Characterization F. PETER
of human
cytochrome
P450
enzymes
GUENGERICW
Department of Biochemistry and Center Tennessee 3 7232-0146, USA
in Molecular
ABSTRACT Many biochemical approaches have been applied to the human cytochrome P450 enzymes, and more than 20 different gene products have been characterized with regard to their properties and catalytic specifIcities. The complement of the various cytochrome P450 enzymes in a given individual varies markedly, and dramatic differences may be seen in drug metabolism, pharmacological response, and susceptibility to toxic effects. An understanding of the nature of the individual cytochrome P450 enzymes and their regulation should be useful in determining the most suitable animal models, ascertaining risk from chemicals, and in avoiding undesirable drug interactions. Guengerich, F, P. Characterization of human cytochrome P450 enzymes. FASEBJ. 6: 745-748; 1992.
Toxicology,
Vanderbilt
P450
.
drugs
carcinogens
.
School of Medicine,
P450
2C
P450s
were
and
3A gene
purified
families
from
human
(7-9).
Subsequently,
liver
on
EVER
mans there metabolism these
SINCE DRUGS HAVE been administered to huhas been an appreciation of the role of in influencing the pharmacological effects of
agents.
Oxidation
reactions
are
dominant
in
the
metabolism of drugs, and the cytochrome P4502 mixedfunction oxidase system was recognized early as the chief
contributor. By the 1950s variations in rates of drug metabolism were recognized in humans (1). Subsequently, the variations were attributed in part to the presence of different P450 enzymes and the inducibility of some of these (2). Some undesirable drug interactions were attributed to the untoward effects of P450s (3, 4). Studies with experimental animal models have led the way to an understanding information about human orthologs.
knowledge realization
of the P450 these models However,
enzymes, and much has been applicable with the acquisition
about the experimental animal systems that many aspects could be understood
of the to the of
came the only by
studying humans. Over the years, biochemical measurements had been made on human liver surgical samples (for review see ref 5). Much of this information involved levels of
P450, rates
cytochrome plus
rates
b5, and of some
NADPH-cytochrome
typical
P450-dependent
c reduction catalytic ac-
tivities. Kaschnitz and Coon (6) were able to fractionate human liver microsomes into three components enriched in P450, NADPH-P450 reductase, and phospholipid, and demonstrate catalytic activities when these fractions were recombined. At about this time Conney (2) and associates carried out in vivo studies involving enzyme induction which have formed the basis for some of our current understanding of human P450 function.
PURIFICATION
OF
HUMAN
P450s
AND
enzymes
earnest in the late 1970s. The 0892-6638/92/0006-745/$01
from
human
firstP450s
.50. © FASEB
some of partic-
their animal
orthologs
(11). Subse-
with
quently, cDNAs tion by antibodies
were isolated on the basis of their recogniraised against human and animal P450s
(from expression libraries) and cDNAs for animal P450s (12). These isolated DNA probes allowed genomic clones to be isolated and characterized. Today these human P450s are grouped into families on the basis of their primary sequences when they become available (13). The known P450s are listed
in Table
1. In most cases they have been work,
characterized
particularly through
on the
the work
of Gon-
some genomic sequences have been isolated from acid sequence
is un-
available (P4SOMp.1 and MP-2 are proteins in the P450 2C family that appear to differ slightly from the known sequences) (16). Searches for posttranslational modifications (other than heme incorporation) in human P450s have been essentially nonexistent. Two of the P450s are found in mitochondria instead of the endoplasmic reticulum but there may be more
(13,
17).
CATALYTIC
ACTIVITES
OF
HUMAN
P450s
There are many approaches useful in determining which P450s make major contributions to particular catalytic activities.They include the use of selective inhibitors and antibodies in crude (microsomal) preparations, the correlation of individual catalytic activities and P450s in different preparations, and the measurement of catalytic activities with
purified enzymes and in eDNA-derived expression systems. The advantages and disadvantages of each of these techniques have been discussed elsewhere (15). Suffice it to say that the most reliable conclusions are reached when a battery of these approaches is applied. An abbreviated synopsis the catalytic specificities is presented in Table 2. A few major points will be made specificity.First, this matter of selectivity
regarding
of
catalytic
is very important and can influence the probability that an individual will respond to a chemical in a particular way. The P450 enzymes that are devoted to particular steroid hydroxylations tend to have much more limited specificities (e.g., P450s 7, hAl, 11BI, 17, 19, and 21A2). Further, there is probably less variation in the levels of these enzymes among different humans because
of their critical roles in processing
endogenous
cDNA
CLONING Efforts to purify P450
basis
cross-reactivity
liver,and in a few cases the exact amino ALMOST
the
ular catalytic activities (10). Another approach has involved purification of the enzymes on the basis of immunochemical
zalez and his associates (12), and are available. Some of the proteins
steroids
Nashville,
isolated without regard to any catalytic activities, and these first purified enzymes can now be recognized to be in the
basis of eDNA Key Words: cytochrome human studies
University
liver began
in
to be purified were
‘Address correspondence to: Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
2Abbreviation:
P450, cytochrome
P450. 745
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TABLE
1. Characterized human P450s P450
Level of characterization
Siteof expression
Primarily extrahepatic Liver Liver (Apparently not expressed) Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver, several extrahepatic
1. 2 3. 4.
lAl IA! 2A6 2A7
eDNA, gene Protein, eDNA, Protein, eDNA eDNA
5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
2B6 2C8 2C9 2C10 2C17 2C18 2C19
2D6 2E1
cDNA Protein, (Protein) (Protein) eDNA eDNA eDNA Protein Protein Protein, Protein,
16. 17. 18.
2F1 3A3 3A4
eDNA (Protein) Protein,
eDNA eDNA
Lung Intestine (liver?) Liver, several extrahepatic
19.
3A5
Protein,
eDNA
Liver,
P4SOMP.l
P4SOMp.2
gene
eDNA eDNA eDNA
gene gene
cDNA, eDNA,
tissues
tissues
20. 21.
3A7 4A9
Protein, eDNA cDNA
22. 23. 24.
4B1 7 1 1A1
eDNA eDNA eDNA,
gene
25.
ilBi
cDNA,
gene
several
extrahepatic
tissues Liver (fetal)
Lung Liver Steroidogenic tissues (mitochondria) Steroidogenic tissues
(mitochondria) 26. 11B2 Steroidogenic tissues 27. 17 eDNA, gene tissues Protein, eDNA, gene Steroidogenic 28. 19 Steroidogenic tissues eDNA, gene 29. 21A2 30. For 27 reference, see refs 13-15. Where information is lacking, Liver Protein 31. P450,, the references
are
TABLE
anecdotal
(13).
steroids. Indeed, considerable problems are encountered in those individuals who have genetic P450 21A2 deficiencies (18). Some of the liver microsomal P450s, on the other hand, can tolerate a great deal of diversity in the substrates they accommodate. P450 2E1 and particularly P450 3A4 are unique in this regard. P450 2El oxidizes a variety of lower molecular weight substrates that have few chemical characteristics in common (19). P450 3A4 accommodates a great variety of substrates, even as large as erythromycin and cyclosporin (20). However, its active site should not be viewed as a loose pocket because of the high regioselectivity seen in certain oxidations (e.g., steroids). P450 3A5, which is highly related to P450 3A4, catalyzes some but not all of the same reactions (21). Understanding the structure-activity relationships that underlie the catalytic specificity in these proteins will be a challenging task. Ultimately, physical techniques will need to be brought to bear on the problem. However, it should be kept in mind that recent work with catalytic antibodies has provided elegant evidence for the hypothesis that what determines catalytic specificity is not just the complementarity of the protein to the substrate but the complementarity to the transition state. That is, to quote Kraut (22), catalysis is specificity. Good examples in the P450 field are lacking, but this is an area for future consideration. In many cases there is a conservation of catalytic specificity among the human P450s and their animal orthologs. For instance, this appears to be the general case in the P450 1A1, 1A2, and 2E1 protein families (12, 15). However, apparent discrepancies are sometimes seen because another protein present in the animal tissue may have an unusually high activity toward a particular substrate [e.g., nifedipine (23)]. However, even among very closely related proteins there may be considerable catalytic differences. For instance, rat P450 2Al has testosterone 7a-hydroxylation as a markedly characteristic activity (24) whereas the very similar human P450 2A6 (69% identity) is devoid of this activity (25); both are efficient coumarin 7-hydroxylases. Therefore the catalytic specificity in this protein family seems to be unusually sensitive to small changes, and indeed Lindberg and Negishi (26) have shown that a single change in the sequence of a mouse P450 2A family member can alter its catalytic specificity totally. It also appears that some gene families
2. Some typical P450 substrates and inhibitors Substrates
P450 P450 P450 P450 P450 P450
1AI 1A2 2A6 2B6 2C8, 9, 10 2C18
4S#{176}Mp.i,
Polycyclic
hydrocarbons
Arylamines,
phenacetin,
Coumarin,
Mephenytoin,
MP-2
7,8-Benzoflavone, arylamides
ellipticine
7,8-Benzoflavone
7-ethoxycoumarin
Tolbutamide, Mephenytoin?
P450 2D6
Inhibitors
Sulfaphenazole
hexobarbital hexobarbital
Debrisoquine
and many
related
amines
Quinidine,
some other
alkaloids
P450 2El
Ethanol,
N-nitrosodialkylamines,
vinyl monomers,
Dihydropyridines,
P450 3A3, 4, 5, 7
Disulfiram,
chlorzoxazone
several
steroids,
4-methylpyrazole
Gestodene,
troleandomyein,
naringenin,
cimetidine
cyclosporin, lovastatin, aflatoxins,many
other drugs Cholesterol Cholesterol I 1-Deoxycortisol
P450 7 P450 11AI P450 11BI P450 17 P450 19 P450 21A2 “For referen 746
Vol. 6
Pregnenolone
Testosterone, androstenedione l7-Hydroxyprogesterone Ce,
see
January
refs
Several
drugs
and ligands
14, 15.
1992
The FASEB Journal
GUENGERICH
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such as human P450 2C are extremely complex and that catalytic specificity is not well conserved among the members (16).
ENZYME INDUCTION, STIMULATION
INHIBITION,
AND
These three phenomena are all considered together because of the nature of the consequences, even though they are mechanistically quite distinct. Several human P450s are known to be inducible by drugs, usually entities other than the substrates (Table 3). This induction can be an order of magnitude or more. Further, alterations of P450 levels can also be the result of dietary influences and smoking (2). It has been possible to demonstrate the induction of human P450 enzymes in cell culture (27). However, the mechanisms underlying the regulation of P450 genes are complex and will probably be best elucidated in models derived from experimental animals. The suppression of transcription of P450s has been demonstrated in animals, but to date the issue has not been addressed in humans. Some major P450s are not expressed in fetal liver (14, 16, 28). Many inhibitors of P450s are known (Table 2). Some are not particularly selective whereas others are. The significance of these becomes apparent in consideration of drug-drug interactions (vide infra). Further, some P450s such as the aromatase P450 19 are targets in drug therapy (29). Some of the known inhibitors are natural products found in the diet (e.g., naringenin and other flavonoids). The inhibitors may be classified on the basis of their action and include some that act competitively (quinidine, sulfaphenazole), compounds that ligand the heme (cimetidine), compounds whose oxidation products bind the heme (troleandomycin) or adduct the protein (chloramphenicol), and true mechanism-based inactivators (gestodene, disulfiram). Of course, whenever two drugs are administered that compete for the same enzyme site and metabolism is not limited by blood flow, then one can be considered an inhibitor of the other. Direct stimulation of P450 catalytic activities has been demonstrated in human liver microsomal preparations (2). This stimulation may be important for several reasons. 1) In vivo stimulation by such chemicals has been documented in animal models. 2) Many chemicals which show this effect are present in foods. 3) Such stimulation has been demonstrated in human liver microsomes in the activation of carcinogens such as the potent hepatocarcinogen aflatoxin B, (30). 4) Relatively low concentrations of some of the flavones are required to elicit the response. Huang et al. (31) found that the flavones could lower the apparent Km of P450 for NADPH P450 reductase in reconstituted enzyme systems. Schwab et al. (32) found that the Km values of rabbit and human liver
TABLE
3. lnducers of human P450 enzymes”
P450
1A1
P450
lA2
Cigarette Cigarette cruciferous
P450 2C8, 9, 10
smoking smoking,
charbroiled
vegetables barbiturates
Rifampicin, Rifampicin
P450 2E1
Ethanol,
P450 3A4
Rifampicin, barbiturates, troleandomycin
HUMAN
P450s
Q,);
CONCERNS
ABOUT
DRUG
INTERACTIONS
With all of the possibilities involving induction, inhibition, and stimulation, and the many forms of P450, it should not be surprising that adverse drug interactions might result. This is a matter of particular concern in the elderly, where many prescriptions may be encountered simultaneously. Adverse
reactions (and even death) have been experienced
in individuals genetically deficient in P450 2D6 who use certain drugs (35). When phenacetin cannot be oxidized by P450 1A2 in the normal O-deethylation pathway, N-deacetylation can occur and the product phenetidine can lead to methemoglobinemia (36). Induction of P450 3A4 can lead to enhanced oxidation of the oral contraceptive 17cr-ethynylestradiol, and menstruation and unanticipated pregnancies have been experienced in women using rifampicin or barbiturates (4, 37). In a similar manner, the up- or downregulation of P450 3A4 by drugs can influence the metabolism of the immunosuppressive agent cyclosporin and its effects. Prescreening of patients with a noninvasive assay (for enzyme activity) has the potential to facilitate rational therapy (38). Recently the consumption of grapefruit juice has been shown to have a dramatically marked effect in reducing the clearance of dihydropyridine drugs-this effect may be explained by the inhibition of P450 3A4 by the flavonoid naringenin (39). These are just a few examples of the possible complications
terms
that may
ultimately be understood
in
of P450s.
FUTURE
STUDIES
INVOLVING
HUMAN
P4SOS
In the future more basic information about the human P450 enzymes may be expected to become available. Ultimately, a desirable goal is the prediction of catalytic specificity of human P450s toward new drugs and chemicals from structural models. The time required until such an approach becomes feasible cannot be predicted. However, there are noninvasive assays that can be used to estimate how much of a particular P450 is present in an individual (15, 38). Further, in the cases of P450 2D6 and P450 21A2 there is extensive knowledge regarding the genetic analysis of deficiencies. Knowledge about P450s will be of use to physicians and pharmacists in avoiding undesirable drug interactions. Further, the comparison of animal and human P450s may lead
P45OMP.,, MP2
“See refs14, 15.
microsomes for the oxidations of estradiol and progesterone were lowered in the presence of the chemical, and binding studies with a rabbit P450 and a model ligand are consistent with this view (33). The generality of these explanations is not yet clear. What does seem established is that allosteric interactions must be involved, with a separate binding site on the enzyme for the stimulating chemical. We have recently demonstrated that yeast recombinant (human) P450 3A4 oxidizes aflatoxin B1 to both the 8,9-oxide and the 3cr-alcohol (aflatoxin 7,8-benzoflavone stimulates the former reaction and inhibits the latter, thus shifting the metabolism from detoxication to activation (34).
isoniazid(?)
meat,
to rational choices of experimental animal models in the process of safety assessment of drug candidates. Such knowledge may also be useful in improving methods of risk assessment - for instance, knowledge regarding rates and the extent of variation in rates of drug oxidation by humans can be inserted into physiologically based pharmacokinetic models when a critical step has been identified in animal models (40).
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This work was supported, in part, by U.S. Public grants CA 44353 and ES 00267.
Health
Service
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748 Vol. 6 January 1992 The FASEBJournal GUENGERICH www.fasebj.org by Kaohsiung Medical University Library (163.15.154.53) on September 12, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumbe