xenobiotic-metabolizing cytochromes p450 in ontogeny...
TRANSCRIPT
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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