Download - Molecular genetics of the P-450 superfamily

Phannac. Ther. Vol. 45, pp. 1-38, 1990 0163-7258/90 $0.00 + 0.50 Printed in Great Britain. 1989 Pergamon Press plc

Specialist Subject Editor: W. KALOW

MOLECULAR GENETICS OF THE P-450 SUPERFAMILY

FRANK J. GONZALEZ

Laboratory of Molecular Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892, U.S.A.

1. INTRODUCTION

1.1. GENERAL CHARACTERISTICS OF P-450S

Genes of the P-450 superfamily code for a group of enzymes that share the following characteristics: (i) they contain a noncovalently bound heme; (ii) they are intrinsic membrane proteins firmly bound to intracellular membranes; and (iii) they use reducing equivalents from NADPH (and sometimes NADH) and an atom of oxygen derived from atmospheric oxygen to oxygenate substrates. The reducing equivalents are transferred to P-450 via a second enzyme. These physicochemical parameters suggest that P-450s share common structural features, and indeed they do (vide infra).

Mammalian P-450s can be divided into two major classes based on their intracellular location and the enzyme from which they receive electrons. The mitochondrial P-450s, side-chain cleavage enzyme and steroid 1 l fl-hydroxylase are found in the mitochondria of the adrenal cortex and are involved in steroid synthesis. These enzymes are synthesized by membrane-free polyribosomes (Nabi et al., 1983) as a large precursor (Du Bois et al., 1981) and are then transported into the organelle concomitant with the cleavage and removal of an NH2-terminai extrapeptide (Du Bois et al., 1981; Morohashi et al., 1984). The mitochondrial P-450s receive electrons from the iron sulfur protein adrenodoxin via NADPH-adrenodoxin oxidoreductase. The great majority of P-450s are of the second class, found primarily in the endoplasmic reticulum membrane. These enzymes are synthesized on membrane-bound polyribosomes (Gonzalez and Kasper, 1980) and inserted directly into the lipid bilayer via the signal sequence recognition system (Bar-Nun et al., 1980; Sabatini et al., 1982). Electrons are donated to the endoplasmic reticulum-bound P-450s via the flavoprotein NADPH-P-450 oxidoreductase and, in some cases, cytochrome b 5. It is of interest that the bacterial P-450cam is a soluble enzyme that receives electrons from the iron-sulfur protein putidaredoxin via a flavoprotein oxidoreductase (Gunsalus and Sligar, 1977), indicating that this enzyme resembles the mitochondrial P-450s.

Other authors have reviewed the extensive literature on the purification of P-450s (Lu and West, 1980; Black and Coon, 1986; Waxman, 1986; Distlerath and Guengerich, 1987; Guengerich, 1987) and their roles in steroid metabolism (Fevold, 1983; Coon and Koop, 1983; Hall, 1985, 1986; Jefcoate, 1986), drug metabolism (Distlerath and Guengerich, 1987; Guengerich, 1987) and carcinogen activation (Kadlubar and Hammons, 1987). Recent reviews have also focused on the molecular biology of P-450s (Adesnik and Atchison, 1986; Nebert and Gonzalez, 1987, 1988; Gotoh and Fujii-Kuriyama, 1988; Gonzalez, 1988).

1.2. BACKGROUND AND METHODOLOGIES

An understanding of the molecular biology of P-450s has been greatly aided by the tremendous progress in the 1970s and early 1980s in purifying many forms of P-450s and preparing highly specific antibodies against these enzymes. During this period, cloning

2 F.J. GONZALEZ

RNA polymerase-~ promoter r~ /

J ~ p r ~ N e A

/ Antibody

- - m R N A ~ Reverse transcriptase/DNA polymerase

I ds cDNA

~ --- ~.gt 11 DNA/DNA ligase

I CA arms

" • P450 partial amino acid sequence

m I ~ , oligon/ucleotide or cDNA probe

FIG. 1. Construction of cDNA-expression libraries in the phage vector 2gt 11 and isolation of specific cDNAs using antibody and oligonucleotide probes.

technology also developed rapidly, and it became possible to construct cDNA libraries from a particular source of mRNA and to use specific antibodies to characterize cDNA clones.

The procedure most commonly used to isolate cDNA clones is depicted in Fig. 1. RNA is isolated from the appropriate tissue source, preferably a tissue that contains a high level of the P-450 of interest. Double-stranded cDNA is then synthesized from the mRNA and inserted into the appropriate vector. The most popular vector is 2 gt 11 (Young and Davis, 1983). This vector allows the direct production of P-450 proteins in bacteria. The proteins can then be detected with specific antibody probes. Alternatively, the cDNAs can be isolated by screening with oligonucleotide probes. These procedures are used to identify and separate (clone) a single cDNA from a population of thousands of diverse cDNAs.

Once the cDNAs for a particular P-450 are isolated and characterized by DNA sequencing, they can be used to isolate their corresponding genomic clones. Genomic libraries are constructed from total DNA isolated from lymphocytes or other tissues. DNA is partially digested with a restriction enzyme, and fragments ranging from 15 to 25 kilobase parts in length are inserted into a 2 phage cloning vector such as 2 EMBL 3 (Kaiser and Murray, 1985). The libraries are then screened by plaque hybridization to isolate the appropriate gene.

The P-450 cDNA and genomic clones can be used to study a variety of problems in pharmacology and biochemistry, as illustrated in Fig. 2. The cDNAs can be used to determine the primary amino acid sequence of P-450s and to establish evolutionary relationships between these enzymes. Probes can be developed from the cDNAs to study P-450 gene induction mechanisms. Enzymatically active P-450s can be generated using cDNA expression technology. Finally, the structure of P-450 genes can be determined, including intron-exon junctions and identification of important gene regulatory domains. These regulatory domains can be used to isolate and characterize trans-acting factors, such as receptors that are required for the control of gene expression.

2. NOMENCLATURE

A nomenclature system was implemented in 1987 to categorize the growing number of P-450 primary amino acid sequences (Nebert et al., 1987). This system is based entirely on global alignments of complete amino acid sequences. Briefly, the P-450 gene superfamily is subdivided into families, subfamilies and individual P-450s. Families are designated by Roman numerals and subfamilies by capital letters. Individual P-450s are denoted by Arabic numerals. Two P-450s that demonstrate <40% amino acid similarity represent members of separate gene families. P-450s that display > 59% similarity are assigned to the same gene subfamily.

Molecular genetics of the P-450 superfamily 3

~.EMBL ~ g e n o m i c

libraries

isolated genes

l promoter characterization expression

i) transcription of i) enzymology heterologous gene ii) immunochemistry

ii) DNA binding assays for iii) carcinogen and receptors and factors drug metabolism

iii) in vitro transcription assays

<3 l iver

~.gtl 1 libraries , - a n t i b o d i e s or

isolated cDNAs

cDNA-directed

oligonucleotide probes

sequence of P450 i) evolutionary comparisons ii) catalogue, develop nomenclature iii) identify important sequences

RNA hybr idizat ion blots

i) study mRNA levels after induction and during development

DNA hybridizat ion blots

i) study gene family complexity ii) search for restriction fragment

length polymorphisms

FIG. 2. The use of cDNA and genomic clones to study problems in pharmacology and biochemistry.

It should be noted that this nomenclature system does not depend on P-450 catalytic activities or function. It is now apparent that P-450s in separate subfamilies can have the same catalytic activities. This feature is in keeping with the long-established overlapping substrate specificities of P-450s, particularly those in the P-450II gene family.

Another inherent problem in P-450 nomenclature is the assignment of orthologous genes between species. For instance, certain P-450II gene subfamilies have many members, and several of these genes have recently diverged. Gene conversion events have also scrambled many P-450 genes within a particular species. The net result of recent gene duplications and gene conversion events is that individual species contain their own unique P-450 genes. This phenomenon will be illustrated below. A scheme for the formation of P-450 gene families, subfamilies and gene conversion is shown in Fig. 3.

Chromosome assignments have been made for human and mouse P-450 genes. These have also been named according to standard nomenclature rules for genetic loci (Nebert et al., 1989). Human and mouse loci are denoted by C Y P followed by an Arabic numeral designating the P-450 family, a letter for the subfamily and an Arabic numeral for the gene. A list of sequenced P-450s compiled from both published and unpublished data through July 1989 is shown in Table 1.

ANCESTRAL P450 GENE ~ r ' ' l

SPECIES A / ~ SPECIES B

~ gene duplication ~ gene duplication

~ gene duplication ~W mutation and divergence

gene conversion I gene loss

FIG. 3. Evolution of the P-450 gene superfamily. A hypothetical scheme is shown in which two species, A and B, are formed. Gene duplications occur independently in both species. A second gene duplication has occurred in species A while a mutation has eliminated a gene in species B. Within the species A family a gene has diverged from the other two due to changes in sequence and natural selection. This gene, represented as a black rectangle, was then involved in a gene conversion with a neighboring gene. After a period of time, species A and B have evolved their own distinct set of P-450 genes. The reason for this may have been dietary habits or constraints and the need of each organism to possess enzymes capable of detoxifying harmful plant poisons.

4 F.J . GONZALEZ

TABLE 1. P - 4 5 0 s S e q u e n c e d b y J u l y 1989

Protein Trivial name Species Reference

IAI c rat Yabusaki et al., 1984 P~ mouse S. Kimura et al., 1984b

S. Kimura et al., 1987b Pi human Jaiswal et al., 1985a

Jaiswal et al., 1985b Quattrochi et al., 1985

form 6 rabbit Okino et al., 1985 Kagawa et al., 1987

IA1 trout Heilmann et al., 1988 IA2 d rat Kawajiri et al., 1984

Haniu et al., 1986b P3 mouse S. Kimura et al., 1984a

S. Kimura et al., 1984b P2 mouse Kimura and Nebert, 1986 P3 human Jaiswal et al., 1986 form 4 human Quattrochi et al., 1985

Quattrochi et al., 1986 LM4 rabbit Fujita et al., 1984

Okino et al., 1985 Ozols, 1986 Kagawa et al., 1987

IIAI A1 rat Nagata et al., 1987 IIA2 A2 rat Matsunaga et al., 1988 IIA3 A3 rat S. Kimura et al., 1989c

15/~ mouse Squires and Negishi, 1988 P-450(1) human Phillips et al., 1985 IIA3 human Yamano et al., 1989b

IIBI b rat Fuji i-Kuriyama et al., 1982 Gotoh et al., 1983 Yuan et al., 1983 Kumar et al., 1983

IIB2 e rat Fuji i-Kuriyama et al., 1982 Yuan et al,, 1983

IIB3 IIB3 rat Labbe et al., 1988 IIB4 LM2 rabbit Heinemann and Ozols, 1983

Tarr et al., 1983 b15 Komori et al., 1988 b54 Komori et al., 1988 B0 Gasser et al., 1988 B1 Gasser et al., 1988

IIB5 HPI rabbit Komori et al., 1988 b52 Komori et al., 1988 B2 Gasser et al., 1988

lIB6 LM2 human Miles et al., 1988 IIB7 hl IBl human Yamano et al., 1989c lIB8 hIIB2 human Yamano et al., 1989c lIB9 pF26 mouse Noshiro et al., 1988 I1BI0 pF3/46 mouse Noshiro et al., 1988 I1CI PBcl rabbit Leighton et al., 1984 IIC2 PBc2,K rabbit Leighton et al., 1984

pHP2 lmai et al., 1988 IIC3 PBc3 rabbit Leighton et al., 1984

3b Ozols et al., 1985 IIC4 PBc4 rabbit Zhao et al., 1987

1-88 Johnson et al., 1987 IIC5 form 1 rabbit Tukey et al., 1985 I1C6 PBI rat Gonzalez et al., 1986a

pTF2 Friedberg et al., 1986 PB1 H. Kimura et al., 1988

IIC7 f rat Gonzalez et al., 1986a pTFI Friedberg et al., 1986 f H. Kimura et al., 1988

IIC8 form 1 human Okino et al., 1987 IIC2 S. Kimura et al., 1987a mp-12 Ged et al., 1988

I|C9 mp-4 human Ged et al., 1988 Yasumori et al., 1987

IIC1 S. Kimura et al., 1987a Meehan et al., 1988a

C o n t i n u e d

TABLE l - -Con t inued

Protein Trivial name Species Reference

IICI0 mp-8 human Ged et al., 1988 IICI 1 h,16a rat Yoshioka et al., 1987

Morishima et al., 1987 Zaphiropoulos et al., 1988

IIC12 i,15fl rat Zaphiropoulos et al., 1988 IICl3 g rat Taken from Nebert et aL, 1989 IICl4 pHP3 rabbit Imai, 1987 IICl5 b32-3 rabbit Imai et aL, 1988 IIDI dbl rat Gonzalez et aL, 1987 IID6(IIDI) dbl human Gonzalez et aL, 1988c

16a mouse Wong et al., 1987 IID2 db2 rat Gonzalez et al., 1987 IID3 db3 rat Matsunaga et al., 1989 IID4 db4 rat Matsunaga et aL, 1989 IID5 db5 rat Matsunaga et al., 1989 IID7 human Author's laboratory, unpublished data IID8 human Author's laboratory, unpublished data IIEl j human Song et al., 1986

rat Song et al., 1986 3a rabbit Khani et aL, 1987

IIE2 gene 2 rabbit Khani et aL, 1988 IIFI human Author's laboratory, unpublished data IIGl olfl rat Nef et aL, 1989 IIH1 PBI5 chicken Hobbs et al., 1986 IIIAl pcnl rat Gonzalez et al., 1985b IIIA2 pcn2 rat Gonzalez et aL, 1986b IIIA3 HLp human Molowa et aL, 1986 IIIA4 nf human Beaune et al., 1986

pcnl human Gonzalez et al., 1988a IIIA5 hpcn3 human Aoyama et al., 1989b IIIA6 3c rabbit Dalet et al., 1988 IVAI LA,ol rat Hardwick et al., 1987 IVA2 LA,o2 rat S. Kimura et al., 1989a IVA3 LA,o3 rat S. Kimura et al., 1989b IVA4 p-2 rabbit Matsubara et al., 1987 IVBI isozyme 5 rabbit Gasser and Philpot, 1989

IVBI human Nhamburo et al., 1989 VIAl house fly Feyereisen et al., 1989 XIAI llfl cow Chua et al., 1987

Morohashi et al., 1987b XIBI scc human Chung et al., 1986b

cow Morohashi et al., 1984 Chashchin et al., 1986

XVIIAI 17~t cow Zuber et al., 1986a human Chung et al., 1987

Bradshaw et al., 1987 XIXAI arom human Simpson et aL, 1987

S. Chen et al., 1988 XXIAI c21A mouse Chaplin et al., 1986

c21 cow Chung et al., 1986a John et al., 1986b Yoshioka et al., 1986

c21 pig Haniu et al., 1987 XXIAIP (pseudogene) human Higashi et al., 1986

White et al., 1986 XXIA2 c21B human Higashi et al., 1986

White et al., 1986 Matteson et al., 1987

XXIA2P (pseudogene) mouse Chaplin et al., 1986 XXVI 26-OH rabbit Andersson et al., 1989 LI 14DM S. cerevisiae Kalb et al., 1987

C. tropicalis C. Chen et al., 1988 LII alk C. tropicalis Sanglard and Loper, 1989 CI cam I s . putida Haniu et al., 1982

Unger et al., 1986 CII BM-3 B. subtilis Ruettinger et al., 1989 Adrenodoxin human Picado-Leonard et al., 1988

cow Okamura et al., 1987 Adrenodoxin reductase cow Hanukoglu et al., 1987

human Solish et al., 1988

All sequences were derived from cDNAs or proteins, except for IICI0, IID4, IID6, IID7, XXIAI, XXIA2, LI, LII, CI and CII, which were sequenced from genomic clones.

6 F .J . GONZALEZ

3. EVOLUTION

P-450s are found throughout the animal and plant kingdoms. In plants, these enzymes are probably involved in a multitude of biosynthetic pathways in the formation of pigments and complex alkaloids. To date, no P-450 cDNAs or genes have been isolated from plants. P-450 genes have, however, been found and characterized in bacteria (Wen and Fulco, 1987; Unger et al., 1986) and yeast (Kalb et al., 1987).

An evolutionary tree (Fig. 4) of the P-450 gene superfamily can be generated by comparing primary amino acid sequence data (Nebert and Gonzalez, 1987; Nelson and Strobel, 1987; Gotoh and Fujii-Kuriyama, 1988). Using the species divergence time generated from fossil evidence (Shoshani et al., 1985) and the amino acid differences between a P-450 sequence in two species, the evolutionary distance and unit evolutionary period (UEP) can be calculated. The UEP is the time in millions of years required for a 1% change in amino acid sequence. P-450 UEPs range from 2.3 to 4.2 (Nelson and Strobel, 1987), a value that suggests that P-450s are rapidly changing. However, it is difficult to predict the evolutionary constraints controlling protein divergence rates.

If the phylogenetic tree is examined and correlated with catalytic activities of P-450s, several suggestions can be made concerning P-450 evolution. The earliest P-450s to emerge (or the oldest P-450 genes) are those that now metabolize steroids and fatty acids. The fatty acid-metabolizing P-450IV family and the steroid-inducible P-450III genes diverged more than 1 billion years ago. It appears, therefore, that the earliest P-450s evolved to maintain membrane integrity through the metabolism of lipids and steroids (Nebert and Gonzalez, 1985). The steroid biosynthetic enzymes in the P-450XVII, XIX and XXI families diverged from drug metabolizing enzymes about 900 million years ago. The P-450I and P-450II gene families formed about 800 million years ago and these genes are now responsible for the metabolism of drugs and carcinogens. Finally, about 400 to 600 million years ago, the P-450II gene family expanded into eight gene subfamilies (Table 1). It has been suggested that this increase in the number of P-450 genes was related to the emergence of mammals onto land several million years after plants were established

P , -

[ I I I

3.0 217 2.4 211 118 1.5 112 019 016 013 0 EVOLUTIONARY DISTANCE

FIG. 4. A P-450 phylogenetic tree. All P-450 amino acid sequences available through October 1988 in all species were aligned and the evolutionary distances were calculated as described (Nelson and Strobel, 1987). The relationship between evolutionary distance and millions of years ago can be found in Nelson and Strobel (1987). The oldest P-450 gene formed about 3 x 109 years ago and the most recent formed about I x 106 years ago. Individual P-450s within each family and subfamily are listed in Table 1. Note that some families and subfamilies found in Table 1 are not

included in this tree.


(Nelson and Strobel, 1987). Plants, therefore, had time to develop compounds that were toxic to predatory animal species. The presence of detoxifying enzymes allowed animals to survive in this hostile environment.

It has become apparent that different species have developed their own unique sets of P-450s, particularly those within the P-450II gene family. This evolutionary phenomenon is the result of recent gene duplications and gene conversion events. For instance, five genes have been identified in the rat P-450IIB subfamily, although others might exist. The P-450IIB1 and P-450IIB2 genes are highly similar in nucleotide sequence and the P-450s encoded by these genes demonstrated 97% amino acid similarity. The high degree of similarity between these genes indicates that they duplicated about 10 million years ago (Fig. 4). This was long after the formation of the rodent-human species lines, which occurred about 75 million years ago. It is improbable that humans have the equivalent of the rat IIB1 and IIB2 genes, unless a similar gene duplication occurred in humans 10 million years ago. Gene conversion has also served to homogenize P-450 genes within a particular species. Gene conversions are those events that result in the transfer of a segment of one gene to another gene. The transferred segment may include exon sequences only, or it may also include intron sequences. In general, the net result of this process is that genes become more like one another. Gene conversion is illustrated in Fig. 3 and has been described for the P-450I (Adesnik and Atchison, 1986), P-450IIA (Matsunaga et al., 1988), P-450IIB (Atchison and Adesnik, 1986), P-450IID (Matsunaga et al., 1989), and P-450III (Gonzalez et al., 1986b) gene families and subfamilies.

It is unclear what role gene conversions have had in P-450 evolution. Analysis of P-450 sequences that have resulted from conversion events reveals that these events can occur throughout the coding region of the gene. There does seem to be an increase in gene conversion near the portion of the P-450 sequence that is associated with the heme iron at the enzyme's active site. This is most evident in the rat P-450IID gene subfamily, in which a region of the sequence surrounding the cysteine-containing peptide, that is, the fifth ligand to the iron, is completely conserved among five genes (author's laboratory, unpublished data). Gene conversions are probably occurring all the time, but only a few are fixed in the gene pool. The selective forces involved in maintaining a converted P-450 gene could be changes in enzymatic activities or the stabilization of enzymatic activity, which result in an increased tolerance to plant toxins through metabolic inactivations.

4. STRUCTURE

P-450s are all hydrophobic, intrinsic membrane proteins that are tightly associated with intracellular membranes. The exceptions are the soluble bacterial enzymes. Based on several lines of evidence, including (i) extrapolation from the crystal structure of the bacterial P-450cam (Poulos et al., 1986); (ii) data derived from studies investigating the mechanism by which P-450s are inserted into the membranes (Finidori et al., 1987; Sakaguchi et al., 1987; Szczesna-Skorupa et al., 1988); (iii) immunochemical studies using antibodies against sequence-derived P-450 peptides (De Lemos-Chiarandini et al., 1987); and (iv) hydrophobicity profile comparisons of P-450 sequences (Nelson and Strobel, 1988), models have been proposed for the structure and orientation of P-450s in the intracellular membranes (Gotoh and Fujii-Kuriyama, 1988; Nelson and Strobel, 1988). The P-450 protein is probably a globular structure that is embedded into the membrane lipid bilayer via its amino-terminus peptide. The bulk of the enzyme is exposed to the cytoplasmic surface of the endoplasmic reticulum, and the heme iron is parallel or at a slight angle to the membrane. This orientation is ideal for the P-450's interaction with hydrophobic and hydrophilic substrates and for cooperative association with the NADPH-P-450 oxidoreductase. The latter enzyme is also anchored to the membrane via a peptide segment with a trypsin-labile connection to a soluble catalytic flavin-containing protein.

8 F, J. QONZALEZ

5. GENE STRUCTURE AND REGULATION OF P-450S INVOLVED IN DRUG, CARCINOGEN, STEROID AND

FATTY ACID METABOLISM

The P-450s within the P-450I, P-450II, and P-450III gene families metabolize drugs and carcinogens. Some of these enzymes also hydroxylate steroids such as testosterone, estrogen and progesterone. This is probably a minor catabolic pathway, of questionable physiological significance, for the elimination of steroids. The P-450IV gene family codes for fatty acid hydroxylases that metabolize lauric acid, palmitic acid, arachidonic acid and prostaglandins. The major metabolite produced by these enzymes is the terminal co hydroxy derivative, with about half to one-third as much of co- I. The P-450IV gene may produce fatty acid or prostaglandin metabolites that are physiologically important. This question remains to be resolved.

5.1. THE P-450I GENE FAMILY

The P-450I gene family consists of two members, IA1 and IA2, in most species examined to date. IA1 is detected only after treatment with inducers such as 3-methylcholanthrene (MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). This P-450 has a high level of catalytic activity toward the polycyclic aromatic hydrocarbon benzo[a]pyrene (Johnson and Muller-Eberhard, 1977; Negishi and Nebert, 1979; Goldstein et al., 1982; Guengerich et al., 1982; Ryan et al., 1982; Kuwahara et al., 1984; Sakaki et al., 1984). IA2, on the other hand, is constitutively expressed in liver, can also be induced by MC and TCDD and has a high level of catalytic activity toward arylamines (Johnson et al., 1980; McManus et aL, 1984). This enzyme can activate several heterocyclic amine promutagens derived from pyrolysates of proteins (Kamataki et al., 1983; Goldstein et al., 1982; McManus et al., 1988; Aoyama et al., 1989a). Both IA1 and IA2 have overlapping specificities and low levels of catalytic activity toward other substrates such as 7-ethoxyresorufin. These enzymes may, therefore, play a very important role in carcinogen activation.

The IA1 and IA2 genes each have seven exons (Gonzalez et al., 1984a, 1985a) and probably lie in tandem on mouse chromosome 9 (Tukey et al., 1984; Hildebrand et al., 1985a) and human chromosome 15 (Hildebrand et al., 1985b). Both genes have been isolated and sequenced from rat, mouse and man (Table 2). Unlike many other P-450 genes, the IA genes have a noncoding first exon.

The mechanism of regulation of the IA1 gene has been extensively studied and reviewed (Whitlock, 1987; Nebert and Gonzalez, 1987; Gotoh and Fujii-Kuriyama, 1988; Nebert and Jones, 1989; Gonzalez, 1988). This gene is induced in most tissues examined (Kimura et al., 1986), including lymphocytes (Jaiswal et al., 1985b) and placenta (Song et al., 1985). Transcription of the IA1 gene is governed by interaction of t rans-ac t ing factor(s) with DNA sequence elements lying within 1 kilobase upstream of the RNA polymerase II start site. These elements have been identified as inducible enhancers that increase transcription of the IA 1 gene only in the presence of inducer (Jones et al., 1986a,b; Neuhold et al., 1986; Sogawa et al., 1986; Jaiswal et al., 1987). Inducible enhancers can act regardless of their orientation or position within or around the IA1 gene. The consensus sequence of the most potent element is

5 ' C G T G C T G T C A C G C T A 3 ' C CT C A

and has been termed the xenobiotic regulatory element (XRE) (Fujisawa-Sehara et al., 1987). Multiple copies of this element result in an additive augmentation of IAl promoter activity in the presence of the inducer MC.

Studies using mutant cell lines that lack a functional receptor for MC and TCDD have suggested that the inducible enhancer or XREs are regulated by an inducer-receptor complex (Gonzalez and Nebert, 1985; Jones et al., 1986a; Sogawa et al., 1986). Direct binding experiments have demonstrated that a protein factor found in nuclear extracts

Molecular genetics of the P-450 superfamily

TABLE 2. Lis t o f S e q u e n c e d P - 4 5 0 Genes

Name Species Exons Reference

IA I rat 7

mouse human

IA2 rat 7 mouse human

IIAI rat 9 IIA2 rat lIB1 rat 9 IIB2 rat

IIC2 rabbit IIC6 rat 9 IIC11 rat IID6(IIDI) human 9 IID2 rat IID3 rat lID4 rat IID5 rat IID7 human IID8 human IIEI rat 9

rabbit human

IIE2 rabbit 9 IVA I rat 13 IVA2 rat 12 XIB1 human 9 XVIIAI human 8 XXIA 1 human 10

XXIA2

Sogawa et al., 1984 Hines et al., 1985 Gonzalez et al., 1985a Jaiswal et al., 1985b Kawajiri et al., 1986 Sogawa et al., 1985 Gonzalez et al., 1985a Quattrochi et al., 1986 Author's laboratory, unpublished data Author's laboratory, unpublished data Suwa et al., 1985 Mizukami et al., 1983a Atchison and Adesnik, 1983 Govind et al., 1986 H. Kimura et al., 1989 Morishima et al., 1987 Author's laboratory, unpublished data Author's laboratory, unpublished data Author's laboratory, unpublished data Author's laboratory, unpublished data Author's laboratory, unpublished data Author's laboratory, unpublished data Author's laboratory, unpublished data Umeno et al., 1988b Khani et al., 1988 Umeno et al., 1988a Khani et al., 1988 S. Kimura et al., 1989a S. Kimura et al., 1989a Morohashi et al., 1987a Picado-Leonard and Miller, 1987 White et al., 1986 Higashi et al., 1986 Rodriques et al., 1987 White et al., 1986 Higashi et al., 1986 Rodriques et al., 1987

binds specifically to XRE elements in vitro (Durrin and Whitlock, 1987; Fujisawa-Sehara et al., 1987; Denison et al., 1988a; Fujisawa-Sehara et al., 1988). Furthermore, the presence of TCDD (Denison et al., 1988a) or MC (Fujisawa-Sehara et al., 1988) is absolutely required for factor binding. In fact, addition of MC to mouse hepatoma cell cytosol results in factor binding to XRE in vitro (Fujisawa-Sehara et al., 1988). Direct evidence for the interaction of the TCDD receptor with the XRE was obtained using a 1251_labeled ligand for the receptor. Specific binding of the ~25 I-ligand to the factor that interacts with the XRE strongly suggests that this element is the TCDD receptor complex (Denison et al., 1988b). However, definitive evidence for the binding of the TCDD receptor to the XRE awaits receptor purification or cloning and expression of the receptor gene. To this end, a novel photoaffinity label has been developed for the receptor (Poland et al., 1986). This ligand binds a 95-kilodalton protein in mouse cytosol, which is encoded by a gene found on chromosome 12 (Poland et al., 1987). Using this ligand as a marker, the receptor has been purified over 20,000-fold from mouse liver (Perdew and Poland, 1988). Antibody or peptide sequences derived from this material should be useful in isolating the TCDD receptor gene.

Regulation of the IA2 gene is poorly understood. This gene is constitutively expressed in liver but is still activated by inducer administration (Gonzalez et al., 1984b; Kimura et al., 1986; Silver and Krauter, 1988; Soderkvist et al., 1988). The IA2 mRNA is also stabilized by MC and TCDD. In contrast to IAI, little IA2 expression is found in extrahepatic tissues. Because IA2 is not expressed in cell culture, we do not know the nature of the DNA elements that are required for gene activation. Transfection experiments using promoter-driven expression vectors, so valuable in the elucidation of the IAI

10 F.J . GONZALEZ

regulatory elements (Whitlock, 1987; Gotoh and Fujii-Kuriyama, 1988; Nebert and Jones, 1989), have not been possible in the absence of an appropriately responsive cell.

5.2. THE P-450IIA GENE SUBFAMILY

The P-450IIA subfamily has been most extensively characterized in rat at the protein level. P-450IIAl was purified (Ryan et aL, 1982; Waxman et al., 1983; Nagata et al., 1987) and found to specifically hydroxylate testosterone at the 7~ position with a minor metabolite at the 6~ position. A second P-450, IIA2, was purified and found to have a more relaxed specificity toward testosterone hydroxylation (I. Jansson et al., 1985; Matsunaga et al., 1988). Testosterone metabolites hydroxylated at the 15~, 15fl, 7ct, 16~, 6fl and 2ct positions were identified, in addition to an unidentified metabolite.

The cDNA to IIA1 was isolated from rat and sequenced (Nagata et al., 1987). Two other rat cDNAs, IIA2 (Matsunaga et al., 1988) and IIA3 (S. Kimura et al., 1989c), have also been characterized. IIA1 and IIA2 demonstrate 88% cDNA-deduced amino acid sequence similarity, and extensive areas of high amino acid similarity interspersed with areas of low similarity were uncovered, suggesting that gene conversion has occurred in the rat IIA subfamily (Matsunaga et al., 1988). cDNA from a third P-450IIA subfamily member, IIA3, was isolated from a lung library, and its deduced amino acid sequence was 71% and 73% similar to IIA1 and IIA2, respectively (S. Kimura et al., 1989c). The sequence comparison data suggest that IIA3 diverged from the ancestor to IIA1 and IIA2 prior to the formation of the latter two genes. The rat IIAl and IIA2 genes have also been isolated and have nine exons (author's laboratory, unpublished results). Significant nucleotide similarities exist throughout the introns and exons of these genes.

A mouse P-450 was isolated and its cDNA was cloned and sequenced. This enzyme, designated P-45015~, is an active testosterone 15~-hydroxylase (Squires and Negishi, 1988). Two related cDNAs were identified and termed 15~ type I and type II; these demonstrated 98% cDNA-deduced amino acid sequence similarity. The type I and type II P-450s exhibit 90% amino acid sequence similarity to rat IIA3 and only 70% and 75% similarity to rat IIA1 and IIA2, respectively, further suggesting that they are orthologous counterparts to rat IIA3. The mouse gene cluster is located on chromosome 7 (S. Kimura et al., 1989c).

The P-450IIA subfamily genes are regulated very differently. IIA1 production is increased in young male and female rats, but its gene is suppressed in males at the onset of puberty (Waxman et al., 1985; Nagata et al., 1987). IIA2, however, is never expressed in females, and its gene is activated only when males reach puberty (Matsunaga et aL, 1988). This sex-specific expression is controlled, in part, by pituitary hormone secretions (Waxman et al., 1985). In addition, IIA1 production is induced by MC, while IIA2 is not induced by this compound. Neither enzyme is found in lung, kidney, or intestine, suggesting that their genes are specifically expressed in liver. In contrast, IIA3 is found only in rat lung, and expression of its gene is also induced by MC (S. Kimura et al., 1989c).

In contrast to rat, mouse IIA genes are expressed in kidney as well as liver (Squires and Negishi, 1988). The type I 15ct-hydroxylase is expressed in male kidney and the type II 15ct-hydroxylase is expressed in female kidney. Both enzymes are expressed in male and female liver. Even though rat IIA3 is orthologous to these mouse P-450s, it is not expressed in liver or kidney.

A partial human P-450 cDNA, designated P-450(1) in the IIA family, has been isolated and sequenced (Phillips et al., 1985). A cDNA representing the full coding sequence of a IIA family member has recently been isolated (Miles et al., 1989; Yamano et al., 1989b). Its deduced amino acid sequence is 85% similar to rat IIA3 and only 69% and 65% similar to IIA 1 and IIA2, respectively, suggesting that the human enzyme is orthologous to IIA3. It is still unclear how many IIA genes exist in man. The human IIA subfamily is found on the long arm of chromosome 19 (Davis et al., 1986).


5.3. ThE P-450IIB GENE SUBFAMILY

The P-450IIB genes are noteworthy because they can be induced by phenobarbital. Two enzymes, IIBI and IIB2, have been extensively studied in rat (Guengerich et aL, 1982; Ryan et al., 1982; Waxman and Walsh, 1982; Wilson et al., 1984). These enzymes have similar broad, overlapping specificities toward a number of substrates and the IIBl form has a two- to ten-fold higher level of activity (depending on the substrate) than IIB2 when the enzymes are purified and reconstituted. The cDNAs and genes were isolated and sequenced, and the IIB1 and IIB2 P-450s were found to possess 97% cDNA-deduced amino acid sequence similarity (Fujii-Kuriyama et al., 1982; Kumar et al., 1983; Suwa et al., 1985). A third P-450 of the IIB family was identified through cDNA cloning and designated IIB3 (Labbe et al., 1988). The cDNA-deduced amino acid sequence of this P-450 exhibits 77% similarity to the sequences of IIBI and IIB2, indicating that it separated from the precursor to IIB1 and IIB2 approximately 75 million years ago. Sequence comparison data and the known speciation times of rodent-man and rat-mouse (75 and 20 million years ago, respectively) suggest that the equivalent of IIB1 and IIB2 may not be found in mouse or human, whereas the genes corresponding to IIBI (or IIB2) and IIB3 may be present in both man and mouse. In fact, P-450 cDNAs corresponding to the IIB subfamily have been identified in mouse (Stupans et al., 1984; Noshiro et al., 1988) and man (Phillips et aL, 1987; Miles et al., 1988; Yamano et al., 1989c). Rabbits also have a subfamily of at least two IIB genes that exhibit approximately 95% amino acid similarity (Gasser et al., 1988; Komori et al., 1988). Several different cDNAs were isolated and sequenced, yet it is unclear whether some of these represent allelic variants from an outbred rabbit strain, or whether they are derived from two distinct genetic loci.

The rat IIB1 and IIB2 genes each contain nine exons (Atchison and Adesnik, 1983; Mizukami et al., 1983a, b; Suwa et al., 1985). The IIB subfamily has been localized to mouse chromosome 7 (Simmons and Kasper, 1983; Noshiro et al., 1988) and human chromosome 19 (Miles et al., 1988; Santisteban et al., 1988).

The mechanisms by which the IIB genes are regulated are poorly understood. Pheno- barbital administration to rats causes a rapid transcriptional activation of the IIB1/IIB2 genes (Atchison and Adesnik, 1983; Hardwick et al., 1983b). The IIB3 gene, on the other hand, is constitutively expressed and cannot be induced by phenobarbital (Labbe et al., 1988). In the absence of a stable hepatoma cell line in which expression can be induced, little information is available concerning the sequence requirements for IIB1 and IIB2 gene activation. Furthermore, a receptor for phenobarbital has not yet been adequately characterized.

Even though the IIB1 and IIB2 genes are very similar, marked differences exist in their expression. IIB2 is constitutively expressed in liver but absent in lung, kidney and testis regardless of treatment (Omiecinski, 1986). Further, IIB2 is constitutively expressed in small intestine but its expression cannot be induced in this tissue (Traber et al., 1988). In contrast, IIB1 is absent in liver until phenobarbital treatment and is constitutively expressed in lung and testis. However, phenobarbital treatment does not induce gene expression in these latter tissues. In small intestine, IIBI is constitutively expressed and its expression can be easily induced with various agents (Traber et al., 1988). These data suggest that the rat IIB1 and IIB2 genes contain different tissue-specific enhancers and inducer control elements.

5.4. THE P-450IIC GENE SUBFAMILY

The P-450IIC subfamily is generally thought to represent a class of constitutively expressed genes. These enzymes are noted for their sex-specific and developmentally regulated expression in rats; the subfamily includes an adult female-specific P-450 (P-450i/15fl), two adult male-specific P-450s (P-450h/16~ and P-450g) and two enzymes expressed in both males and females (P-450PB-1 and P-450f). These enzymes have been

12 F.J. GONZALEZ

purified and studied by several laboratories (see Guengerich, 1987; Gonzalez, 1988, for a review). In general, IIC enzymes have broad overlapping specificities toward a number of chemicals, although some demonstrate high levels of catalytic activity toward steroids such as testosterone.

Several rat IIC cDNAs have been characterized. The cDNAs for P-450PB-1, designated IIC6, and P-450f, designated IIC7, have been isolated and sequenced (Friedberg et al., 1986; Gonzalez et al., 1986a; H. Kimura et al., 1988). cDNAs of the male-specific P450h/16e, designated IICI1 (Yoshioka et al., 1987; Zaphiropoulos et al., 1988), and the female-specific P450i/15/?, designated IIC12 (Zaphiropoulos et al., 1988), have also been characterized. The amino acid sequence similarities between these P-450s range from 68% to 75%, indicating that their genes diverged from a common ancestor more than 75 to 100 million years ago (Fig. 3),

Several P-450 cDNAs from the rabbit have also been characterized. These include four forms from a phenobarbital-induced liver library designated IICI through IIC4 (Leighton et al., 1984); P-450 1 (IIC5), a progesterone 21-hydroxylase (Tukey et al., 1985); and pHP3 and b32-3, designated IIC14 and IICI5, respectively (Imai, 1987; Imai et al., 1988). Therefore, at least seven P-450s appear to be expressed in the rabbit.

Two P-450 cDNAs have been isolated from human liver libraries and sequenced. These have been designated IIC8 (Kimura et al., 1987a; Okino et al., 1987) and IIC9 (Kimura et al., 1987a; Umbenhauer et al., 1987; Yasumori et al., 1987; Meehan et al., 1988a). The IIC9 corresponds to the enzymes P-450Mp ~ and P-450MP_2, isolated from human liver (Shimada et al., 1986). These enzymes catalyze the oxidation of the drug S-mephenytoin, and a deficiency in this metabolism has been found in the human population (Inaba et al., 1984; Kupfer and Preisig, 1984; Wedlund et al., 1984).

The IIC2 (Govind et al., 1986) and IICll (Morishima et al., 1987) genes were isolated from rabbits and mice, respectively. Both genes contain nine exons with intron-exon junctions occurring at the same positions as in the liB genes. The IIC gene cluster has been localized to human chromosome 10 (Okino et al., 1987; Riddell et al., 1987; Meehan et al., 1988a) and mouse chromosome 19 (Meehan et al., 1988b).

The mechanism by which the IIC genes are regulated during development has been extensively investigated in the rat. The IIC6 and IIC7 genes are transcriptionally activated just before male and female rats reach puberty (Gonzalez et al., 1986a). Serum testosterone levels probably do not play a role in the regulation of these genes (Waxman et al., 1985). In contrast the IICII and IIC12 genes are controlled, to some extent, by testosterone. IICll is expressed in adult males only if they receive testosterone during the neonatal period (Morgan et al., 1985; Waxman et al., 1985; Dannan et al., 1986; Yamazoe et al., 1986a; Kato et al., 1986). Expression of IIC12 is only partially dependent on estradiol, since ovariectomy only reduces its expression in adults to the levels seen in immature females (Dannan et al., 1986). The effect of neonatal sex hormone exposure appears to be due, in part, to growth hormone secretions. Neonatal androgen exposure determines the adult pattern of growth hormone secretion, which is pulsatile in males and constant in females (J. Jansson et al., 1985). Hypophysectomy of males results in the loss of IICI 1 mRNA, which is partially restored upon intermittent injection of growth hormone (Mode et al., 1987; Zaphiropoulos et al., 1988). If these rats are given a continuous infusion of growth hormone, the IICII mRNA level remains suppressed, but IIC12 mRNA, or the female-specific gene product, is increased (Zaphiropoulos et al., 1988). Time-course experiments further suggest that other intermediary protein factors may play a role in the control of these genes, since periods of 2 and 6 days of growth hormone treatment are required to achieve an increase in IICI 1 and IIC12 mRNAs, respectively (Zaphiropoulos et al., 1988). These data also suggest that the increases in IICll and IIC12 may be due to posttranscriptional events such as mRNA stabilization. It is also noteworthy that other P-450s, including IIB1 and IIB2 (Yamazoe et al., 1987), IIA2 (Waxman et al., 1988b) and IIIA2 (Waxman et al., 1985; Kato et al., 1986; Yamazoe et al., 1986b) are regulated by pituitary hormone levels. Interestingly, the adult male-specific IIA2 is further elevated in hypophysectomized adult males and is also expressed in hypophysectomized adult females


(Waxman et al., 1988b). These data suggest that the mechanisms controlling sex-specific expression of P-450s vary between genes.

5.5. THE P-450IID GENE SUBFAMILY

P-450s of the IID subfamily were purified from rat (Larrey et al., 1984; Gonzalez et al., 1987), mouse (Harada and Negishi, 1984) and human (Distlerath et al., 1985; Gut et al., 1986a). The rat and human enzymes carry out the oxidation of the drugs debrisoquine and bufuralol. These activities are associated with a common human defect in drug oxidation known as the 'debrisoquine/sparteine polymorphism' (Idle and Smith, 1979; Eichelbaum, 1986). The defect was originally identified in population studies, where it was found that 5-10% of individuals given subtherapeutic doses of debrisoquine failed to hydroxylate the compound. Subsequent studies showed that this polymorphism results from the absence of the debrisoquine 4-hydroxylase IIDI protein (Gonzalez et al., 1988b).

Studies in rats revealed two immunochemically related P-450s and two cDNAs with 73% cDNA-deduced amino acid similarity, suggesting the presence of a subfamily of l iD P-450s (Gonzalez et al., 1987). Furthermore cloning studies showed that at least five genes, designated IID1, IID2, IID3, IID4 and IID5, are present in rat and that these genes have between 75% and 95% DNA-deduced amino acid similarity (Matsunaga et al., 1989). Four of the rat genes have been cloned and found to be located head to tail on a 60-kilobase pair segment of DNA. At least four of the five rat genes are believed to be expressed in the liver and kidney (author's laboratory, unpublished data). Multiple gene conversions were found to have occurred in the rat l i d genes and have resulted in a marked conservation of the cysteine peptide region at the active site of the enzymes.

A cDNA for male-specific testosterone 16~-hydroxylase was cloned and sequenced (Wong et al., 1987) and found to belong to the IID gene family. The cDNA-deduced amino acid sequence of the 16~-hydroxylase clone demonstrated 82%, 72%, 78% and 70% similarity to rat IIDI, IID2, IID3 and IID4, respectively. Multiple l iD genes were also detected by cloning and Southern blot analysis (Wong et al., 1987).

In contrast to the rat, humans have three genes that lie in tandem and exhibit from 92% to 97% cDNA-deduced amino acid similarity to each other (author's laboratory, unpublished data). One of the human genes, designated IID8, was found to have several gene-inactivating mutations. A second gene, IID7, had a single base deletion, resulting in a protein-coding frame shift, mRNAs for IID7 and IID8 were not detected in several human liver RNA samples. The third gene, liD1, codes for the debrisoquine 4-hydroxylase (Gonzalez et al., 1988b,c). This was verified by direct cDNA cloning and expession. Analysis of human liver samples, where debrisoquine 4-hydroxylation was undetectable, further revealed the presence of mutant alleles of the IID1 gene that produced defectively spliced RNA transcripts (Gonzalez et al., 1988b). Further studies established that two or more mutant IIDI genes can be detected in lymphocyte DNA (Skoda et al., 1988). The liD gene clusters were localized to mouse chromosome 15 (Gonzalez et al., 1987) and human chromosome 22 (Gonzalez et al., 1988c).

5.6. THE P-450IIE GENE SUBFAMILY

A unique form of P-450 was purified from rabbits (Koop et al., 1982; Koop and Coon, 1984), rats (Ryan et al., 1985; Patten et al., 1986; Favreau et al., 1987) and man (Wrighton et al., 1987). In rodents, production of this enzyme is induced by ethanol and the enzyme carries out the metabolism of ethanol (Koop et al., 1982), acetone, acetoacetate and acetol (Casazza et al., 1984; Koop and Casazza, 1985), and N-nitrosodimethylamine (Patton et al., 1986; Wrighton et al., 1987; Thomas et al., 1987). The human enzyme, both purified and reconstituted (Wrighton et al., 1987) and expressed from its cDNA (Umeno et al., 1988a), also catalyzes demethylation of N-nitrosodimethylamine. The activation of this

14 F.J. GONZALEZ

compound to unstable intermediates may play a role in nitrosamine-induced cancer (Hong and Yang, 1985).

A cDNA, designated IIE1, has been isolated from rats (Song et al., 1986), rabbits (Khani et al., 1987) and man (Song et al., 1986). Only a single liE gene has been identified in rats (Umeno et al., 1988b) and man (Umeno et al., 1988a). In contrast, two genes have been identified in rabbits, both of which are expressed (Khani et al., 1988). The DNA-deduced amino acid sequences of the two rabbit genes demonstrated 97% similarity, indicating that they diverged from a common ancestor only about 10 million years ago. Since rodents and man diverged about 75 million years ago, the gene duplication in rabbits occurred after the formation of the rabbit and human species. The two rabbit genes and the rat and human liE genes all contain approximately 10 kilobase pairs and 9 exons like other genes in the P-450II gene family. The IIE genes have been localized to mouse chromosome 7 (Umeno et al., 1988b) and human chromosome 10 (Umeno et al., 1988a).

The mechanism by which IIEI is regulated is quite distinct from that of other P-450s. Administration of ethanol, acetone, pyrazole or methylpyrazole to rats causes a four- to nine-fold increase in IIE1 protein synthesis without an increase in IIEI mRNA (Song et al., 1986, 1987; Johansson et al., 1988). The same result has been obtained with rabbit IIE1 (Khani et al., 1987). Interestingly, 4-methylpyrazole induced increased levels of mRNA coding for IIBI and/or IIB2, yet did not induce the IIE1 mRNA (Song et al., 1986). Similarly, ethanol preferentially induced IIB1 and/or IIB2 mRNAs, and this increase was due to transcriptional activation (Johansson et al., 1988). These studies indicate that the liB and l ie subfamily P-450s are induced by entirely different mechanisms. The liB genes are transcriptionally activated by ethanol treatment, while the IIEI gene is unaffected. Recent results have shown that acetone treatment stabilizes the liE protein, and the lack of turnover of the protein results in its induction (Song et al., 1989).

In contrast to the results with ethanol, IIE1 mRNA levels are markedly elevated when rats are starved (Hong et al., 1987) or made diabetic (Song et al., 1987). This increase appears to result from posttranscriptional mRNA stabilization (Song et al., 1987). Induction of IIE1 was also found in spontaneously diabetic rats, and this increase was reversed by insulin administration (Favreau et al., 1987; Bellward et al., 1988; Dong et al., 1988). The mechanism by which the diabetic state of fasting stabilizes IIE1 mRNA is unknown but poses an intriguing question. Certain signals in the IIE1 mRNA sequence may specifically govern its degradation relative to other liver mRNAs. Of interest was the finding that acetone treatment and starvation have an additive effect on IIE1 induction (Johansson et al., 1988).

5.7. TIlE P-450III GENE FAMILY

A P-450 that is induced by the synthetic glucocorticoid pregnenolone 16~-carbonitrile (PCN) was first purified from rats (Elshourbagy and Guzelian, 1980) and designated PCNI (Gonzalez et al., 1986b). A male-specific P-450 in the P-450III family was also purified (Guengerich et al., 1982; Waxman, 1984; Halpert, 1988) and designated PCN2 (Gonzalez et al., 1986b). For unknown reasons, these enzymes have been very difficult to reconstitute into an active form for substrate specificity studies. Recent studies, using extracted microsomal lipids as a lipid source for reconstitution, resulted in high activity of the PCN2 P-450 (Yamazoe et al., 1988). Immunochemical studies using polyclonal antibodies to the PCN-induced P-450 indicated that it is involved in the metabolism of testosterone at the 6fl position (Waxman et al., 1985; Nagata et al., 1987) and in ethylmorphine N-demethylation (Elshourbagy and Guzelian, 1980), erythromycin N-demethylation (Wrighton et al., 1985), and mephenytoin 4-hydroxylation (Shimada and Guengerich, 1985). The related human P-450, designated P450•v, was purified and shown to be involved in testosterone 6fl-hydroxylation, 17ct estradiol 2- and 4-hydroxylation, d-benzphetamine demethylation, aldrin epoxidation and nifedipine oxidation (Guengerich et al., 1986a). The major form of purified rat P-450 responsible for nifedipine


oxidation is the male-specific IIC12 (Guengerich et al., 1986a). Dehydroepiandrosterone 3-sulfate, benzo[a]pyrene, 7-ethoxycoumarin and testosterone are metabolized by either P-450Nv, purified from human fetal liver, or a related P-450 (Kitada et al., 1987a,b).

Rat and human P-450III genes appear to be regulated differently. In rats, the adult male-specific PCN2 is absent from the livers of adult females. In contrast, adult male and female human liver specimens were all found to contain a P-450 immunochemically related to PCN2 (Guengerich et al., 1986b; Gonzalez et al., 1988a; Waxman et al., 1988a). Another interesting contrast between rats and humans is the finding that a human enzyme of the P-450III gene family is expressed in fetal liver (Kitada et al., 1985), whereas rat PCN2 expression is activated only after birth (Gonzalez et al., 1986b).

The multiplicity of the P-450III gene family was established by cDNA cloning studies. In rats, the PCN-inducible PCNl cDNA was cloned (Hardwick et al., 1983a; Gonzalez et al., 1985b) also designated P-450p (Wrighton et al., 1985), and later studies established the existence of the constitutively expressed PCN2 that is regulated very differently from PCN1 (Gonzalez et al., 1986b). A single P-450 cDNA, designated P-450 3c, was isolated from rabbits and sequenced (Dalet et al., 1988). P-450 3c is constitutively expressed in rabbits, suggesting that it may correspond to rat PCN2 (Dalet et al., 1986, 1988); however, the amino acid sequence of P-450 3c demonstrates equal degrees of similarity to the sequences of PCN1 and PCN2 (Dalet et al., 1988). Western and Southern blot analyses suggest that two or more P-450s may exist in rabbits (Dalet et al., 1985; Wrighton et al., 1985) although a second rabbit P-450III gene or cDNA has not yet been cloned. In humans, three P-450III genes have been identified in liver. These were designated HLp (Molowa et al., 1986), P-450NF (Beaune et al., 1986), and hPCN3 (Aoyama et al., 1989b). The full cDNA corresponding to P-450NF was also cloned and sequenced (Gonzalez et al., 1988a). HLp and P-450NF demonstrated 98% amino acid sequence similarity to each other and 90% similarity to hPCN3, indicating that the former two genes were recently formed. The 10% differences between the sequences of hPCN3 and HLp/P-450NF suggests that they diverged about 30-40 million years after the formation of the rodent-human lineage. Therefore, we would not expect to find hPCN3 or the precursor to HLp and P-450NF in rodents. There may exist two P-450IIIs in rats, one to three in rabbits, and three in humans. The gene structure of the P-450III family has not been reported, although the human and mouse P-450III loci have been localized to chromosome 7 (Gonzalez et al., 1987; Riddell et al., 1987; Brooks et al., 1988) and chromosome 6 (Simmons et al., 1985), respectively.

The regulation of PCNI and PCN2 in rats has been extensively investigated. The PCN1 gene is transcriptionally activated by glucocorticoids (Simmons et al., 1987). Expression of this enzyme is totally dependent on the presence of an inducer, since PCN1 mRNA is not detected in untreated rats (Gonzalez et al., 1986b). PCN2, on the other hand, is constitutively expressed in adult male rats and transiently expressed in young females (Gonzalez et al., 1986b). Furthermore, expression of this gene is not induced by PCN treatment, while both PCN1 and PCN2 mRNA levels are elevated by phenobarbital. Clearly, the PCN1 and PCN2 genes are regulated by both overlapping and distinct control elements.

Posttranscriptional events also play a role in the regulation of the P-450III enzymes in rat and rabbit. In rabbit, administration of triacetyloleandomycin (TAO) caused a marked stabilization of P-450 3c mRNA in the absence of an increase in transcription (Dalet et al., 1988). In rats, TAO also increased the translatable mRNA presumed to code for the PCNl mRNA (Watkins et al., 1986). Since antibodies to PCNI probably cross- react with the highly homologous PCN2 protein (Gonzalez et al., 1986b), it is possible that this mRNA could be coding for PCN2. The effect of TAO on mRNA in rat, however, was not apparent in primary hepatocyte cultures (Watkins et al., 1986). Dexamethasone, on the other hand, increased mRNA levels in rats and in in vitro cultures. This finding further suggests that TAO and dexamethasone induce the P-450III proteins via different mechanisms: TAO via mRNA stabilization (Watkins et al., 1986) and dexamethasone primarily through transcriptional activation (Simmons et al., 1987).

JPT 45/I--B

16 F.J. GONZALEZ

Interestingly, in a culture system in which hepatocytes are plated on ~matrigel' (Kleinman et al., 1986), TAO increased the level of mRNA for PCN2 and/or PCN1 (Schuetz et al., 1988). It is entirely possible that TAO stabilizes the PCN2 mRNA that is constitutively expressed in adult male rats, whereas dexamethasone increases transcription of only the PCN1 gene. Indeed, the effects of TAO and dexamethasone on immunoreactive protein are additive (Watkins et al., 1986).

In addition to stabilizing the PCN1/PCN2 mRNA(s), TAO also stabilizes the P-450III protein(s) against degradation. Administration of TAO to rats increases the degradation half-life of the protein from 14 hr to 60 hr (Watkins et al., 1986). Dexamethasone, on the other hand, did not alter the turnover rate of the PCNI/PCN2 protein. It was postulated that TAO may exert its effect on protein degradation by binding to the active site of the protein (Watkins et al., 1986).

5.8. THE P-450IV GENE FAMILY

A P-450 induced by hypolipidemic drugs such as clofibrate was purified from rats and found to metabolize lauric acid at its to position (Tamburini et al., 1984). This enzyme is also active in the metabolism of arachidonic acid (Bains et al., 1985). Western immuno- blot analysis using an antibody to the enzyme, designated P-450LAco, revealed the presence of two proteins of 51,500 and 52,000 kilodaltons (Hardwick et al., 1987). The P-450LA¢o corresponded to the 51,500-kilodalton protein; the nature of the second protein is unknown, but it may be an immunochemically related member of the same gene family (S. Kimura et al., 1989b).

The cDNA of the rat P-450LAco designated P-450IVAl, was isolated and sequenced and found to represent the first member of the P-450IV gene family (Hardwick et al., 1987). A second cDNA isolated from rat, designated IVA3, demonstrated 72% cDNA-deduced amino acid similarity to IVA1 (S. Kimura et al., 1989b). A third rat P-450IV gene, designated IVA2, has been identified through the isolation and sequencing of genomic clones, but its expression has not been verified (S. Kimura et al., 1989a). A P-450 expressed in lung was purified from pregnant rabbits (Williams et al., 1984) and rabbits treated with progesterone (Yamamoto et al., 1984). The cDNA for this enzyme, designated P-450p-2, was isolated and sequenced (Matsubara et al., 1987) and found to share 72% of the cDNA-deduced amino acid sequences of IVA1 and IVA2.

The gene for rat IVAI has been isolated and possesses 13 exons (S. Kimura et al., 1989a). The large number of exons in the P-450IV family, compared to the P-450I family (seven exons) and the P-450II family (nine exons), supports the suggestion that this family of genes evolved early in the evolution of the P-450 gene superfamily (Nelson and Strobel, 1987), assuming that exon loss has occurred during the evolution of the eukaryote genome (Gilbert et al., 1986).

Clofibrate administration results in a rapid transcriptional activation of the IVA1 gene (Hardwick et al., 1987). This gene may be a member of a gene battery, including genes coding for peroxisomal enzymes (Reddy et al., 1986) that are induced by hypolipidemic agents. It is likely that this induction response is mediated through the action of a receptor (Lalwani et al., 1983, 1987). The IVA1 gene is induced in both liver and kidney (Hardwick et al., 1987). In contrast, the IVA2 gene is induced only in liver. Unlike the IVAi gene, IVA2 is constitutively expressed in kidney (S. Kimura et al., 1989b). Neither rat gene is expressed in the lungs of pregnant rabbits, yet rabbits express the P-450p-2 prostaglandin co-hydroxylase, an enzyme of the P-450IV family that is present in pregnant animals and animals injected with progesterone. This induction results from an increase in P-450p-2 mRNA (Matsubara et al., 1987). Progesterone also increases the level of P-450p-2 mRNA in liver. The P-450p-2 gene, or a related gene, is also expressed in kidney and placenta of untreated nonpregnant rabbits (Matsubara et al., 1987). The significance of the species-specific expression of P-450p-2 in pregnant rabbit lung is unclear. However, the high level of activity of the P-450IV enzymes toward fatty acids suggests that they may carry out physiologically important hydroxylation reactions.


The cDNAs for a P-450 in the IVB subfamily have recently been cloned and sequenced from man (Nhamburo et al., 1989), rat and rabbit (Gasser and Philpot, 1989). The human IVB1 gene is not appreciably expressed in liver but is constitutively expressed in lung and probably other tissues. Interestingly, the rabbit IVB1 enzyme is capable of metabolizing and activating 2-aminofluorene to a mutagenic metabolite (Robertson et al., 1981) whereas the human counterpart is incapable of catalyzing this activity (Nhamburo et al., 1989). These results indicate a potentially important species difference in carcinogen metabolism in pulmonary tissue.

6. P-450s INVOLVED IN STEROID SYNTHESIS

Four mammalian P-450 gene families produce enzymes that are almost exclusively involved in the steroid biosynthetic pathways. These P-450s do not contribute, to any degree, to the oxidation and elimination of foreign compounds. Furthermore, these gene are expressed in extrahepatic tissues, and their expression reflects the steroid biosynthetic pathways of these tissues.

The P-450XI gene family codes for two mitochondrial P-450s, steroid l lfl-hydroxylase (XIAI) and cholesterol side-chain cleavage enzyme (XIB1). The human (Chua et al., 1987) and bovine (Morohashi et al., 1987b) XIAI cDNAs have been cloned and sequenced, and the structure of the bovine XIA1 gene has also been reported (Hashimoto et al., 1989). The bovine XIB1 cDNA was cloned and the cDNA-deduced amino acid sequence was 520 residues, demonstrating that the enzyme was translated with a 39-residue 'extrapeptide', similar to other mitochondrial proteins (Morohashi et al., 1984) and the XIA1 gene (Hashimoto et al., 1989). The human XIB1 cDNA has been characterized (Chung et al., 1986b), and its gene has been isolated and sequenced and found to contain nine exons (Morohashi et al., 1987b). The promoter region of the XIB1 gene was found to be responsive to cAMP (Inoue et al., 1988). These studies follow from earlier work demonstrating that the peptide hormone ACTH, which acts through cAMP, augments transcription of the XIBI gene (John et al., 1986a,b). The XIA1 and XIB1 genes have been localized to human chromosomes 15 (Chung et al., 1986b) and 8 (Chua et al., 1987), respectively.

The bovine (Zuber et al., 1986a) and human (Bradshaw et al., 1987; Chung et al., 1987) XVIIA1 cDNAs have been cloned and sequenced. Expression of the bovine cDNA provided confirmation of studies with the purified enzyme, which showed that XVIIA 1 has both steroid 17~-hydroxylase and 17,20-1yase activities (Zuber et al., 1986b). The human XVIIA1 gene was found to contain eight exons and to span about 7 kilobase pairs (Picado-Leonard and Miller, 1987). The gene is located on human chromosome 10 (Matteson et al., 1986) and is regulated by ACTH in the adrenal gland.

The most actively studied P-450 in the steroidogenic pathway is the progesterone 21-hydroxylase (XXIA1). Deficiency in this activity is the major cause of congenital adrenal hyperplasia. Although several enzymes in the steroidogenic pathway are known to be defective in this inherited condition, mutations affecting XXIA1 expression are by far the most common. The clinical and molecular biology aspects of this disorder, which occurs in about 1 in 7,000 births, have been reviewed elsewhere (Miller and Levine, 1987; White et al., 1987a,b).

The P-450XXI locus consists of two genes arranged in tandem (Higashi et al., 1986; White et al., 1986) on the short arm of human chromosome 6 (White et al., 1985). One gene, designated XXIA1, is nonfunctional, whereas the second gene, designated XXIA2, is normal. The nonfunctional XXIA1 gene is flanked by genes of the HLA major histocompatibility complex, the C4A and C4B genes, located upstream and downstream of XXIA1, respectively. The normal XXIA2 gene is located downstream of the C4B gene. The XXI locus has also been cloned from mouse (Chaplin et al., 1986) and cow (Chung et al., 1986a). Unlike the human gene, the leftmost mouse gene (relative to the direction of transcription) is active (Parker et al., 1985). The fact that both mice and humans have two genes suggests that these genes were formed by gene duplication more than 75 million

18 F.J. GONZALEZ

years ago. However, the high degree of similarity in the nucleotide sequences of these genes suggests that they evolved by concerted evolution, by a mechanism such as gene conversion and/or unequal crossing over (Higashi et al., 1986, 1988a).

One of the mechanisms thought to give rise to defective XXIA2 genes in humans is gene conversion between the XXIA1 and XXIA2 genes (Donohoue et al., 1986; Matteson et al., 1987; Higashi et al., 1988a,b; Miller, 1988). Three mutant XXIA alleles were directly cloned from deficient patients, sequenced, and subjected to functional analysis by COS cell-mediated expression (Higashi et al., 1988b). Two genes were found to carry base changes that cause aberrant splicing of their pre-mRNA. The other allele was found to have missense mutations. All mutations could be accounted for by possible gene conversions between the XXIA1 pseudogene and the normal XXIA2 gene (Higashi et al., 1988b). Gene conversion has also been detected in normal individuals (Higashi et al., 1988a,b), however, these events are only deleterious when nonfunctional mutated regions of the XXIAI gene are converted to the XXIA2 gene. Gene conversions are probably very frequent in close, tandemly arranged genes such as those in the XXI and l i d loci.

Two other interesting mutations that result in detrimental amino acid changes in the XXIA2 P-450 have been uncovered. A Ser-269 to Thr and Asn-494 to Ser mutation apparently results in the production of an enzyme lacking progesterone 21-hydroxylase activity (Rodriques et al., 1987). However, the involvement of these amino acid changes in the activity of the enzyme was not directly demonstrated. Interestingly, the change at codon 269 was also found in some alleles of the XXIAI gene, suggesting that gene conversion between XXIA1 and XXIA2 had occurred (Rodriques et al., 1987). Another interesting mutation in the XXIA2 protein was an Ile-172 to Asn change that probably also resulted from a gene conversion event (Amor et al., 1988). When the mutant allele was transfected into adrenal cells, normal levels of mRNA were produced, further suggesting that this amino acid change was crucial. The study of natural P-450 mutations might provide insight into which amino acid residues are important for enzymatic activities.

Another cause of steroid 21-hydroxylase deficiency is deletion of the XXIA2 gene. In many instances, this gene has been found to be deleted along with the C4B gene (White et al., 1984, 1988; Carroll et al., 1985; Shiroishi et al., 1987). However, other investigators did not find a high frequency of gene deletion (Matteson et al., 1987).

The aromatase P-450 (P-450XIXA1) catalyzes the aromatization of C19 androgens to form C18 estrogens. The enzyme was purified from human placenta (Nakajin et al., 1986), and several peptides were sequenced (S. Chen et al., 1988). Oligonucleotides were used to isolate a cDNA clone that contains 80% of the coding sequence (S. Chen et al., 1988). Others have also isolated the human cDNA using specific antibody probes; however, the complete sequence of this clone has not been reported (Evans et al., 1986). The XIXA1 gene was mapped to human chromosome 15 (S. Chen et al., 1988). Isolation and structural analysis of genomic clones have not been reported.

7. YEAST P-450 GENES

The yeast lanosterol 14c¢-demethylase; P-45014DM, isolated from S a c c h a r o m y c e s cerevisiae, has been extensively studied. This enzyme is very important clinically because it is inhibited by antifungal agents such as ketoconazole and itraconazole (Yoshida and Aoyama, 1987). A form of the enzyme that lacks demethylase activity has been characterized (King et al., 1985; Aoyama et al., 1987). The substrate binding and heme environment of the variant enzyme were markedly different from that of the wild-type enzyme. The amino acid residue changes in the mutant P-45014DM have not been determined.

The P-45014DM gene was isolated by transforming yeast with a gene library in an expression vector, using ketoconazole as a selection agent (Kalb et al., 1986). A single plasmid was isolated that, when introduced into yeast, increased resistance to ketoconazole and elevated P-450 levels. The complete sequence of the P-45014DM gene (P-450LI) has


been determined from this plasmid (Kalb et al., 1987). The P-450LI protein sequence was found to be more similar to the mammalian P-450s, including a hydrophobic amino- terminal putative membrane-binding domain. The orthologous gene was also isolated from the strain Candida tropicalis and its sequence was found to be 66% similar to that of P-450LI (C. Chen et al., 1988).

A P-450 induced by n-alkanes has also been found in Candida tropicalis (Wiedmann et al., 1986). This induction was found to result from an increase in mRNA (Weidmann et al., 1986; Sanglard et al., 1987). The gene for P-450alk was isolated from an expression library using specific polyclonal antibody (Sanglard et al., 1987). The sequence of the P-450alk gene was determined, and surprisingly, the deduced amino acid sequence was less than 30% similar to that of P-45014DM (C. Chen et al., 1988). Therefore, this P-450 represents the second yeast gene family, designated P-450LII.

8. BACTERIAL P-450 GENES

A P-450 isolated from Pseudomonas putita, designated P-450cam, is the best characterized of the P-450 enzymes. This P-450, which converts camphor to 5-exohydroxycamphor, was used in early work to study oxygen metabolism and electron transfer pathways; mechanisms common to all heme-containing P-450s (Gunsalus and Sligar, 1977; Unger et al., 1986). This enzyme's solubility renders it more amenable to study than the eukaryote P-450s. P-450cam is not a membrane-bound enzyme, and this feature has allowed investigators to determine the crystal structure of its substrate-free and substrate-bound forms (Poulos et al., 1986). This enzyme requires flavoprotein putidaredoxin reductase and the iron-sulfur-containing protein putidaredoxin for the transfer of electrons from NADPH into its active site (Unger et al., 1986). Similarly, the mitochondrial P-450s require the iron-sulfur protein adrenodoxin that is similar to the bacterial putidaredoxin, indicating that the P-450XI family is similar to the bacterial P-450cam.

The gene encoding for P-450cam was isolated, sequenced, and designated camC (Unger et al., 1986). Interestingly, the gene coding for the putidaredoxin reductase (camA) was found just 22 base pairs downstream from the camC gene, suggesting the presence of an operon. The mechanism by which the camC gene is induced by camphor has not been determined.

A second bacterial P-450 with a very unusual structure was isolated from Bacillus megaterium and characterized. This enzyme, designated BM-3, is a self-contained monooxygenase system that catalyzes hydroxylation of long-chain fatty acids without the requirement of another electron-transferring protein (Wen and Fulco, 1987). BM-3 is a 120-kilodalton enzyme that appears to be comprised of half heme-containing P-450 and half NADPH-P-450 oxidoreductase (Narhi and Fulco, 1987). The BM-3 gene has recently been cloned and sequenced, and the data demonstrate that it is a member of a new (P-450CII) bactrial gene family (Ruettinger et al., 1989). Interestingly, this gene is induced by barbiturates through a transcriptional mechanism that involves cis- and trans-acting elements (Wen et al., 1989).

9. THE P-450 SUPPORT GENES

P-450s alone are enzymatically inactive in the absence of an electron transport enzyme. Microsomal P-450s use the flavoprotein NADPH-P-450-oxidoreductase (OR) as a conduit to collected electrons from NADPH. Only a single form of OR exists and this enzyme is able to interact with all microsomal type P-450s. The OR is an intrinsic membrane protein, tightly bound to the endoplasmic reticulum lipid bilayer, via a hydrophobic amino-terminal peptide. The enzyme can, in fact, be solubilized in an active form by trypsin treatment of microsomes. The trypsin labile site resides between the membrane-binding domain and the catalytic domain of OR (Black and Coon, 1986). Although solubilized OR can effectively reduce cytochrome c, only the intact membrane- binding enzyme is able to reduce P-450. OR contains one molecule of flavine mono-

20 F.J. GONZALEZ

nucleotide (FMN) and one molecule of flavin dinucleotide (FAD) as noncovalently bound cofactors.

The OR cDNA was isolated from rat (Gonzalez and Kasper, 1982), and its complete cDNA-deduced protein sequence was determined (Porter and Kasper, 1985; Murakami et al., 1986). The OR sequence was also determined in other species, including yeast (Yabusaki et al., 1988), pig (Haniu et al., 1986a), rabbit (Katagiri et al., 1986), trout (Urenjak et al., 1987) and man (Yamano et al., 1989a). The pig and trout sequences were determined by direct protein sequencing. Putative binding domains for FAD, F M N and NADP H were originally postulated based on comparisons with the sequences of other enzymes that use these cofactors (Haniu et al., 1986a; Katagiri et al., 1986; Porter and Kasper, 1986). These binding sites were maintained at a high level of amino acid similarity when the yeast sequence was analyzed, even though the overall amino acid resemblance of the yeast enzyme to the other four enzymes was only about 33 % (Yabusaki et al., 1988).

The OR cDNA has been expressed in both bacteria (Porter et al., 1987) and yeast (Murakami et al., 1986). The enzyme expressed in bacteria was able to transfer electrons to purified P-450 in a reconstituted system. Likewise, the OR produced in yeast was fully active. A mammalian monooxygenase system was reconstituted in yeast through simultaneous expression of P-450 and OR (Murakami et al., 1986). Furthermore, when the OR cDNA was directly connected to the open reading frame of the P-450 cDNA, a self-catalytic monooxygenase enzyme was formed (Murakami et al., 1987). This genetically engineered protein has similar properties to the self-sufficient natural bacterial BM-3 P-450 (Wen and Fulco, 1987).

The OR gene is coordinately regulated with several P-450 genes. Administration of phenobarbital results in a transcriptional activation of the OR gene and the P-450IIBI/IIB2 genes (Hardwick et al., 1983b). The P-450IIIAI and IIIA2 genes are also stimulated by phenobarbital (Gonzalez et al., 1986b). The OR gene is not, however, significantly activated by other P-450 inducers such as MC or dexamethasone.

The support genes associated with the mitochondrial XIA1 and XIB1 P-450s are adrenodoxin and adrenodoxin reductase. The iron-sulfur protein adrenodoxin transfers electrons from adrenodoxin reductase directly to the mitochondrial P-450s. These proteins are expressed in adrenal cortex, testicular Leydig cells, ovary, placenta, and specific regions of brain. The adrenodoxin reductase cDNA was isolated from cow (Hanukoglu et al., 1987) and humans (Solish et al., 1988). These probes were used to determine the presence of a single gene coding for the enzyme in both cow and man, similar to the microsomal OR. The adrenodoxin reductase gene was localized to human chromosome 17 (Solish et al., 1988).

The adrenodoxin cDNA has also been cloned from man (Picado-Leonard et al., 1988) and cow (Okamura et al., 1987) and sequenced. These studies confirmed that the protein is processed from a 19-kilodalton precursor upon transport into the mitochondria. Interestingly, adrenodoxin is produced from a single gene, yet several mRNAs are found (Okamura et al., 1987; Picado-Leonard et al., 1988). Two mRNAs are produced from alternate promoters; one of these transcripts yields an mRNA incapable of producing a functional protein (Kagimoto et al., 1988). The second intron of the same gene has a functional promoter that transcribes a variable mRNA that produces adrenodoxin. These normal and variant mRNAs are present in a ratio of 9:1 in cow adrenocortical cells. It has yet to be determined whether this unusual gene is found only in cows or is present in other species. This may give a clue to the relevance of the unusual mRNA. Multiple polyadenylation sites have, however, been observed in cow (Okamura et al., 1987) and man (Picado-Leonard et al., 1988).

10. S UMMARY OF INTERSPECIES DIFFERENCES IN P-450 EXPRESSION

It should now be apparent that there are tremendous interspecies variations in the expression of P-450 genes. This variability is due to both qualitative differences in P-450s and quantitative differences in levels of expression. For instance, recent gene duplications


that are species specific have resulted in species-specific P-450 genes. Among the gene pairs that have recently formed through gene duplications are the IIA1 and IIA2, IIB1 and IIB2, and IIIA1 and IIIA2 genes. These gene pairs were formed about 40, 12 and 40 million years ago, respectively. Since mammalian radiation occurred about 75-85 million years ago, forming the rabbit, rodent and human species lines, rabbits and humans would not be expected to have orthologous counterparts to these rat genes. However, given that the rat and mouse lineages diverged about 20 million years ago (Shoshani et al., 1985), we might expect the mouse to have counterparts to the IIA1/IIA2 and IIIA1/IIIA2 genes.

The fact that a gene exists in two species does not, however, guarantee that it is regulated in a similar manner or that it has the same substrate specificity in both species. A simple examination of the liD locus in mouse and rat illustrates these points. The amino acid similarities between four rat IID P-450s, deduced from nucleotide sequences, indicates that the liD subfamily was formed around 100 million years ago (Matsunaga et al., 1989). Another duplication occurred about 15-20 million years ago, forming a fifth rat gene. Mice, therefore, should possess at least four genes and perhaps five genes. Indeed, multiple genes in the liD locus were found in mice (Wong et al., 1987). One of these mouse genes has been extensively characterized (Wong et al., 1987). The mouse IID P-450 is expressed only in males and hydroxylates testosterone at the 16ct position. In contrast, none of the rat P-450 genes is specifically expressed in males and, furthermore, no liD P-450, either purified or expressed through cDNA, has been found to hydroxylate testosterone. The mouse and rat, therefore, appear to have evolved different regulatory pathways for liD gene expression and different enzymatic activities for the IID P-450 proteins. These species-specific differences could have been generated by conversion events among the IID subfamily genes. The environmental constraints influencing the evolution and selection of different regulatory pathways and enzymatic activities are unknown, but it is reasonable to suspect that diet played a role.

11. POLYMORPHIC EXPRESSION OF RODENT P-450 GENES

Polymorphic expression of P-450s can be caused by either a defect in the regulatory factors that govern transcription of the genes or direct mutations of the P-450 genes. Several P-450 expression polymorphisms have been detected in rats and mice.

The most well-studied rodent polymorphism is the mouse A h (aryl hydrocarbon) locus defect. A polymorphism in expression of inducible benzo[a]pyrene hydroxylase activity was detected by screening a large number of mouse strains (Nebert and Gelboin, 1969). In these experiments, mice strains were uncovered that were refractive to induction by MC. Later studies in which responsive and nonresponsive mice were bred led to the identification of the A h locus (Gielen et al., 1972; Thomas et al., 1972; Thomas and Hutton, 1973). The defect in the nonresponsive mice was found to be associated with the TCDD receptor (Poland and Glover, 1975). The receptor in these mice apparently has a decreased affinity for the usual polycyclic aromatic hydrocarbon ligands; however, nonresponsive mice can become responsive to high concentrations of the potent inducer TCDD (Poland et al., 1974). Other defective mouse strains have been found, and these too have a modified receptor. In no case has a nonresponse to polycyclic aromatic hydrocarbons in vivo been shown to be associated with a defective IA1 gene. However, variant mouse hepatoma cell lines that possess mutant structural genes for IA1 have been selected (Hankinson et al., 1985; S. Kimura et al., 1987b). These findings imply that IA1 may have a crucial physiological role in the intact animal. Further support for the importance of IA1 is its ubiquitous nature; the gene and/or its inducible enzymatic activity has been detected in numerous species.

In contrast to the omnipresence of IAI, enzymes in the P-450II family appear to be highly polymorphic in rodents. Two-dimensional electrophoretic analysis established the presence of multiple alleles for the IIBI and IIB2 proteins (Rampersaud and Walz, 1983). A rat polymorphism for expression of a P-450IIB gene was also uncovered using

22 F. J, GONZALEZ

this technique (Rampersaud and Walz, 1987). Screening of 20 rat strains revealed the presence of a IIB2 expressionless rat strain, Marshall 520/N. The mechanism by which the gene was extinguished, however, was not established in this study. Other investigators found a Japanese colony of Sprague-Dawley rats that did not express phenobarbital- induced IIB2 (Hashimoto er al., 1988). However, phenobarbital readily activated the IIB1 gene, suggesting that the mutation was in the IIB2 structural gene. Breeding studies established that this defect is a recessive trait and the result of a mutation in a single gene. The homozygous mutant rat produces IIB2 mRNA, but its level is markedly diminished. Examination of the DNA sequence surrounding the promoter region of the mutant gene did not reveal any obvious difference from the wild-type gene. It is possible that a mutation exists in a regulatory region further upstream from the immediate transcription start site of the IIB2 gene (Hashimoto et al., 1988). Unfortunately, little information is available on the cis-acting elements responsible for control of transcription of the lib genes.

A polymorphism was also detected in the mouse liB gene subfamily (Noshiro et al., 1986). A testosterone 16~-hydroxylase was found to be repressed in the 129/J female compared to 10 other mouse strains. The enzyme is not significantly expressed in males of any strain. This trait was inherited as an autosomal recessive and results from a lack of 16e mRNA expression. There were no differences in the phenobarbital-induced 16c~ expression among the various strains.

An interesting rat polymorphism was also detected for the expression of P-450g (Bandiera et al., 1986; McClellan-Green et al., 1987). This enzyme was found to be expressed only in males (Ryan et al., 1984) and is a member of the P-450IIC gene subfamily. P-450g was found to be expressed in Sprague-Dawley and Long-Evans rats only when males reached puberty (Bandiera et al., 1986; McClellan-Green et al., 1987). The enzyme was not, however, appreciably expressed in Fisher rats at any stage of development (McClellan-Green et al., 1987). Two populations of adult male Fisher and Long-Evans rats were observed; one group expressed high levels of the protein and the other expressed low levels. Both groups had the same amount of mRNA, suggesting an impairment of a posttranscriptional process in the low level expressors (McClellan-Green et al., 1987). The precise mechanism governing this bimodal expression of P-450g is still unclear.

A urine phenotyping test uncovered a deficiency in the metabolism of the antihyper- tensive drug debrisoquine in the DA rat strain (A1-Dabbagh et al., 1981). This rat was proposed as a model for the human polymorphism in debrisoquine metabolism (Mahgoub et al., 1977). Enzyme kinetic studies on debrisoquine metabolism using microsomes revealed that multiple enzymes contribute to this metabolic pathway in rat (Kahn et al., 1985). This was also supported by studies on purified rat P-450s, in which at least two enzymes were shown to hydroxylate debrisoquine at the fourth position (Larrey et al., 1984). However, rat P-450s, designated dbl (Gonzalez et al., 1987) and UT-H (Larrey et al., 1984), were purified which have high catalytic activities for debrisoquine 4-hydroxylase and activities for other related compounds. Using antibodies to these various preparations, conflicting results were obtained concerning the quantity of the putative dbl/UT-H protein in DA rat liver; Larrey et al. (1984) found lower levels of UT-H, whereas Gonzalez et al. (1987) found similar levels of dbl, when female DA rats were compared to Sprague Dawley and Lewis rats. dbl mRNA were also found not to vary between these rat strains (Gonzalez et al., 1987).

Caution must be observed when interpreting the results of studies in which antibodies and cDNA probes are used to quantitate levels of protein and mRNA. For instance, rats clearly have more than one immunologically related IID P-450. Two proteins and cDNAs, dbl and db2, were isolated and characterized from rats (Gonzalez et al., 1987). Later cloning studies established the presence of five genes in the rat IID subfamily; four of these genes are transcribed in liver and kidney (Matsunaga et al., 1989). The cDNA-deduced amino acid sequences for these four enzymes revealed a significant degree of similarity. Therefore, antibodies to liD1 would be expected to cross-react with at least


three other proteins on immunoblots. In fact, two P-450s, IID1 and IID5, exhibit 95% amino acid similarity (Matsunaga et al., 1989). These results suggest that earlier mRNA analysis using the dbl probe may have been detecting IID1 and IID5 mRNAs. Further studies using oligonucleotide probes revealed that the active debrisoquine 4-hydroxylase (IID I) gene was not expressed in either male or female DA rats; only the very similar IID5 gene is expressed in the DA rat (Matsunaga et al., 1989). The low levels of activity observed in DA rats were probably related to other P-450s. Humans appear to have only a single enzyme with a low K m debrisoquine 4-hydroxylase activity. These results would support the contention that this rat strain is not a good model for the human 'poor metabolizer' phenotype.

12. SUMMARY AND FUTURE PROSPECTS

12.1. REGULATION OF P-450 GENES

A large amount of information has accumulated concerning the mechanism by which the IA1 gene is regulated. In summary, the gene is controlled by the TCDD receptor, which binds to upstream DNA sequences. The gene is also controlled by an autoregulatory loop mediated by the enzymatic activity of the IAI P-450 (Gonzalez and Nebert, 1985). In addition, a repressor has been implicated in the control of this gene. Further studies are needed to define the repressor and the autoregulatory loop. The receptor must also be either fully purified or cloned, in order to define the nature of the signal that augments IA1 transcription. Unfortunately, regulation of IA2 is still a 'black box', and the development of responsive cell culture lines or primary hepatocytes will be necessary to study this gene. There is little information concerning the sequence and receptors required for regulation of the phenobarbital-, clofibrate- and steroid-inducible P-450s. Finally, the control of developmentally regulated and sex-specific P-450 genes must be defined, especially the role of pituitary hormones in mediating levels of gene activity.

12.2. PRODUCTION OF P-450S IN CELL CULTURE USING cDNA-DIRECTED EXPRESSION

One of the most critical concerns of pharmacology is to define the effect and metabolic consequences of drugs. As discussed in the previous sections of this chapter, there is tremendous variability between individual species in terms of their P-450 gene composition and expression. Humans, for instance, appear to have their own unique set of P-450 genes; this is particularly evident in the P-450II family. It has been well established, however, that P-450s have significant overlapping specificities toward many substrates. Some compounds are only metabolized by a single form of P-450. In this latter instance, substantial intra- species differences have been demonstrated. In any case, extreme caution must be observed when attempting to extrapolate drug metabolism data from rodents to man.

Human systems to study drug metabolism must be developed. To do so, we must have a thorough understanding of the human P-450s and other enzymes involved in drug clearance. The use of human liver specimens has allowed the purification and study of human P-450s (Distlerath and Guengerich, 1987). It is likely, because of the lack of a consistent supply of human liver tissue and the marked heterogeneity of human specimens, that only the most abundant human P-450s will ever be purified. The use of antibodies and cDNA probes derived from rats will, however, allow an increasing number of less abundant enzymes to be studied. The human P-450 cDNAs can be isolated, inserted into eukaryotic expression vectors and used to express active enzyme in cell culture (Aoyama et al., 1989b). Enzymatic activities and substrate specificities can be analyzed for each form of human P-450. Furthermore, the human enzymes can be assessed for reactivities toward carcinogens, in particular, their abilities to activate carcinogens to electrophilic mutagens.

Several systems have been developed to express P-450s from their cDNAs. Among the most popular are the yeast expression system and the monkey kidney cell (COS cell)-based

24 F.J. GONZALEZ

system. The yeast system has been used to express rat IAI (Oeda et al., 1985), IA2 (Sakaki et al., 1987; Shimizu et al., 1988), IIAI (Nagata et al., 1987), XXIA2 (Higashi et al., 1988b) and IVAI (Hardwick et al., 1987). Rabbit pHP3 has also been expressed in yeast. The advantage of the yeast system is that it is simple, rapid, inexpensive and produces a stable population of cells harboring a single P-450 in the absence of an endogenous background. Tremendous variability exists in the levels of expression obtained by various laboratories, and some mammalian P-450s cannot be stably expressed in yeast. The COS cell system has been used to express P-450s in the XVII and XIB1 gene families (Zuber et al., 1986b, 1988). This system has also been used to express human IID1 (Gonzalez et al., 1988b) and PCNI (Gonzalez et al., 1987) genes. The advantages of this system are that it is a higher eukaryote cell vector and it is simple and rapid. The disadvantage is the low level of P-450 production and the fact that its expression is restricted to COS cells. In addition, the system is transient, and the cells containing the expressed enzyme cannot be propagated. For each

r - . . -Smal ,TG

TKL~~sC11 ~ TKR / Ampr~"-''~ ~ .

WILD TYPE ~'~ VECTOR ~VACCINIAVIRUS(W' TKL~

~~HoAm / TRANsFEcTION ologous Recombination

(inactivation of W TK gene)

r

~ TKR

Budr, TK- cells and /J-gaP Selection

Budr, TK" cells and ~J-gal ÷ Recombinant Virus

P4~ '~ infect cells

) 2., days b P450 production

FIG. 5. Scheme for constructing vaccinia virus recombinants containing P-450 cDNAs. See the text for details.


experiment, DNA must be transfected into the COS cells, a process that is cumbersome and tedious.

One of the most promising systems is the vaccinia virus expression system (Smith and Moss, 1984; Chakrabarti et aL, 1985). This system allows the production of large quantities of fully active enzyme in a variety of mammalian cells. Like the COS system, the vaccinia system is transient; cells infected with recombinant virus perish within several days of infection. The infection process is quite simple, however, compared to DNA transfection using the COS cell system. The major disadvantage of the vaccinia system is that it is time consuming to construct recombinant viruses. Therefore, site-directed mutagenesis studies in which large numbers of cDNAs must be assayed would not be practical. In this case, the newer vaccinia T7 system (Fuerst et aL, 1986) would be useful, although levels of expression in this system are quite low compared to the standard system. The procedure for constructing vaccinia recombinants is illustrated in Fig. 5. Briefly, the cDNA containing a full coding region of the P-450 is placed into the insertion vector pSC11 in the correct orientation, relative to the P75 vaccinia promoter. This plasmid is then introduced into CV-I cells together with the wild-type vaccinia virus. Spontaneous recombination between the virus and plasmid DNA occurs by virtue of the homologous thymidine kinase (TK) gene sequences located on both virus and plasmid. The plasmid integrates into and inactivates the viral TK gene. Viruses that have an inactivated TK gene will survive on TK- cells in the presence of the suicide substrate 5-bromodeoxyuridine. The recombinant viruses can also be selected based on their production of blue color transferred from the plasmid-born Lac Z gene (producing fl-galactosidase). Finally, recombinant viruses producing P-450 are defined by their production of P-450.

12.3 HUMAN VARIABILITY IN DRUG AND CARCINOGEN METABOLISM

Over the past l0 years, at least two well-defined polymorphisms in drug oxidation have been discovered. The clinical aspects of the debrisoquine/sparteine (Idle and Smith, 1979; Eichelbaum, 1986) and mephenytoin (Inaba et aL, 1984, 1986; Kupfer and Preisig, 1984) polymorphisms have been extensively studied. The number of individuals who lack the ability to metabolize debrisoquine and mephenytoin ranges from 5% to 10% and from 1% to 5%, respectively, in Caucasians. In Oriental subjects, however, the percentage of individuals who cannot metabolize mephenytoin is as high as 18%, whereas no deficient debrisoquine metabolizers have been identified (Nakamura et aL, 1985). These studies suggest that significant ethnic differences exist in drug metabolism.

The human debrisoquine 4-hydroxylase P-450 (IIDl) has been isolated (Distlerath et al., 1985; Gut et al., 1986a), and its cDNA has been cloned and sequenced (Gonzalez et al., 1988b,c). The human mephenytoin hydroxylase (IIC9) has also been isolated (Shimada et al., 1986; Gut et aL, 1986b) and its cDNA cloned and sequenced (Umbenhauer et aL, 1987; Kimura et aL, 1987a). Isolation of the IID l cDNA has led to the complete characterization of the genetic defect responsible from the debrisoquine/sparteine polymorphism and to the direct phenotyping of mutant IID l alleles by analysis of lymphocyte DNA (Gonzalez et al., 1988b; Skoda et al., 1988).

It is entirely possible that other P-450 genes are polymorphically expressed in humans. Studies addressing this question should be possible with the availability of cloned human cDNAs. The study of human P-450 polymorphisms can be addressed at two levels. First, the levels of P-450s and/or important drug and carcinogen oxidation reactions can be assessed using human liver specimens maintained in liver banks. This can be achieved with antibodies against human and rodent enzymes. If liver specimens which lack a particular P-450 are identified, the mutant genes in the same liver can be identified and characterized using cDNA probes.

REFERENCES

ADESNIK, M. and ATCHISON, M. 0986) Genes for cytochrome P-450 and their regulation. CRC Crit. Rer. Biochem. 19:247 305.

26 F.J. GONZALEZ

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JPT 4 5 / I ~

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