Toxicology and Applied Pharmacology 207 (2005) S57 – S61

www.elsevier.com/locate/ytaap

Review

Novel extrahepatic cytochrome P450s

Maria Karlgren*, Shin-ichi Miura, Magnus Ingelman-Sundberg
Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, SE-171 77 Stockholm, Sweden

Received 15 July 2004; revised 9 December 2004; accepted 9 December 2004

Available online 28 June 2005

Abstract

The cytochrome P450 enzymes are highly expressed in the liver and are involved in the metabolism of xenobiotics. Because of the
initiatives associated with the Human Genome Project, a great progress has recently been seen in the identification and characterization of
novel extrahepatic P450s, including CYP2S1, CYP2R1, CYP2U1 and CYP2W1. Like the hepatic enzymes, these P450s may play a role in
the tissue-specific metabolism of foreign compounds, but they may also have important endogenous functions. CYP2S1 has been shown to
metabolize all-trans retinoic acid and CYP2R1 is a major vitamin D 25-hydroxylase. Regarding their metabolism of xenobiotics, much
remains to be established, but CYP2S1 metabolizes naphthalene and it is likely that these P450s are responsible for metabolic activation of
several different kinds of xenobiotic chemicals and contribute to extrahepatic toxicity and carcinogenesis.
D 2005 Elsevier Inc. All rights reserved.

Keywords: Extrahepatic P450; Vitamin D; Naphtalene; Arachidonic acid; all-trans retinoic acid; Lung toxicity

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S57
CYP2S1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S58
CYP2R1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S58
CYP2U1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S59
CYP2W1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S60
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S60
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S61
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S61

Introduction extrahepatic tissues. The hepatic P450 enzymes belonging

to the cytochrome P450 families 1 – 3 are mainly involved in
The cytochrome P450 enzymes constitute a superfamily the metabolism of foreign compounds, and are of major
of heme-containing monooxygenases. In the human genome importance for metabolism of drugs and other xenobiotics.
approximately 57 cytochrome P450 genes that encode The importance of extrahepatic P450s for drug clearance is
active enzymes as well as 58 pseudogenes have been relatively small compared to the hepatic P450s. By contrast,
identified (Nelson et al., 2004). Many of the previously the known extrahepatic P450s often exhibit important
well-characterized mammalian P450s are expressed primar- endogenous functions, e.g., in the regulation of the levels
ily in liver, and only at much lower or undetectable levels in of steroid hormones, bile acids, lipids and other signaling
molecules.
* Corresponding author. Fax: +46 8 337 327. Many P450s are expressed in the respiratory tract,
E-mail address: Maria.Karlgren@imm.ki.se (M. Karlgren). including nasal mucosa, lung, and trachea. This region is
0041-008X/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.taap.2004.12.022
S58 M. Karlgren et al. / Toxicology and Applied Pharmacology 207 (2005) S57 – S61

exposed to both inhaled and blood-borne xenobiotic

compounds, and is therefore an important target in extra-
hepatic xenobiotic metabolism. A common subsets of P450s
are expressed preferentially in the respiratory tract, for
example, the P450 genes located on the CYP2 gene cluster
on chromosome 19, including CYP2A13 (Su et al., 2000)
and CYP2F1 (Nhamburo et al., 1990). Both CYP2A13 and
CYP2F1 are important for the bioactivation of xenobiotics.
CYP2A13 has been shown to activate the tobacco-specific
nitrosamine, NNK, which has been suggested to play a role
in human tobacco-related cancers (Su et al., 2000), and
CYP2F1 has been shown to metabolize naphthalene (Lanza
et al., 1999), a pulmonary toxicant in mice (Plopper et al.,
1992).
The brain contains very low P450 levels and the
amount in brain homogenate is only about 0.5– 2% of
that in liver microsomes (Hedlund et al., 2001). The P450
levels vary between different brain regions, with an
exceptionally high overall P450 content in cerebellum
(Warner et al., 1988). There are a few P450s that are
predominantly expressed in brain. These include CYP7B,
expressed in particular in hippocampus of rat and mouse Fig. 1. Unrooted phylogenetic tree of human cytochrome P450s. CYP2S1,
(Stapleton et al., 1995), CYP26B, expressed in cerebellum CYP2R1, CYP2U1 and CYP2W1 are shown in bold.
and pons of the human brain (White et al., 2000) and
CYP46, showing a widespread expression in brain (Lund An unusual feature of CYP2S1 is that it is induced by
et al., 1999). These three brain-specific enzymes are not dioxin in both mouse and human (Rivera et al., 2002). Such
involved in the metabolism of foreign compounds, but are an induction via the Ah-receptor (AhR) by dioxin or
important for endogenous functions like in the regulation polycyclic aromatic hydrocarbons (PAHs) usually affects
of neurosteroids, cholesterol and vitamin A metabolism the expression of members within the CYP1 family. This
(Hedlund et al., 2001). opens the possibility that CYP2S1 participates in the
Because of the completion of the sequence of the metabolism of aromatic hydrocarbons. Naphthalene has
humane genome and the initiatives related to this project, been shown to be metabolized into reactive intermediates by
there has recently been a remarkable progress in the the murine enzyme Cyp2f2 (Shultz et al., 1999) and by the
identification and characterization of novel P450 genes, human homologue CYP2F1 (Lanza et al., 1999). CYP2S1 is
which have been found to be expressed mainly in closely related to CYP2F1 (47.2% identity), and is
extrahepatic tissues. Here, we present some of the lately expressed in lung. It has also been shown to be induced
identified cytochrome P450s with potential importance for by dioxin and PAHs trough activation of the AhR. Indeed,
metabolic activation of xenobiotics. we found that CYP2S1 is capable to metabolize naphtha-
lene. CYP2S1 was heterologously expressed in yeast and
examination of the CYP2S1-dependent activity of the
CYP2S1 microsomes revealed that the enzyme converted naphtha-
lene into two different metabolites, as revealed by HPLC
CYP2S1 is a newly identified cytochrome P450 enzyme analysis of the incubates (Fig. 2). This finding indicates that
localized on chromosome 19q13.2 close to the previously CYP2S1 might play a role in naphthalene-induced lung
known CYP2 cluster. The other P450s in that cluster are cytotoxicity (Buckpitt et al., 2002).
also the enzymes with highest identity to CYP2S1 (Fig. 1).
When the CYP2S1 mRNA expression was investigated,
extrahepatic tissues such as the respiratory and gastro- CYP2R1
intestinal tracts were found to contain significant amounts.
The CYP2S1 protein was also detected by Western blotting The CYP2R1 gene is localized on chromosome 11p15.2
analysis in human lung (Rylander et al., 2001). In another and has five exons. Thus, it differs from most other
study CYP2S1 mRNA and protein were found in human members of the CYP2 family, carrying nine exons. CYP2R1
skin, where it was shown to be induced by ultraviolet is also highly conserved across species with human
radiation, coal tar, and all-trans retinoic acid (Smith et al., CYP2R1 showing 89% and 66% sequence identity at the
2003). All-trans retinoic acid was found to be a substrate of amino acid level to mouse and fugu fish CYP2R1,
CYP2S1. respectively.
 M. Karlgren et al. / Toxicology and Applied Pharmacology 207 (2005) S57 – S61 S59

conserved across species, indicate that it might constitute

the previously unknown hepatic microsomal 25-hydroxy-
lase. Further genetic evidence was recently provided when a
mutation in the second exon of the CYP2R1 gene was
described in a patient with vitamin D deficiency (Cheng et
al., 2004). This caused an amino acid exchange at position
99 where a leucine was replaced by a proline. Experiments
using heterologous expression of the corresponding cDNA
showed that the mutated CYP2R1 enzyme was not capable
to metabolize vitamin D3 (Cheng et al., 2004). Any role of
CYP2R1 in the metabolism of xenobiotic chemicals remains
to be shown.

CYP2U1

Fig. 2. Naphthalene metabolism by CYP2S1. Microsomes from yeast The novel human cytochrome P450 CYP2U1 is closely
transfected with CYP2S1 or empty vector were incubated with naphthalene related to CYP2R1 (Fig. 1) Like CYP2R1, CYP2U1 is
for 1 h at 37 -C. After incubation, the reaction products were subjected to highly conserved across species, with human CYP2U1
HPLC analysis. The two peaks labeled M1 and M2 in the CYP2S1 showing a 58% and 78% identity to fugu fish and murine
chromatogram (blue) are metabolites not seen in the chromatogram for the
negative control (red).
CYP2U1, respectively. Another similarity to CYP2R1 is
that both genes only contain five exons. CYP2U1 is
different in its primary structure as compared to other
The CYP2R1 mRNA tissue distribution has been CYP2 family members. Alignment of the CYP2U1 amino
examined both in humans and mice. In human, we found acid sequence to the closest relatives CYP2R1 and
the highest CYP2R1 mRNA levels in pancreas followed by CYP2D6, as well as to rabbit CYP2C5, reveals about 50
liver and kidney (M. Oscarson, unpublished observations). extra amino acids in the NH2-terminal part, compared to
In mouse, CYP2R1 mRNA is seen preferentially in liver these other CYP2 enzymes. One region of about 20 amino
and testis, but lower expression levels are evident in a acids is located at the very NH2-terminus and an insertion of
number of other tissues (Cheng et al., 2003). 25 amino acids is located after the NH2-terminal membrane-
The mouse CYP2R1 cDNA was cloned from a cDNA spanning region (Fig. 3). A comparison between the mouse
library of CYP27A1 deficient mice. CYP27A1 is one of the and fugu fish CYP2U1 shows that the very NH2-terminal
two hepatic vitamin D 25-hydroxylases. CYP2R1 was sequence has evolved later in time than the sequence
shown to 25-hydroxylate both forms of vitamin D, vitamin inserted after the membrane-spanning region. It is possible
D2 and D3 (Cheng et al., 2003). These findings, the high that these additional sequences affect membrane incorpo-
expression in liver and the metabolism of both vitamin D2 ration, intracellular targeting or structure of the CYP2U1
and D3, together with the fact that CYP2R1 is highly enzyme.

Fig. 3. Alignment of the N-terminal amino acid sequences from human, mouse, and fugu fish CYP2U1, human CYP2R1 and CYP2D6 and rabbit CYP2C5.
The approximate amino acids of the trans-membrane region are indicated by asterisks.
S60 M. Karlgren et al. / Toxicology and Applied Pharmacology 207 (2005) S57 – S61

The CYP2U1 mRNA has a similar expression pattern in found in a cDNA library from the human hepatoma cell line
rat and human, showing high levels in especially human HepG2. The CYP2W1 gene is located on chromosome 7 and
thymus but also in human heart and brain. In rat, a CYP2U1 shows the typical family 2 structure with nine exons. The
transcript was observed in thymus and brain (Karlgren et al., predicted 490 amino acid sequence displays between 33 and
2004). We examined the tissue distribution of the CYP2U1 42% identity with CYP2 family members.
protein in rat. The protein was detected in brain and thymus, Northern blot and mRNA dot –blot analysis demonstra-
whereas no expression was seen in lung, small intestine, ted no significant expression in adult human tissues like
colon, liver, kidney, spleen, skeletal muscle and heart. In liver, kidney, intestine, trachea, lung, stomach, small
contrast to the results obtained by quantification of the intestine and spleen nor in the fetal tissues brain, heart,
CYP2U1 mRNA, the rat CYP2U1 protein expression seems kidney, liver, spleen, thymus and lung. Northern blot
to bee much higher in brain than in thymus, with analysis of mRNA isolated from the human cell line HepG2
particularly high expression in the limbic structures and in showed a major transcript of 2.4 kb. Western blotting
the cortex (Karlgren et al., 2004). experiments revealed that no significant expression was seen
The high conservation across species as well as the in human liver microsomes, but that high expression was
selective tissue distribution suggests that CYP2U1 has an seen in the transformed cell line HepG2 and other trans-
endogenous function. Recently, CYP2U1 has been shown formed tissues (M. Karlgren, unpublished observations).
to metabolize arachidonic acid (AA) and other long chain
fatty acids (Chuang et al., 2004). Examples of other AA
metabolizing P450s are enzymes belonging to the CYP4 Conclusions
family, which like CYP2U1 is expressed in brain. AA is
metabolized by CYP2U1 into the two metabolites 19- and Here, we have discussed four newly identified extra-
20-HETE. In brain, 20-HETE has previously been shown hepatic cytochrome P450 enzymes. An overview of these
to serve as an endogenous vasoconstrictor (Roman, 2002). enzymes can be seen in Table 1. Even if there has been a
The physiological function of CYP2U1 remains to be progress in the characterization of extrahepatic cytochrome
elucidated, but considering the selective expression in P450 enzymes, this is still an unexplored area. The
brain and the ability to metabolize AA, CYP2U1 might extrahepatic P450 enzymes are important in the metabolism
play a role in the regulation of brain blood flow. of endogenous compounds, like vitamin D3 metabolism,
Xenobiotic chemicals as substrates for CYP2U1 has to which involves the newly identified CYP2R1. Besides
be searched for. endogenous functions, the P450 enzymes are important in
the metabolism of foreign compounds and therefore it is
particularly interesting to know how the extrahepatic P450s
CYP2W1 contribute to the tissue-specific metabolism of xenobiotics.
Since most of the drugs metabolized by P450s have
A part of the CYP2W1 sequence was first reported in the extrahepatic tissues as targets the extrahepatically expressed
year 2000 (GenBank Accession no AK000366). It was P450s may affect the effectiveness of drug treatment. Other

Table 1
Novel extrahepatic cytochrome P450s
CYP2S1 CYP2R1 CYP2U1 CYP2W1
Chromosome 19q13.2 11p15.2 4q25 7p22.3
Number of exons 9 5 5 9
Highest identity to CYP2B6, CYP2U1 CYP2R1 CYP2D6
CYP2A6,
CYP2A13
Conserved No Yes Yes No
Tissue distribution mRNA: trachea, lung, fetal lung, mRNA: pancreas, liver, mRNA: thymus, fetal thymus, mRNA: HepG2 cells
stomach, small intestine, kidney, testis heart, brain
spleen, skin
Protein: lung, skin Protein: brain, thymus Protein: HepG2 cells
Activity Metabolize all-trans retinoic acid, 25-hydroxylate vitamin D Metabolize aracidonic Metabolize aracidonic
naphthalene acid acid
Inducers Dioxin, PAH, UV-light nd nd nd
Polymorphic Yes Yes nd nd
References (Rylander et al., 2001; (Cheng et al., 2003; (Nelson, 2003;
Rivera et al., 2002; Nelson, 2003; Chuang et al., 2004;
Smith et al., 2003) Cheng et al., 2004) Karlgren et al., 2004)
Data are from references listed in the table, http://drnelson.utmem.edu/CytochromeP450.html, http://www.imm.ki.se/CYPalleles/, as well as unpublished
observations. Abbreviation: nd, not determined.
 M. Karlgren et al. / Toxicology and Applied Pharmacology 207 (2005) S57 – S61 S61

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