DRUG METABOLISM REVIEWS

Vol. 36, Nos. 3 & 4, pp. 549–568, 2004

Xenobiotic-metabolizing Cytochromes P450 inOntogeny: Evolving Perspective

Dharamainder Choudhary,1,2 Ingela Jansson,1 Mansoor Sarfarazi,1,2

and John B. Schenkman1,*

1Department of Pharmacology and Molecular Ophthalmic Genetics Laboratory,2Department of Surgery, University of Connecticut Health Center,

Farmington, Connecticut, USA

ABSTRACT

While much is known about inducibility of the xenobiotic-metabolizing forms of

cytochrome P450, the Family 1–3 enzymes, less well understood is the purpose for

the presence of some of these forms in the developing conceptus. Many cytochrome

P450 forms are present in the embryo and fetus, like the anabolic forms in Families 5

and higher, and are known to produce molecules with specific functions, e.g.,

cholesterol, steroids, and their metabolites necessary for normal physiological

functions. As we gain greater understanding of the cell cycle and its regulation, and

the roles of nuclear receptors in modulating transcriptional activities, a picture begins

to emerge in which cytochrome P450 forms appear as molecule-altering enzymes

producing and eliminating ligands associated with nuclear receptor activities. For

these CYP enzymes to exert a developmental action, a controlled spatial and temporal

expression pattern would be essential. Studies now indicate the existence of such

temporal control on the appearance of a number of these enzymes and the necessary

coenzyme, NADPH-cytochrome P450 reductase.

Key Words: Cytochrome P450; CYP; Retinoic acid; Developmental P450s;

Eicosanoids.

*Correspondence: John B. Schenkman, Department of Pharmacology, University of Connecticut

Health Center, Farmington, CT 06030, USA.

549

DOI: 10.1081/DMR-200033447 0360-2532 (Print); 1097-9883 (Online)

Copyright D 2004 by Marcel Dekker, Inc. www.dekker.com

PROLOGUE

It was 39 years ago that I (JBS) first met Herbert Remmer, when he came to the

Johnson Foundation at the University of Pennsylvania for a brief stay. I found him to

be a charming person and a dedicated scientist. His interests were really in toxicology;

a field of which I knew little. My own training had been in biochemistry, and I was

learning spectroscopy and drug metabolism while at the Johnson Foundation. I put

myself at his disposal, and Herbert set out to teach me Pharmacology. It was fun

working with him. We did hexobarbital sleeping times and a number of other studies

including some on drug disposition. At the same time, he joined me in examining the

spectral interaction between drug substrates and this newly discovered hemoprotein in

liver microsomes, cytochrome P450. It was an exciting time, and several publications

resulted from the collaboration (Remmer et al., 1966, 1967, 1968; Schenkman et al.,

1967a,b). Herbert invited me to spend some time with him in Tubingen, Germany,

which I did in 1968, where we again interacted in interesting studies (Greim et al.,

1970). Over the years Herbert proved to be a good friend, and we interacted not only in

meetings, but also traveled together, sightseeing in Germany and hiking in Austria. I

will miss him. The following contains a synopsis of studies that are a continuation of

my interest in the drug-metabolizing enzymes, showing a change, somewhat, from our

direction in the area of xenobiotic metabolism and induction, to endobiotic metabolism

by cytochrome P450 forms, and a discussion of other studies suggestive of possible

roles of certain forms of the ‘‘xenobiotic-metabolizing’’ cytochromes P450 in

development. I am joined in this endeavor by my colleague, Dr. Ingela Jansson, who

became a member of my group in 1973, while at Yale, and a more recent addition to

our group, Dr. Dharamainder Choudhary, currently a postdoctoral fellow, plus my

friend and colleague, Professor Mansoor Sarfarazi.

INTRODUCTION

Early studies on cytochrome P450 (CYP) forms focused on the metabolism of

xenobiotics, i.e., on drugs and chemicals of external origin. Such studies were mainly

restricted to cytochrome P450 Families 1–3, the xenobiotic-metabolizing CYP

enzymes. However, a number of studies were concerned with metabolism of

endobiotics, chemicals of intermediary metabolism involved in homeostasis, and

developmental control [see reviews, Kupfer, (1993); Zimniac and Waxman, (1993), and

Section VI: Biosynthetic Forms of cytochrome P450, (Schenkman and Greim, 1993)].

Our studies (Schenkman lab) were concerned with metabolism of steroid hormones by

constitutive forms of rodent cytochromes P450 (Cheng and Schenkman, 1983, 1984;

Jansson et al., 1985), and the influence of pathophysiological conditions on the

microsomal population of CYPs (Favreau and Schenkman, 1987; Favreau et al., 1987;

Thummel and Schenkman, 1990; Thummel et al., 1991). Subsequently, other

investigators concerned with teratologies and fetal toxicities began to examine

neonates, fetuses, and embryos for ability to metabolize xenobiotics. In the process,

it was found that fetal tissues could metabolize such compounds, and that prior

exposure to xenobiotics while in utero resulted in enhanced abilities to metabolize these

chemicals (Juchau, 1997; Juchau et al., 1998). As a result, the presence of these

550 Choudhary et al.

metabolic enzymes in fetal and embryonic tissue was conceptualized in a framework of

drug metabolism and detoxification. In the last decade, interest has turned to the

presence and role of CYP enzymes in the developing conceptus and their roles in

disease phenotypes.

EXPRESSION OF CYPs IN ONTOGENY

Earlier studies considered the presence of CYP enzymes in the embryo and fetus to

be a kind of adaptive response toward exposure to environmental challenges. CYP1A1

was detected in human prenatal hepatic tissue, but was not seen in prenatal rodents

(Juchau et al., 1998), although reportedly found in prenatal rabbit liver (Rich et al.,

1993); and it was speculated that it would not be constitutively expressed, but would

only appear as the result of exposure to chemical inducers. In contrast, CYP1A2

orthologs were not detected prenatally even after exposure to xenobiotics (Juchau et al.,

1998). Orthologs of another Family 1 member, CYP1B1, were found in a number of

human and rodent prenatal tissues, especially hormone-producing tissues (Juchau et al.,

1998). A recent review indicates the detection of at least fourteen different CYP

transcripts from human Family 1–3 during ontogeny (Schenkman et al., 2003). The

presence of CYP1A1, CYP2A6/13, CYP2E1, CYP2J2, CYP3A5, and CYP3A7 proteins

was also shown in human fetal samples, using immunoblot analysis (Carpenter et al.,

1996; Chen et al., 2003a; Gu et al., 2000; Hakkola et al., 2001; Johnsrud et al., 2003;

Shimada et al., 1996; Stevens et al., 2003). It is difficult to conclude a developmental

significance of expression of these CYPs in the embryo and fetus, as humans are

constantly exposed to a variety of environmental and dietary chemicals. To date, of the

23 Family 1, Family 2, and Family 3 CYP forms in humans, the 14 appearing during in

utero development have been explained in terms of xenobiotic induction and related to

xenobiotic metabolism and toxicity.

Other studies, however, have suggested a number of forms of cytochrome P450

may be present constitutively in the conceptus. Constitutive appearance of specific

CYPs have been observed in laboratory animal lines in a number of reports during

ontogeny in the absence of exposure to xenobiotic agents (Itoh et al., 1994; Keeney

et al., 1998; Ma et al., 1999; Rich and Boobis, 1997; Rich et al., 1993). Further, in a

study monitoring 40 of the 93 forms of mouse cytochrome P450, it was found that 27

forms appeared during the in utero period at one or more developmental stages in the

absence of external xenobiotic challenge (Choudhary et al., 2003). The appearance of

the different forms showed temporal specificities, with as few as fourteen appearing at

7 days post conception (E7), and as many as twenty-one forms appearing by E17. The

specific expression pattern of these different Cyp forms suggests the possibility of their

association with some critical functional role during ontogeny. Interestingly, the

number of ‘‘xenobiotic-metabolizing’’ Family 1–3 forms seen at E7, E11, E15, and

E17, were 4, 7, 8, and 9 respectively, with some forms appearing at one developmental

stage, then disappearing, to be replaced by another from the same Cyp family. For

example, Cyp1a1 was expressed early, at E7, but was absent from the subsequent

developmental stages examined. Cyp1b1 was not present at E7, first appearing at E11,

and remaining throughout the subsequently monitored stages (Choudhary et al., 2003).

It is possible that the expression of the different CYPs is the result of activation of

Xenobiotic-metabolizing Cytochromes P450 in Ontogeny 551

different nuclear receptors (NRs); these are known to concomitantly trans-activate a

range of different genes in response to the binding of ligands, including the differential

expression of groups of CYP isoforms (Nebert, 1991). Such effects are apparent under

certain pathophysiological conditions, such as in induction of diabetes and response to

insulin (Favreau and Schenkman, 1988; Favreau et al., 1987), or changes during

hypophysectomy and responses to male pattern and female pattern of growth hormone

(Morgan et al., 1985). In the same context, lesional psoriatic skin was shown to possess

elevated levels of CYP2S1, compared with non-lesional skin (Smith et al., 2003). That

CYPs present in the conceptus may have developmentally important roles is also

implied by reports on the presence of NADPH-cytochrome P450 reductase in discrete

areas of the embryo (Keeney and Waterman, 1999), and the early death of embryos in

the NADPH-cytochrome P450 reductase null mouse (Otto et al., 2003; Shen et al.,

2002), with associated elevated levels of retinoic acid and severe inhibition of

vascularogenesis (Otto et al., 2003).

ORTHOLOGOUS FORMS OF CYPs AND CONSERVEDFUNCTION HYPOTHESIS

Based on amino acid sequence identity, catalytic activities, and evolutionary origin,

some CYPs in different species have been assigned the same designation and are

considered to be orthologous forms (Nebert et al., 1991). The presence of the CYP

orthologs in different phyla and classes of vertebrates also points to a common

evolutionary origin and the probability of association with conserved functions. A

number of orthologous rat and mouse CYP forms are compared with their human

orthologs in Table 1. Specific physiological roles are known for most of the

cytochrome P450 forms in CYP Families 5–51, and a number of these forms have been

reported in different human fetal tissues (Pezzi et al., 2003). These CYPs are involved

in homeostasis and include the many anabolic steps pertaining to developmentally

important steps in steroid metabolism. Several of the CYP orthologs, including

CYP2W1, CYP4X1, and CYP20, are considered to be orphan CYPs, as no known

constitutive function has been associated with them. While Families 1–3 have

generally been considered as xenobiotic metabolizing enzymes, orthologous forms are

found in Families 1 and 2, but not in Family 3 (Table 1). The Family 3 isoforms all

metabolize steroid hormones as well as xenobiotics, converting them into more polar

and thus, more readily excreted metabolites (Waxman et al., 1991). All three CYP

forms found in Family 1, e.g., CYP1A1, CYP1A2, and CYP1B1 have orthologs in

other vertebrate species. Two of these forms, as indicated above, were also seen during

in utero development in the mouse (Choudhary et al., 2003). Until recently, only one

orthologous form was known in Family 2, CYP2E1. However, newly discovered

orthologous forms resulting from the human and mouse genome sequencing projects

include several from Family 2, CYP2R1, CYP2S1, CYP2U1, and CYP2W1. These

forms have yet to be studied in detail. Two of them (CYP2R1 and CYP2U1) have

orthologs in vertebrate species as diverse as pufferfish (Fugu), human, and mouse

(Nelson, 2003). Recently, CYP2U1 was reported to possess fatty acid o- and (o-1)-

hydroxylase activity (Chuang et al., 2004), and CYP2R1 was reported to have Vitamin

D3 hydroxylase activity (Cheng et al., 2003). A search of the UNIGENE database

552 Choudhary et al.

Table 1. Identity between orthologous CYPs and associated endogenous substrates.

CYPs

(Accession numbers)

% Identity with human ortholog

SubstrateRat Mouse

CYP1A1 (NP_000490) 81 (P00185) 80 (NP_034122) Retinoid and

estrogen signaling

CYP1A2 (NP_000752) 74 (NP_036673) 72 (NP_034123) Retinoid and

estrogen signaling

CYP1B1 (NP_000095) 80 (NP_037072) 81 (NP_034124) Retinoid and

estrogen signaling

CYP2E1 (NP_000764) 79 (NP_113731) 78 (NP_067257) Fatty acid signaling

CYP2R1 (AAQ23114) 88 (XP_341910) 88 (AAQ23115) Vitamin

D3 25-hydroxylase

CYP2S1 (NP_085125) 75 (XP_218347) 76 (NP_083051) Retinoid signaling

CYP2U1 (NP_898898) 81 (XP_227677) 85 (XP_131188) Fatty acid signaling

CYP2W1 (NP_060251) 73 (XP_221971) 74 (XP_144624) Orphan

CYP4B1 (NP_000770) 86 (NP_058695) 85(NP_031849) Fatty acid signaling

CYP4X1 (NP_828847) 70 (NP_663708) 71 (XP_144006) Orphan

CYP5A1 (NP_001052) 80 (NP_036819) 81 (NP_035669) Thromboxane synthase

CYP7A1 (NP_000771) 82 (NP_037074) 81 (NP_031850) Cholesterol

7a-hydroxylase

CYP7B1 (NP_004811) 64(XP_342219) 65 (NP_031851) Oxysterol 7a-hydroxylase

CYP8A1 (NP_000952) 84 (NP_113745) 86 (NP_032994) Prostacyclin synthase

CYP8B1 (NP_004382) 75 (NP_112520) 75 (NP_034142) Sterol 12a-hydroxylase

CYP11A1 (NP_000772) 76 (NP_058982) 75 (NP_062753) P450scc

CYP11B1 (NP_000488) 68 (NP_036669) 67 (XP_139430) 11b-hydroxylase

CYP11B2 (NP_000489) 69 (NP_036670) 69 (NP_034121) Aldosterone synthase

CYP17A1 (NP_000093) 67 (NP_036885) 67 (NP_031835) 17a-hydroxylase

CYP19A1 (NP_000094) 77 (NP_058781) 79 (NP_031836) Aromatase

CYP20A1 (NP_803882) 82 (NP_955433) 83 (XP_129747) Orphan

CYP24A1 (NP_000773) 82 (CAA42093) 82 (NP_034126) 1a,25-dihydroxyvitamin

D3 24-hydroxylase

CYP26A1 (NP_000774) 92 (NP_569092) 94 (NP_031837) Retinoic acid hydroxylase

CYP26B1 (NP_063938) 96 (NP_851601) 96 (NP_780684) Retinoic acid hydroxylase

CYP26C1 (NP_899230) Not identified 81 (XP_140712) Retinoic acid hydroxylase

CYP27A1 (NP_000775) 72 (CAA68822) 72 (NP_077226) Sterol 27-hydroxylase

Vitamin

D3 25-hydroxylase

CYP27B1 (NP_000776) 82 (NP_446215) 82 (XP_125908) 25-OH-vitamin

D3-1a-hydroxylase

CYP39A1 (NP_057677) 67 (XP_236983) 74 (NP_061375) Oxysterol 7a-hydroxylase

CYP46A1 (NP_006659) 95 (XP_343109) 95 (NP_034140) Cholesterol

24-hydroxylase

CYP51A1 (NP_000777) 93 (NP_037073) 92 (NP_064394) Lanosterol

14a-demethylase

Xenobiotic-metabolizing Cytochromes P450 in Ontogeny 553

revealed CYP2R1, CYP2U1, and CYP2W1 are expressed in embryonic cDNA clones

(BX440857.1, AK018458.1, and AU119534.1, respectively). Of the 30 orthologous

forms listed in Table 1, most are expressed during development (Bean et al., 2001;

Choudhary et al., 2003; Keeney et al., 1995a,b; Nelson, 2003; Pezzi et al., 2003;

Trofimova-Griffin and Juchau, 1998, 2002; Tahayato et al., 2003), as might be ex-

pected from their putative endogenous substrates.

CONSTITUTIVE EXPRESSION OF CYP1B1 ANDROLE IN DEVELOPMENT

Over the past 7 years, our interest has been focused on a Family 1 form of

cytochrome P450, CYP1B1. There are orthologous forms of this hemoprotein in

vertebrate species from bony fish to rodent and human. This form of cytochrome P450

took on importance when a null CYP1B1 genotype in humans was linked to a disease

phenotype, primary congenital glaucoma (PCG) (Sarfarazi et al., 2003; Stoilov et al.,

1997, 1998). Individuals homozygous for null CYP1B1 genotype developed the

disease, which, if not surgically corrected to relieve pressure in the anterior chamber,

results in blindness. The defect was reported to be in the trabecular meshwork, which

serves as a filter for the fluid of the anterior chamber. Mice, made homozygous for a

null Cyp1b1 gene, were subsequently found to have similar defects of the trabecular

meshwork (Libby et al., 2003). A number of individuals in families afflicted with PCG

were found to have point mutations in their CYP1B1 gene, with deduced sequences

indicating single amino acid substitutions rather than truncation or lack of expression of

the protein (Sarfarazi and Stoilov, 2000; Stoilov et al., 1997). Individuals homozygous

for these mutations in the CYP1B1 gene showed incomplete penetrance in the disease

phenotype. When the cDNA of CYP1B1 was mutated to express two of these mutant

proteins in an Escherichia coli heterologous expression system, the two mutant forms

of cytochrome P450, G61E and R469W, were found to have considerably diminished

activities compared with the wild type protein. The former, G61E, with a mutation in

the hinge region of the protein, had a greatly diminished stability, while R469W was

stable, but had only 30% catalytic efficiency of the wild type protein (Jansson et al.,

2001). Both defects would result in diminished function in vivo. It was suggested that

environmental factors inducing (up-regulating) the enzyme level in utero might

compensate for the lowered activities of the mutant enzymes and thereby provide an

ability to escape the disease phenotype.

Comparison of the primary structures of the orthologs of rat, mouse, and human

CYP1B1 indicates identical amino acid sequences generally above 80% in alignments,

with a few exceptions (Table 1). Mouse and human CYP1B1 orthologs had an 81%

sequence identity. Further, examination of the substrate recognition sites [SRSs, (Gotoh,

1992)] of Family 1 forms (Lewis et al., 2003), indicated greater than 90% sequence

identities in the six SRS regions between mouse (Cyp1b1) and human (CYP1B1)

orthologs. The primary sequence identity between mouse Cyp1b1 and human CYP1B1

was far greater than the sequence identities between human CYP1B1 and human

CYP1A1 or CYP1A2. This conservation of structure of CYP1B1 orthologs, and the

similarity of trabecular meshwork defects in mouse and human with CYP1B1-null

genotypes, indicated a strong conservation of function of CYP1B1 monooxygenases. It

554 Choudhary et al.

also suggested there might be a conservation of endogenous substrate(s) utilized by the

monooxygenase enzymes and associated with normal eye development. Such an

endobiotic would either be formed or inactivated by CYP1B1 monooxygenase activity

in vivo. After such considerations, we began to investigate the metabolism of

endobiotics by human and mouse CYP1B1 orthologs. What sort of compound might be

associated with a role for CYP1B1 in normal eye development? Such a compound

would probably be lipophilic, like other CYP substrates, and further, would in some

manner activate nuclear receptors to influence transcriptional events.

ENDOBIOTICS AND NUCLEAR RECEPTOR LIGANDS

Suggestions have appeared periodically indicating roles for the drug metabolizing

enzymes in regulating growth, morphogenesis, and homeostasis (Nebert, 1991; Otto

et al., 2003; Rich and Boobis, 1997; Schenkman et al., 2003; Stoilov et al., 2001). If

CYP forms act to influence tissue development, such action would be expected via

synthesis or catabolism of lipophilic ligands that can function in activation of nuclear

receptors (NRs), or in signal transduction pathways influencing NRs with a role in

tissue development. Small biomolecules like retinoids, steroids, and fatty acids and

their metabolites, can serve as specific ligands for activation of the NRs (Table 2). In

our studies, we examined the abilities of the human and mouse CYP1B1 orthologs to

metabolize a number of these lipophilic endogenous compounds; we chose members of

three classes of compounds involved in NR activation, retinoids, lipids and steroids

(Fig. 1). We reasoned that if CYP1B1 is involved in a conserved developmental

function, the substrate molecule associated with its function might also be conserved

Table 2. CYP-asssociated NR ligands.

Endobiotic ligands Nuclear receptors affecteda

All trans retinoic acid RAR (NR1B1-3)

9-cis retinoic acid RXR (NR2B)

Fatty acids PPAR (NR1C1-3)

HNF4 (NR2A1,2)

ROR (NR1F1-3)

HETEs and EETs PPAR (NR1C),

Estradiol ER (NR3A1,2)

Pregnenolone, progesterone PXR (NR1I2)

Lithocholic acid PXR (NR1I2)

Chenodeoxycholic acid FXR (NR1H4)

1a,25-dihydroxyvitamin D3 VDR (NR1I1)

Cortisol GR (NR3C1)

Aldosterone MR (NR3C2)

Progesterone PR (NR3C3)

Dihydrotestosterone AR (NR3C4)

aSelected from (Willson and Moore, 2002).

Xenobiotic-metabolizing Cytochromes P450 in Ontogeny 555

for the mouse and human orthologs. The basis for choice of substrates and our results

are indicated below.

RETINOID METABOLISM

All-trans-retinoic acid (RA) is a potent active morphogen that functions as a ligand

for nuclear receptor RAR. It can isomerize to form 9-cis-retinoic acid, a ligand of

another nuclear receptor, the retinoid X receptor (RXR). An excess (Kessel, 1992) or

deficiency (White et al., 1998) of RA results in abnormal development of vertebrate

organisms (Clagett-Dame and DeLuca, 2002). RAR and RXR form homodimers as well

as heterodimers, and RXR partners with a number of other nuclear receptors (CAR,

PXR, LXR, FXR, etc.) [see reviews, Clagett-Dame and DeLuca, (2002); Duester,

(2000)]. Embryogenesis is tightly regulated by spatial and temporal expression of

enzymes involved in the synthesis and degradation of RA (Haselbeck et al., 1999).

During embryogenesis, sharp boundaries of RA-containing and RA-lacking regions are

seen (Maden, 1999; Maden et al., 1998; Niederreither et al., 1999), generated by

retinaldehyde dehydrogenase-2 (Raldh2) and removed by CYP26 (McCaffery et al.,

1999), respectively, in discrete regions of the embryo. The latter step is considered to

be mediated by specific CYP26 family enzymes (CYP26A, CYP26B, CYP26C), which

catalyze the 4- and 18-hydroxylation and inactivation of the morphogen (Fujii et al.,

1997; Tahayato et al., 2003; Taimi et al., 2004; White et al., 1997, 2000). Raldh2– / –

embryos show abnormalities and die at mid-gestation (Niederreither et al., 1999).

Similarly, Cyp26a1– / – embryos die in mid to late gestation period, and also show

many abnormalities (Abu-Abed et al., 2001; Sakai et al., 2001), attesting to the role and

Figure 1. Endogenous substrate metabolism by Family 1–3 CYPs.

556 Choudhary et al.

functional significance of CYP26 in ontogeny. Recently, a number of CYP Family 1–3

members have been shown to be capable of retinoic acid hydroxylation, in particular,

CYP3A7, CYP3A5, CYP2C8 and CYP2C18 were effective in the 4-hydroxylation of

RA (Marill et al., 2000). The 18-hydroxylation of RA was also carried out by

CYP4A11, CYP3A7, and CYP1A1, but at less than 10% the rate of 4-hydroxylation

(Marill et al., 2000). CYP2S1 has, similarly, been shown to be capable of RA

hydroxylation (Smith et al., 2003). To date, there has not been a comparison of the

efficiency of RA oxidation by CYP26 and other CYPs. It would be of interest to

determine the temporal and spatial arrangement of CYP26 and other CYPs capable of

RA oxidation with the enzymes of RA synthesis in order to assess the significance of

Family 1–3 metabolism of RA in development.

Synthesis of RA from retinol (vitamin A) involves two oxidations and is catalyzed

by several retinoid dehydrogenases, as indicated above (Duester, 2000). However, a

number of Family 1–3 CYP forms have also been shown to be effective in catalyzing

one or both steps in synthesis of RA. Family 1 enzymes readily catalyze the first

oxidative step, ROL to RAL, as well as the subsequent step, RAL to RA (Chen et al.,

2000; Roberts et al., 1992; Zhang et al., 2000). The exact impact and significance of

these CYPs in development will depend upon their regional and temporal expression

pattern relative to that of retinoid dehydrogenases. To date, comparisons of the extent

of contribution of the different CYPs to the synthesis of RA in a tissue or region of the

developing embryo or fetus have not been reported, although a number of these forms

of Cyp do appear during in utero development of mice (Choudhary et al., 2003).

In comparing retinoid metabolism of Cyp1b1 with its human ortholog, it became

apparent that the mouse form had lower specific activities with these endogenous

substrates than human CYP1B1 (Choudhary et al., 2004). Human CYP1B1 had a

catalytic efficiency more than 10-fold higher than the mouse ortholog with retinol as

substrate and more than 25-fold higher with retinal as substrate. The lower efficiencies

of Cyp1b1 were mainly the result of much higher Km values for the retinoids.

Interestingly, neither mouse nor human ortholog was able to oxidize retinoic acid,

indicating that if retinoid metabolism is the conserved function of CYP1B1 orthologs, it

involves the synthesis and not the degradation of the ligand, retinoic acid.

EICOSANOID METABOLISM

Fatty acids can constitute an important part of the cellular signaling cascade.

Arachidonic acid is released from the membrane phospholipids by hormonally and

transcriptionally regulated phospholipase A2 action. It is metabolized by three distinct

pathways; by cyclooxygenase, which forms prostaglandins and thromboxanes; by

lipoxygenase, which synthesizes leukotrienes and mid-chain hydroxyeicosatetraenoic

acids (HETEs); and by cytochromes P450, which form HETEs (mid-chain and

terminal) and epoxyeicosatrienoic acids (EETs) (Capdevilla et al., 2000). The

arachidonic acid metabolites possess strong biological activities, including mediating

release of peptide hormones and vasoconstriction (20-HETE), vasodilatation (EETs)

(Fleming, 2001), and ion transport (Maier and Roman, 2001). The terminal hydroxy-

arachidonate metabolite, 20-HETE, causes depolarization and constriction of vascular

smooth muscle cells. 20-HETE was also shown to activate p38MAP kinase in vascular

smooth muscle cells (Kalyankrishna and Malik, 2003), influencing signaling pathways

Xenobiotic-metabolizing Cytochromes P450 in Ontogeny 557

that can terminate in transcriptional events and influence transactivational responses.

The presence of a 20-HETE receptor has been proposed (Yu et al., 2003) that may be

responsible for activating a signal cascade. EETs interact with membrane receptors,

exert a fibrinolytic effect by activating membrane associated Gas protein in endothelial

cells (Node et al., 2001), and have specific high affinity binding sites on monocyte

membranes (Wong et al., 2000) and vascular smooth muscle plasma membranes

(Snyder et al., 2002). Binding of EETs to the latter membranes inhibits induction of

aromatase in vascular smooth muscle. EETs constitute a major proportion of

arachidonic acid metabolites by human CYP1A2, CYP2C8, CYP2C9, CYP2J2 (Daikh

et al., 1994; Rifkind et al., 1995; Wu et al., 1996), and mouse Cyp2b19, Cyp2c29,

Cyp2c38, Cyp2c39, Cyp2j5 (Keeney et al., 1998; Luo et al., 1998; Ma et al., 1999).

The EETs are also substrates for further metabolism, and are converted to 19-OH-EET

and 20-OH-EET by Family 4 enzymes in rat (Cowart et al., 2002).

Polyunsaturated fatty acids, HETEs and the hydroxy metabolites of EETs can bind

to and transactivate human peroxisomal proliferator activated receptors (PPARs) and

affect lipid metabolism (Murakami et al., 1999) and a wide variety of growth responses

(Keller et al., 2000). PPAR is an important NR family, and activation of PPARalpha

can suppress apoptosis and induce proliferation (Roberts et al., 2002). Activation of

PPARgamma, mediates differentiation of some cell types, such as adipocytes, blocks

inflammation, regulates fat metabolism, and influences a number of pathophysiological

conditions, such as diabetes (Na and Surh, 2003), and can also stop neoplastic cell

proliferation (Keshamouni et al., 2004), actions all relevant in cancer as well as in

growth and development. The mid-chain HETE, 15(S)-HETE, was found to be a ligand

for PPARgamma in lung adenocarcinoma cells (Shankaranarayanan and Nigam, 2003).

Another mid-chain hydroxy metabolite, 5(S)-HETE, was shown to promote growth of

prostate cancer cells, and involvement of a G-protein coupled 5-HETE receptor has

been proposed (O’Flaherty et al., 2002). Several other receptors, orphan receptors, such

as ROR (NR1F1-3) and hepatic nuclear factor HNF4 (NR2A1, 2A2), are activated by

fatty acid ligands (Willson and Moore, 2002) (Table 2).

The characterization and involvement of the different eicosanoid metabolites in

transcriptional regulation is still in its infancy and future studies are needed to more

clearly delineate whether their formation is associated with a form of cytochrome P450 in

development. In recent studies (Choudhary et al., 2004), it was found that human

CYP1B1 is capable of forming midchain HETEs. Indeed, over 50% of arachidonate

products produced by this ortholog were midchain HETEs. It had an activity about 10%

of CYP1A1. However, the metabolite patterns of the isoforms differed. CYP1A1

preferentially generated o-terminal region metabolites. Arachidonate proved to be a

particularly poor endogenous substrate for the mouse ortholog, Cyp1b1. The catalytic

efficiency of arachidonate oxidation by the human ortholog was more than 60-fold higher

than by Cyp1b1. This was because the Km for arachidonate was about 0.5 mM with the

mouse ortholog, compared with a value of about 30 mM with the human enzyme.

STEROID METABOLISM

The greatest number of nuclear receptors, like the nuclear hormone receptors of

NR Family 3, appears to be activated by ligands of steroids and sterol classification

558 Choudhary et al.

(Willson and Moore, 2002). For example, estrogen receptors (ER) family, pregnane X

receptor (PXR), liver X receptor, and farnesoid X receptor (LXR and FXR) are all

activated by steroid molecules (Table 2). As noted earlier (Nebert, 1991), almost every

NR ligand that triggers a response also causes an alteration in the steady state

composition of the cytochrome P450 population, which suggested these hemoproteins

serve to maintain homeostatic levels of modulating ligands under conditions imposed

by the NRs. For example, as indicated above, RA activates the retinoid receptor that

influences growth and differentiation during development. In addition, RA also induces

CYP26, a form of cytochrome P450 fairly specific in its ability to inactivate RA to its

4-oxy metabolite (Fujii et al., 1997). The nuclear receptors clearly have a major role in

tissue differentiation and function, and can influence cellular proliferation. Examples

are the effects of estradiol binding to the ER and its proliferative influences in ER-

containing tissues and cancers (Clarke et al., 2003), and the influences of androgen on

AR-containing tissues like the prostate (Cude et al., 1999). For these reasons, the

possibility of a steroid serving as ligands associated with CYP1B1 in normal eye

development was considered, and the metabolism of several of them by human and

mouse orthologs was examined; CYP1B1 metabolizes b-estradiol, testosterone, and

progesterone to metabolites that are more readily excreted (Jansson et al., 2001). The

catalytic efficiency toward these endogenous substrates by CYP1B1 was good.

However, Cyp1b1 had an extremely poor catalytic efficiency with these substrates,

relative to that of CYP1B1.

The lower catalytic efficiency of Cyp1b1 towards the endobiotics examined could

have been a reflection of a lower ability of the mouse ortholog to function as a

monooxygenase. To test this possibility, metabolism of a polycyclic aromatic

hydrocarbon substrate, 7, 12-dimethylbenz(a)anthracene (DMBA), was examined. This

chemical was shown earlier to be a good substrate for the mouse enzyme (Pottenger

et al., 1991), and to be metabolized by Cyp1b1 at a faster rate than by the human

enzyme (Savas et al., 1997). In agreement, Cyp1b1 had a catalytic efficiency for DMBA

metabolism four-fold higher than CYP1B1, indicating its lower efficiency with the

tested endogenous substrates was not due to a lower intrinsic monooxygenase activity.

DEVELOPMENTALLY ACTIVE CYPs INNON-VERTEBRATE PHYLA

Since the discovery that mutations in mammals nullifying the CYP1B1 gene result

in structural abnormalities in eye development, reports have begun to appear in other

phyla showing abnormal development to result from mutations in other CYPs. For

example, dumpy primordia (mature fruiting bodies developed on short stipes) were

observed in the mushroom, Coprinus cinereus, due to a truncating mutation in the eln-2

gene product, CYP502 (Maraguchi and Kamada, 2000). The identity of an associated

endogenous substrate associated with the developmental function of CYP502 is

currently unknown. Shortened leaves, a defect in polar elongation of leaf cells, is seen

in the rotundifolia3 mutant of Arabidopsis thaliana. Cloning of the rot3 gene revealed

it to code for CYP90C1, a putative steroid hydroxylase suggested to be involved in

synthesis of a brassinosteroid hormone (Kim et al., 1998; Tsukaya, 2002). Another

Arabidopsis mutant, bus-1 of A. thaliana, exhibits a bushy phenotype with small

Xenobiotic-metabolizing Cytochromes P450 in Ontogeny 559

crinkled leaves and retarded vascularization. This mutation involves the loss of the

CYP79F1 gene, which participates in glucosinolate synthesis from short chain

methionine derivatives (Chen et al., 2003b; Reintanz et al., 2001). Mutation in the

Daf-9 gene has been shown to cause the entrance of Caenorhabiditis elegans into a

dormant or dauer larval stage (Jia et al., 2002). Daf-9 was found to be a CYP gene,

CYP22, and was suggested to provide a ligand, currently unidentified, for orphan

nuclear receptor DAF-12. In Drosophila, a mutation nullifying the disembodied (dib)

gene was observed to result in inhibition of dorsal closure, midgut morphogenesis, head

involution, and a poorly differentiated embryonic cuticle. The gene encodes CYP302A1

(Chavez et al., 2000), which catalyzes 22-hydroxylation of an intermediate in synthesis

of the insect steroid molting hormone, 20-hydroxyecdysone (20E) (Warren et al., 2002).

The metabolite, 20E, acts as a ligand for a nuclear hormone receptor, EcR, which forms

a heterodimer with nuclear hormone receptor UsP (ultraspiracle, insect ortholog of the

vertebrate RXR) and modulates expression of a wide array of developmental genes

(Riddiford et al., 2000). From the developmental influences indicated above, it is clear

that mutations in some CYP forms result in developmental anomalies, and these are

probably due to changes in concentrations of ligands associated with transcriptional

regulatory pathways. The demonstrated involvement of CYPs in structural development

of organisms of the different phyla provides support for the suggestion of

developmental roles for certain xenobiotic-metabolizing forms of cytochrome P450

which appear during in utero development.

ACKNOWLEDGMENT

Supported in part by grant 1 R01 EY11095 from the NIH.

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