1985; Smith, 1985; Weirich, 1997; Lafont et al., 2005). The Shd N-terminus may provide the explanation as it is thought to carry a dual signal sequence resulting in either microsomal or mitochondrial localization

Fig. 4. Theoretical illustration of ecdysteroid metabolism and action. (A) The sites of terminal hydroxylations mediated by the Halloween gene encoded P450s: a, biosynthetic hydroxylation of ecdysteroid precursors occurring in the prothoracic gland cells of insects and in the Y-organ of crustaceans resulting in the formation of ecdysone (E); b, peripheral hydroxylation at position C-20 creating the active molting hormone, 20-hydroxyecdysone (20E). (B) The biosynthesis, inactivation and action of ecdysteroids modulated through the ligand-specific regulation of the EcR (Ecdysteroid receptor)/USP (Ultraspiracle) heterodimer that controls the transcription of target genes. Note that the first step in the formation of E, the cholesterol 7,8-dehydrogenation to form 7-dehydrocholesterol (7dC) and hydroxylation of E and 20E to the more inactive 26-hydroxyecdysone (26E) and 20,26-dihydroxyecdysone (20,26E), respectively, are likely catalyzed by yet uncharacterized P450s (Grieneisen et al., 1993; Williams et al., 2000b). The formation of the ketodiol (2,22,25-trideoxyecdysone), the substrate of Phantom, from 7dC occurs in the so-called black box reaction (a series of uncharacterized reactions) and is followed by the Halloween P450 catalyzed hydroxylations (see Lafont et al., 2005).

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(Petryk et al., 2003; Rewitz et al., 2006b). P450 enzymes are believed to be involved in two additional enzymatic steps in the biosynthesis and metabolism of ecdysteroids. Biochemical evidence suggests that in insects the first step in the biosynthesis of ecdysteroids, the 7,8-dehydrogenation of cholesterol, and the C-26 hydroxylation of E and 20E, which is believed to be a metabolic inactivation, are catalyzed by P450 enzymes (Grieneisen et al., 1993; Williams et al., 2000b). The specific enzymes involved have not been identified. Ecdysteroids have also been found in non-arthropod invertebrates including nematodes, annelids and molluscs but their origin and function are obscure. It has yet to be demonstrated if these organisms produce ecdysteroids or obtain ecdysteroids through the diet (see Lafont et al., 2005). So far there is no evidence for the existence of ecdysteroidogenic Halloween P450s in non-arthropods. 7.2. Sex steroids The presence and/or metabolism of the vertebrate sex steroids, progesterone and testosterone, have been demonstrated in gonadal tissues of marine invertebrates including molluscs, crustaceans and echinoderms (Schoenmakers, 1979; James and Shiverick, 1984; Livingstone et al., 1989; Ronis and Mason, 1996; Osada et al., 2004). Fig. 5 shows the presumed P450 mediated reactions occurring in vitro in tissues of marine invertebrate species. Estrogens have also been detected in marine invertebrates, and in the Japanese scallop Patinopecten yessoensis estrogens are possibly synthesized by P450 mediated aromatase activity in estrogenic cells of the gonads (Osada et al., 2004). The aromatase (CYP19 in vertebrates), responsible for the conversion of androgens to estrogens, has received considerable attention because tributyltin (TBT) induced imposex in gastropods is associated with increased testosterone levels. Therefore it has been speculated that TBT inhibits P450 mediated aromatase activity which in turn results in elevated levels of testosterone that causes the development of male sex characteristics in female gastropods (see

Gibbs et al., 1991; Alzieu, 2000). However, Ronis and Mason (1996) found only modest loss of aromatase activity in the periwinkle, Littorina littorea, even at high TBT concentrations. In the more TBT susceptible gastropod Buccinum undatum, a significant reduction in aromatase activity was found in imposex affected animals (Santos et al., 2002). In contrast, TBT induced alterations of sex hormone levels were not associated with changes in aromatase activity in the gastropod Bolinus brandaris (Morcillo and Porte, 1999). Currently, it is conjectural whether inhibition of aromatase activity is responsible for imposex development. The disruption of other mechanisms, besides aromatase activity, may be causing the imposex development and sex steroid receptors may be involved (Santos et al., 2005). An ortholog of the vertebrate estrogen receptor identified in the mollusc Aplysia californica is not activated by estrogen, although it has been suggested that the ancient form of this receptor was ligand activated (Thornton, 2003). The lack of affinity of this invertebrate estrogen receptor for its ligand, estrogen, makes the role of the aromatase in this invertebrate species questionable. Testosterone 6β-hydroxylation has been detected in digestive gland microsomes of molluscs (Ronis and Mason, 1996; see Peters et al., 1998). Several different metabolites of progesterone and testosterone are formed in preparations of crustacean tissues including hepatopancreas and testes (Burns et al., 1984; James and Shiverick, 1984; see James and Boyle, 1998). In vertebrates, the position at which the steroid is hydroxylated depends on the specific P450 enzyme involved. For example, in mammals the 6β-position is predominantly hydroxylated by hepatic CYP3 and CYP2, enzymes involved in steroid degradation (see Funae and Imaoka, 1993), whereas the extrahepatic CYP17, involved in steroid biosynthesis, hydroxylates the 17αposition (see Kühn-Velten, 1993). The metabolism of cholesterol into pregnenolone and progesterone, and progesterone into 17α-hydroxyprogesterone (a precursor of testosterone) occurs in the pyloric caeca and gonads of the sea star A. rubens (Schoenmakers, 1979; Schoenmakers and Voogt, 1980). In mammals, these reactions involve the action of CYP11A1 and CYP17 enzymes (see Zimniak and Waxman, 1993). So far there

Fig. 5. P450 catalyzed hydroxylations of sex hormones, observed in tissues of marine invertebrates. 1P450 enzyme(s) known to catalyze these reactions in mammals (see Zimniak and Waxman, 1993). Literature references are shown according to the observations of these reactions in marine invertebrates. ⁎ Indicate positions at which the substrates are hydroxylated.

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is no evidence for the existence of these enzymes in species of marine invertebrates. Whether marine invertebrates synthesize sex steroids or obtain precursors from the diet as well as their roles in reproduction is conjectural. If sex steroids are produced within these organisms it seems likely, in analogy with vertebrates, that P450 enzymes are involved. Verification of such biosynthetic pathways and identification of specific P450 enzymes involved would be a significant step towards better understanding invertebrate physiology. 7.3. Non-steroidal endogenous substrates Capacity for hydroxylations of fatty acids has been demonstrated in invertebrates (see Feyereisen, 1999). Lauric acid hydroxylase activity, which is catalyzed in mammals by CYP4A enzymes, has been observed in digestive gland microsomes of the mussel M. galloprovencialis (Michel et al., 1994). In vertebrates, the hydroxylation of the ω-position of arachidonic acid and the synthesis of eicosanoids involve P450s (Zimniak and Waxman, 1993). These signal molecules are generally found in both vertebrates and invertebrates (see Stanley-Samuelson, 1987; De Petrocellis and Di Marzo, 1994), although their physiological significance in marine invertebrates is not well established. As discussed above, it is plausible that CYP51 is involved in sterol synthesis in polychaetes, echinoderms and some molluscs. 8. P450 mediated metabolism of xenobiotics The major pathway for lipophilic xenobiotic elimination includes P450 mediated metabolism in the vertebrate liver or analogous invertebrate tissues. Marine invertebrate P450 enzyme systems catalyze a number of reactions in vitro with exogenous substrates (Table 2) but knowledge about the specific P450 enzymes involved is lacking. Fig. 6 shows some common P450 catalyzed reactions in tissues of marine invertebrates and the enzyme(s) catalyzing these reactions in mammals and insects. Although P450s in general mediate the first step in the metabolic elimination of lipophilic compounds, this will in some cases generate more toxic and even mutagenic and/or carcinogenic compounds (Chipman et al., 1991; Stegeman and Lech, 1991). The high P450 levels (estimated from CO spectrum) in these tissues probably reflects the presence of several P450s. Most studies in marine invertebrates have focused on P450-dependent biotransformation of PAHs to which the fauna is exposed in contaminated areas. Historically, some assays such as BaPhydroxylase activity have been interpreted as a measure of “total” P450 activity. However, it has long been evident that total or fractioned tissue homogenates contain a pool in which an unknown number of P450s, with broad or narrow and sometimes overlapping substrate specificities are present at highly differing levels. Due to the broad substrate specificity of many xenobiotic metabolizing P450s, more than one P450 enzyme will often contribute to the activity of a measured enzymatic reaction. Generally, crustaceans have high total P450 level and BaPhydroxylase activity compared to polychaetes, molluscs and echinoderms (Livingstone, 1990, 1998). Although the total P450 level in the hepatopancreas of some crustaceans ap-

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proximates vertebrate hepatic levels, the high level is not accompanied by an equally high rate of xenobiotic metabolism (Livingstone, 1998; Solé and Livingstone, 2005). The abundance of P450 enzymes does not necessarily reflect the activity, which also depends on the availability of coenzymes and cofactors such as the P450 reductase, cytochrome b5 and NADPH (Stegeman and Kloepper-Sams, 1987; see Feyereisen, 1999). When comparing P450 abundance and xenobiotic metabolizing activity it should be emphasized that both vary significantly not only between species from different phyla but also between species within the same phylum. For instance, in some polychaetes such as Abarenicola pacifica and Arenicola sp., PAHs are metabolized at a lower rate compared to the polychaetes N. virens and Nereis diversicolor (Payne and May, 1979; Driscoll and McElroy, 1996; Christensen et al., 2002). Information about P450 activity in cnidarians is very limited. NADPH dependent 7-ethoxyresorufin O-deethylase (EROD) activity has been observed in sea anemones (Heffernan and Winston, 1998, 2000). In the sea anemone, Anthopleura elegantissima, EROD activity was 2.3 pmol/min/mg protein. The P450 abundance in another sea anemone Bunodosoma cavernata, was 52 pmol P450/mg microsomal protein (Heffernan and Winston, 1998), which is in the same order of magnitude as for most other marine invertebrates. In whole body homogenates of the annelid polychaete C. capitata, BaP-hydroxylase activity was detected only after exposure to PAHs (Lee et al., 1979; Lee and Singer, 1980). Although the major metabolite of BaP formed in the polychaete N. virens was 3-hydroxybenzo(a)pyrene, 7-hydroxy- and 9hydroxybenzo(a)pyrene and diol derivates were also detected (Fries and Lee, 1984; see Lee, 1998). As a substrate, pyrene has the advantage that only one Phase I metabolite is formed (1hydroxypyrene: Giessing et al., 2003). In N. virens, the hydroxylation of pyrene to 1-hydroxypyrene occurs in gut tissue (Jorgensen et al., 2005b). The P450 enzyme systems of molluscs are widely involved in the biotransformation of xenobiotics (Table 2), although the catalytic properties differ from those of crustacean P450 enzymes. Quinones are the primary metabolites of BaP in molluscs whereas diols and phenols are the main products in crustaceans and echinoderms (see Livingstone, 1989). The metabolites of BaP are crucial for the ultimate toxicity of the compound, since the position at which BaP is attacked determines the carcinogenicity of the product. Animals that primarily metabolize BaP to 7,8dihydrodiol-9,10-oxides, the ultimate carcinogen of BaP, have greater risk of suffering DNA damage (James, 1990). Overall, decapod crustaceans are the most efficient biotransformers of xenobiotics among marine invertebrates (Livingstone, 1998). However, PAHs are also metabolized in non-decapod crustaceans such as the barnacle, Balanus eburneus, capable of metabolizing BaP in microsomes from the digestive gland (Stegeman and Kaplan, 1981). 9. Regulation of P450s Since the early discovery of mammalian CYP1A enzymes and the PAH activated aryl hydrocarbon receptor (AHR),

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Table 2 P450 catalyzed enzymatic reactions with xenobiotic substrates in tissues of marine invertebrates Species Phylum Cnidaria Actinia equina Anthopleura elegantissima Anthopleura xanthogrammica Calliactis parasitica Anemonia sulcata Phylum Annelida (Class: Polychaeta) Capitella capitata Nereis diversicolor Nereis succinea Nereis virens Scolecolepides viridis Phylum Mollusca Cerastoderma edule Crassostrea virginicia Cryptochiton stelleri Illex illecebrosus Littorina sp. Mytilus californianus Mytilus edulis

Tissue

Reaction

Refences

A C C A A

BaP-hydroxylase EROD EROD BaP-hydroxylase BaP-hydroxylase

Solé and Livingstone (2005) Heffernan and Winston (1998) Heffernan and Winston (1998) Solé and Livingstone (2005) Solé and Livingstone (2005)

A A A I A

BaP-hydroxylase BaP-hydroxylase BaP-hydroxylase BaP-hydroxylase, EROD BaP-hydroxylase

see Lee (1981) Driscoll and McElroy (1996); Solé and Livingstone (2005) see Lee (1981) Lee and Singer (1980); McElroy (1990); see Lee (1998) Driscoll and McElroy (1996)

D D D D D D D

BaP-hydroxylase BaP-hydroxylase BaP-hydroxylase BaP-hydroxylase BaP-hydroxylase Aldrin epoxidase BaP-hydroxylase, ECOD, N,N-dimethylaniline N-demethylase BaP-hydroxylase, ECOD, N,N-dimethylaniline N-demethylase, Aminopyrine N-demethylase BaP-hydroxylase BaP-hydroxylase

Solé and Livingstone (2005) see Lee (1981) see Livingstone (1991) see Lee (1981) see Lee (1981) see Livingstone et al. (1989); see Livingstone (1991) Livingstone (1985); see Livingstone et al. (1989); see Livingstone (1991); Peters et al. (1999) see Livingstone et al. (1989); see Livingstone (1991); Michel et al. (1994); Pisoni et al. (2004)

Aniline hydroxylase, Aminopyrine N-demethylase, BaP-hydroxylase, ECOD, EROD, Benzphetamine N-demethylase BaP-hydroxylase, EROD BaP-hydroxylase, Pyrene hydroxylase

Singer et al. (1980); see Livingstone (1991)

Mytilus galloprovicialis

D

Modiolus modiolus Mercenaria mercenaria Phylum Arthropoda (Subphylum: Crustacea) Callinectes sapidus

D D

He, S, Gr, Gi, G, Y

Carcinus aestuarii Carcinus maenas

He, Gi He

Cancer irroratus Homarus americanus

He He

Panulirus argus

He

Maja crispata Balanus eburneus Penaeus aztecus Uca pugnax

He, S D, I, G He He, Gu, Gi, Gr

Phylum Echinodermata Asterias rubens Marthasterias glacialis Stronglyocentrotus sp.

P, G, S P D

BaP-hydroxylase Aldrin epoxidase, BaP-hydroxylase, Aniline hydroxylase Benzphetamine N-demethylase, Aminopyrine N-demethylase, BaP-hydroxylase, EROD, ECOD, Aldrin epoxidase, Aniline hydroxylase BaP-hydroxylase BaP-hydroxylase ECOD Aldrin epoxidase

BaP-hydroxylase BaP-hydroxylase BaP-hydroxylase

see Lee (1981) see Lee (1981)

Fossi et al. (1998) Solé and Livingstone (2005); K.F. Rewitz, unpublished results see Lee (1981) Elmamlouk and Gessner (1976); see Lee (1981); Carlson (1974) see Lee (1981); see Livingstone (1991), see James and Boyle (1998); James et al. (1979) Bihari et al. (1984) Stegeman and Kaplan (1981) see James and Boyle (1998) see Lee (1981)

den Besten et al. (1990, 1993), Solé and Livingstone (2005) den Besten et al. (1990), Solé and Livingstone (2005) see Lee (1981)

A; Whole animal, C; Columnar region, D; Digestive gland, G; Gonads, He; Hepatopancreas, I; Intestine, P; Pyloric caeca, S; stomach, Y; eyestalk, Gi; Gills, Gu; gut, Gr; green gland, EROD; ethoxyresorufin O-deethylase, ECOD; ethoxycoumarin O-deethylase.

eliciting the rapid induction of CYP1A gene expression (see Nebert et al., 2000), evidence for the presence of CYP1A enzymes in invertebrates has been pursued. The majority of investigations have focused on xenobiotic (primarily PAHs) induced P450 activity. Comparative studies have demonstrated that some marine invertebrates are de facto capable of regulating their P450 enzyme levels and to some extent increase P450 mediated metabolism of xenobiotics when exposed to

PAHs. Inhibition of P450 activities may have similarly important implications in ecotoxicology as well as in the physiology but has not been studied with the same vigor. The P450 enzyme systems of vertebrates and invertebrates appear to differ, insofar as the inducibility of invertebrate P450s by PAHs is less prominent. In general, PAHs induce activity and expression of P450s only a few-fold in marine invertebrates (Li et al., 2004; Rewitz et al., 2003, 2004; see James and Boyle,

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1998; see Snyder, 2000). However, the capacity of invertebrates to regulate P450 expression is substantiated by 267-fold upregulation of M. sexta shd within a single day (the first day of wandering) of development (Rewitz et al., 2006b). Mammalian P450 gene expression has been demonstrated to be controlled by a regulatory machinery composed of promoters with various response elements, enhancers and transcription factors (Waxman, 1999; see Nebert et al., 2000). The mechanisms underlying marine invertebrate P450 regulation are currently unknown but it is likely that nuclear receptors are involved. In insects, E and 20E influence P450 activities including the 20-hydroxylation and 26-hydroxylation of ecdysteroids (see Smith, 1985; Koegh et al., 1989; Williams et al., 1997). Although information about the regulation of P450 expression in marine invertebrate by endogenous compounds is virtually absent, it is beyond doubt that future research will

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show that humoral factors modulate the expression of some marine invertebrates P450 genes. Induction of P450 enzymes and P450 mediated activity by xenobiotics has been observed in several phyla (Annelida, Polychaeta; Mollusca; Arthropoda, Crustacea; Echinodermata) of marine invertebrates following exposure to PAHs (Lee et al., 1979; Lee and Singer, 1980; Bihari et al., 1984; Livingstone, 1988; den Besten et al., 1993; Michel et al., 1994; Sparagano et al., 1999). Induction of pyrene hydroxylase activity in gut tissue of the polychaete N. virens, by the substrate itself, was accompanied by an increase in Vmax and a decrease in the apparent Km (Jorgensen et al., 2005b). This indicates qualitative and quantitative alterations i.e. a selective induction of pyrene metabolizing P450 enzyme(s). This is in agreement with upregulation of expression of CYP342A1 encoding a pyrene metabolizing P450, following exposure of this species to PAHs (Rewitz et al.,

Fig. 6. Examples of P450 mediated metabolism of xenobiotics in tissues of marine invertebrates. ⁎Shown are the mammalian/insect P450 catalysts of these reactions (see Correia, 1995; Feyereisen, 1999).

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2004; Jorgensen et al., 2005a). In the polychaete, Abarenicola pacifica, which is considered to be a poor metabolizer of PAHs, no BaP-hydroxylase activity was observed after one week of continuous exposure to sediment-associated oil (Payne and May, 1979). However, in another study, PAH levels increased in A. pacifica for the first two weeks of exposure to contaminated sediment, but then decreased (Augenfeld et al., 1982). This might indicate induction of P450 mediated PAH metabolism only after long-term exposure in this species. Elevated BaP-hydroxylase activity has also been observed in the sea star A. rubens injected with PCBs (den Besten et al., 1993), in the mussel M. galloprovencialis exposed to 3-methylcholanthrene (Michel et al., 1994) and in the crab, Carcinus aestuarii, exposed to environmental contaminants (Fossi et al., 1998, 2000). At present, data are not extensive enough to allow comparison of the inducing efficiency of these xenobiotics but overall the data suggest that P450 enzymes inducible by PAHs are present in the majority of marine invertebrates. This induction is likely to be a significant component of the physiological response leading to elimination of toxic lipophilic compounds. The apparent absence of CYP1A enzymes in marine invertebrates suggests that PAHs cause the induction of other P450s in these animals. In C. elegans, a systematic screen of the complete set of P450s revealed that the CYP35 family enzymes were most effectively induced by PAHs (Menzel et al., 2001). The low degree of induction of marine invertebrate P450s by PAHs, compared to some mammalian P450s, indicates differences between vertebrate and invertebrate signal pathways. An obvious difference and possible explanation is that the invertebrate AHR orthologs of e.g. C. elegans, the clam Mya arenaria and Drosophila lack affinity for typical AHR ligands such as dioxin and βnaphthoflavone (Butler et al., 2001; see Hahn, 2002). This suggests that PAH induction of marine invertebrate P450s is AHR independent, which is in fact true for the PAH mediated induction of CYP35 genes in C. elegans (Menzel et al., 2005). Although the invertebrate AHR does not have affinity for the typical mammalian ligands, it is involved in the regulation of the CYP6B1 promoter in the black swallowtail caterpillar, Papilio polyxenes (Brown et al., 2005). CYP6B1 is a xenobiotic metabolizing enzyme that supports resistance to host-plant protective phytochemicals. This indicates a possible role of the AHR as component of invertebrate detoxification systems and emphasizes the fundamental conservation of signal pathways in vertebrates and invertebrates. In mammals, clofibrate induces CYP4 enzymes involved in the hydroxylation of fatty acids (Simpson, 1997). However, clofibrate did not induce total P450 level and laurate hydroxylase activity in the mussel M. galloprovencialis (Michel et al., 1994). Alterations in the metabolism of [14C]testosterone have also been observed in the freshwater crustacean Daphnia magna after exposure to xenobiotics including the P450 expression modulators phenobarbital, β-naphthoflavone and nonylphenolpolyethoxylates (Baldwin and LeBlanc, 1994; Baldwin et al., 1998). Such alterations of the metabolism of steroids may cause perturbation of endocrine homeostasis. This emphasizes the importance of gaining knowledge about the physiological functions of P450s in marine invertebrates and about the interactions between endog-

enous and exogenous substrates/inducers mutually influencing each other's fate. 10. Development, sex and season In the insect M. sexta, developmental changes in the expression of the phm, dib and sad indicate that the upregulation of these genes is important to support the zenith of ecdysteroid production by the prothoracic glands (Rewitz et al., 2006a). Such changes indicate that developmental expression of P450s may play an important role in the regulation of transient signal molecules. Although the crustacean P450s involved in the biosynthesis of ecdysteroids have not yet been identified, it is likely that they will turn out to be regulated over the molt cycle to support the rapid increase in ecdysteroid levels during premolt (Chang, 1995; Styrishave et al., 2004a). In mammals, developmental alterations in the expression of hepatic P450s, which result in differences in drug metabolizing activities, are governed by humoral factors (Kato and Yamazoe, 1993). We recently demonstrated molt cycle variations in the expression of several P450s in the hepatopancreas of C. maenas (Styrishave et al., 2004a; E. Dam, K.F. Rewitz, B. Styrishave, O. Andersen, unpublished data). In different life stages of shore crabs, high susceptibility to pyrene was accompanied by low hepatopancreatic expression of CYP330A1 and CYP4C39 (Styrishave et al., 2004b). Since the hepatopancreas is the major site of xenobiotic metabolism, such variations, which might be controlled by hormones, could affect the rate of the metabolism of xenobiotics and result in a stage specific susceptibility of animals exposed to environmental pollutant. Sex-specific expression of P450s has also been observed. In Drosophila, Cyp312a1 is expressed predominantly in males (Kasai and Tomita, 2003) and in the blue crab C. sapidus, P450 protein levels differ in the green gland and pyloric stomach between sexes (Singer and Lee, 1977). Such variations may affect the rate of excretion/elimination of xenobiotics. Since most marine animals express circa-annual variations in their basic physiology and biochemistry, seasonal variations in P450 activity occur. In the blue mussel M. edulis, both BaPhydroxylase activity and CYP1A IPP levels change during season (Wootton et al., 1996; Shaw et al., 2000). The crab M. crispata expressed seasonal variations in BaP-hydroxylase activity that correlated with ambient temperature but not with the reproductive cycle (Bihari et al., 1984). In the hepatopancreas microsomes from shrimp, ethoxycoumarin O-deethylase (ECOD) activity also undergoes seasonal changes (Zapata-Perez et al., 2005). These variations in P450 activities may be important in natural populations as environmental factors are changing. 11. In summary and the future Classical biochemical approaches have established that cellular fractions of marine invertebrate tissues contain P450s that catalyze a variety of different reactions. Despite considerable research, this approach has not resulted in the identification of the specific P450s involved. The membrane-bound nature of these enzymes makes purification notoriously difficult. However,

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molecular techniques open up new possibilities to clone and characterize P450s, even those expressed at low levels. From partially or fully sequenced genomes, it is now evident that P450 gene diversification in marine invertebrates, as in insects, has led to a large number of P450s in each species. It is possible that species and population susceptibility to environmental contaminants (e.g. Capitella sp. display remarkable differences in their ability to cope with environmentally induced stress) depends on genetic variations that determine the capacity of their P450 enzyme systems to rid toxic compounds from the organism. Although P450 enzymes are present ubiquitously within marine invertebrate phyla, the expansion of this family probably varies between species. In other animals, the divergence of a rather small number of truly orthologous P450 genes has been constrained by their physiological importance whereas consecutive gene duplications combined with environmental changes and dietary adaptations may have driven the functional diversification of paralogous P450 genes in species occupying different niches. Such diversification may increase the fitness of species in highly variable or contaminated areas. One of the most important and major challenges will be to functionally characterize the numerous orphan P450s emerging from sequencing projects. Those involved in physiologic functions, such as the metabolism of signal molecules, are worthy of special interest because they represent a key to understanding developmental processes. However, it is also important to characterize the specific xenobiotic metabolizing P450 enzymes as their biotransformation activities are often pivotal for the magnitude of the biological effects of xenobiotics. Investigations of xenobiotically induced gene expression using genomic approached and sensitive techniques such as quantitative real time PCR may in the future unravel stress induced P450s. The integration of biochemical techniques with molecular and genomic technologies will probably facilitate the functional characterization of marine invertebrate P450s. This is critical to our understanding of the basic physiology and biochemistry of these animals and should provide the foundations for discerning interference between environmental contaminants and endocrine aspects of development, growth and reproduction. Acknowledgements We thank Anne Jorgensen and unknown reviewers for constructive comments on the manuscript. This work was supported by The Danish Natural Science Research Council to O.A. References Addya, S., Anandatheerthavarada, H.K., Biswas, G., Bhagwhat, S.V., Mullick, J., Avadhani, N.G., 1997. Targeting of NH2-terminal-processed microsomal protein to mitochondria: a novel pathway for the biogenesis of hepatic mitochondrial P450MT2. J. Cell Biol. 139, 589–599. Alzieu, C., 2000. Impact of tributyltin on marine invertebrates. Ecotoxicology 9, 71–76. Anandatheerthavarada, H.K., Addya, S., Mullick, J., Avadhani, N.G., 1998. Interaction of adrenodoxin with P4501A1 and its truncated form P450MT2

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Further Reading http://drnelson.utmem.edu/CytochromeP450.html A list of annotated P450s from bacteria, fungi, plants and animals. http://p450.antibes.inra.fr/ Information on insect P450s. http://www.earthworms.org/ ESTs from the annelid polychaete N. virens. http://wfleabase.org/ Daphnia genome and gene resources.