Toxicology and Applied Pharmacology 207 (2005) S52 S56 www.elsevier.

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Review

The human genome project and novel aspects of cytochrome P450 research
Magnus Ingelman-Sundberg*
Division of Molecular Toxicology, IMM, Karolinska Institutet, SE-171 77 Stockholm, Sweden Received 16 August 2004; revised 3 January 2005; accepted 3 January 2005 Available online 1 July 2005

Abstract Currently, 57 active cytochrome P 450 (CYP) genes and 58 pseudogenes are known to be present in the human genome. Among the genes discovered by initiatives in the human genome project are CYP2R1, CYP2W1, CYP2S1, CYP2U1 and CYP3A43, the latter apparently encoding a pseudoenzyme. The function, polymorphism and regulation of these genes are still to be discovered to a great extent. The polymorphism of drug metabolizing CYPs is extensive and influences the outcome of drug therapy causing lack of response or adverse drug reactions. The basis for the differences in the global distribution of the polymorphic variants is inactivating gene mutations and subsequent genetic drift. However, polymorphic alleles carrying multiple active gene copies also exist and are suggested in case of CYP2D6 to be caused by positive selection due to development of alkaloid resistance in North East Africa about 10,000 5000 BC. The knowledge about the CYP genes and their polymorphisms is of fundamental importance for effective drug therapy and for drug development as well as for understanding metabolic activation of carcinogens and other xenobiotics. Here, a short review of the current knowledge is given. D 2005 Elsevier Inc. All rights reserved.
Keywords: Ultrarapid metabolizers; Poor metabolizers; Adverse drug reactions; Genetic polymorphism; Vitamin D; Naphtalene; CYP2S1; CYP2R1; CYP2U1

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . Human cytochrome P S450 genes . . . . . . . . . . . . . Evolution of human P 450s and P 450 variants . . . . . . Functional effects of the interindividual variability in CYP Conclusions . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S52 S53 S53 S55 S55 S55 S55

Introduction The cytochrome P 450 enzymes in families 1 3 are responsible for 70 80% of all phase I dependent metabolism of clinically used drugs (Evans and Relling, 2004, Ingelman-

* Fax: +46 8 337 327. E-mail address: maging@ki.se. 0041-008X/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2005.01.030

Sundberg, 2004) and participate in the metabolism of a huge number of xenobiotic chemicals. The polymorphic forms of P 450s are many times responsible for the development of adverse drug reactions. Phillips et al. (2001) showed that 56% of drugs which are cited in adverse drug reaction (ADR) studies are metabolized by polymorphic Phase 1 enzymes of which 86% are P 450s, whereas only 20% of drugs that are substrates for nonpolymorphic enzymes are in the ADR reports. It has been estimated that: (i) ADRs cost the US

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society about 100 billion US$, (ii) they cause more than 100,000 deaths annually in the US and (iii) up to 7% of all hospital admissions are due to ADRs (see IngelmanSundberg, 2001 for references). Knowledge about the cytochrome P 450 system is fundamental both for drug therapy and for drug development as well as for the understanding of mechanisms behind metabolic activation of potentially toxic and carcinogenic compounds.

Human cytochrome P 450 genes The completion of the sequence of the human genome allowed David Nelson to provide evidence for the presence of 57 different active genes encoding cytochrome P 450 (http://drnelson.utmem.edu/CytochromeP450.html) and 58 pseudogenes (Nelson et al., 2004). The number of active genes is much smaller than seen in rice (323 genes), Thale cress (249 genes) or in the mouse (102 genes) but similar to what is observed in the dog (54 genes). The major P 450 forms that are important in human metabolism of xenobiotics are shown in Table 1. Of particular importance for drug metabolism are CYP2C9, CYP2C19, CYP2D6 and CYP3A4, whereas CYP1A1, CYP1A2, CYP1B1, CYP2E1 and CYP3A4 are the most important isoforms responsible for metabolic activation of precarcinogens. Among the genes in families 2 3, some new ones like CYP2R1, CYP2S1, CYP2W1, CYP2U1 and CYP3A43 have appeared as a consequence of the initiatives of the human genome project. The function and importance of those are still incompletely known. CYP2R1 has been shown to be active in the 25Table 1 Relative importance of polymorphisms of human P 450s Enzyme CYP1A1 CYP1A2 CYP1B1 CYP2A6 CYP2B6 CYP2C8 CYP2C9 CYP2C19 CYP2D6 Fractiona 5% 2% 2% 2 4% 1% 10% 5% 25 30% Substrates Carcinogens Drugs carcinogens Carcinogens Nicotine, Drugs, carcin. Drugs Drugs Drugs Drugs Drugs

hydroxylation of vitamin D2 and D3 (Cheng et al., 2003), but hitherto no xenobiotic substrate for this enzyme has been identified. CYP2S1 is an extrahepatic enzyme expressed in lung and in the intestinal tract (Rylander et al., 2001), inducible by dioxin (Rivera et al., 2002) and able to metabolize trans -retinol (Smith et al., 2003). It is also active in the metabolism of naphthalene (Karlgren et al., 2005), whereas CYP3A43 appears to be a pseudoprotein. Although having very tiny activity as being mutated for expression in bacteria (Domanski et al., 2001), the enzyme is inactive when expressed in several different mammalian systems, and furthermore the mRNA expression is insignificant (Westlind et al., 2001). CYP2U1 is preferentially expressed in the brain (Karlgren et al., 2004) and has been shown to metabolize arachidonic acid (AA) and other long chain fatty acids (Chuang et al., 2004). The physiological function of CYP2U1 remains to be elucidated as well as any role in xenobiotic metabolism.

Evolution of human P 450s and P 450 variants The human cytochrome P 450 genes have evolved during a period of 1.5 billion years. A tremendous adaptation to the new terrestrial environment was taken place about 400 million years ago, including several gene duplications and gene modifications. Furthermore, a similar partly parallel expansion and modification of the P 450 genes have been taken place in the Fugu fish and in Zebra fish, both genomes clarified with respect to their P 450 genes recently by David Nelson (http://drnelson.utmem.edu/fishtree.pdf). Alignment

Major allelic variantsb None CYP1A2*1K Rare defect alleles CYP2A6*4 CYP2A6*9 CYP2C8*3 CYP2C9*2 CYP2C9*3 CYP2C19*2 CYP2C19*3 CYP2D6*2xn CYP2D6*4 CYP2D6*10 CYP2D6*17 CYP2D6*41 Rare CYP3A5*3

Clinical effects of the polymorphism None Less expression and inducibility None Altered nicotine metabolism Relatively high Taxol metabolism Side effects Drug dosage Drug dosage Drug efficacy Non response Side effects Drug dosage Drug dosage Drug dosage Not shown Not shown Not shown

Significance polymorphismc + + + + ++ +++ +++ +++ +++ +++ ++ + ++

CYP2E1 CYP3A4 CYP3A5

a b

2 4% 45 50% <1%

Carcinogens, solvents, drugs Drugs, carcinogens Drugs

Contribution to the cytochrome P 450-dependent metabolism of drugs (see Ingelman-Sundberg (2004)). A description of the alleles is found on the human CYP allele nomenclature committee home page: http://www.imm.ki.se/CYPalleles/. c The significance of the polymorphism is based on the number of reports showing impact of the P 450 polymorphism on the pharmacokinetics of substrates for the enzyme in question. The number of + illustrates the relative importance between the different forms of P 450. The table is partly from IngelmanSundberg (2004) with permission from the author and the publisher.

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of the fish P 450 genes with those of human reveals a generally important conservation of the number and type of active P 450 genes, the Fugu and Zebra fish having 17 and 42 members in the CYP2 family, respectively. Interestingly, both CYP2R1 and CYP2U1 are rather conserved in both fish species as compared to human, indicating a fundamental role of these two cytochrome P 450 forms. Modification of the gene expression profile among different human cytochrome P 450s has taken place the last 100,000 years, creating important interindividual differences in the expression of various forms of xenobiotic metabolizing P 450s. At present, many different functional variants of the major genes have been elucidated and amount to between 5 and 46 for the different P 450s (Table 2). The updated information of the polymorphism of the human cytochrome P 450 genes encoding enzymes active in the biotransformation of xenobiotics (Human CYP allele Nomenclature Web site) is present at our server at Karolinska Institutet (http:// www.imm.ki.se/cypalleles). The basis behind these interethnic differences (see Table 2) is the founder effect, the genetic drift caused by migration of populations of a specific genotype but also because of genetic selection (see Ingelman-Sundberg, 2004). An interesting example is CYP2D6. This enzyme has a very high affinity for alkaloids and is not inducible in a conventional mannerno increased gene expression is seen as a result of inducers. During evolution, it is clear that the CYP2D genes have been preserved in rodents, whereas inactivation has occurred within humans. Thus, mice have 9 active genes in this locus, whereas in humans only a one polymorphic gene (CYP2D6 ) remains. This is probably due to the higher need for alkaloid inactivation in mice as compared to humans, having a more restricted diet. However, there are exceptions in humans. Thus, there appears to have been a selection for alleles

containing multiple active CYP2D6 gene copies particularly in North East Africa where up to 30% of the population carry such alleles (Ingelman-Sundberg, 2004, Ingelman-Sundberg et al., 1999). We have hypothesized that this high frequency in this area is caused by dietary selection: subjects with a higher content of CYP2D6 would be able to have a higher capacity for detoxification of plant toxins (alkaloids) and therefore being able to tolerate an expanded panorama of plants during periods of starvation. Subjects without this capability would starve more severely. In support of this hypothesis, we found that the activity of CYP2D6, as monitored using the in vivo probe debrisoquine, was lower in Ethiopians living in Ethiopia of the same specific CYP2D6 genotype, as compared to Ethiopians in Sweden (Aklillu et al., 2002). This might be due to the content of specific alkaloids in the Ethiopian diet that constitutively would interact with the active site of CYP2D6, giving an inhibition of the metabolism of debrisoquine among Ethiopians in Ethiopia, whereas adaptations to the Swedish food by the Ethiopians in Sweden would not give this extent of inhibition. The specific components in the Ethiopian plants responsible for such an inhibitory effect warrant further investigations, and the proposed selection for genes containing multiple copies of the CYP2D6 gene would be defined as the Alkaloid resistance in humans (Fig. 1). A similar genetic adaptation to the environment has occurred during development of insecticide resistance among Drosophila during the time period 1930 to 1960 (Amichot et al., 2004; Daborn et al., 2002). The adaptation of the flies to insecticides has involved the incorporation of an accord element in the upstream region of Cyp6g1 , thereby increasing its expression by 20- to 40-fold (Fig. 1). Furthermore, three key mutations have occurred in the Cyp6a2 gene, causing three missense mutations shifting the

Table 2 Major polymorphic variants of cytochrome P 450 enzymes and their allele frequencies Enzyme No. of functional variants alleles describeda 16 12 Major variant Mutation Consequence Allele frequencyb Caucas CYP2A6*2 CYP2A6*4 CYP2C9*2 Inactive enzyme No enzyme Reduced affinity for P 450 reductase CYP2C9*3 I359L Altered substr spec CYP2C19*2 Aber splice site Inactive enzyme CYP2C19*3 Stop codon Inactive enzyme CYP2D6*2xn Gene dupl Increased activity CYP2D6*4 Defective splic Inactive enzyme CYP2D6*5 Gene deletion No enzyme CYP2D6*10 P34S, S486T Unstable enzyme CYP2D6*17 T107I, R296C, S486T Red affinity for substrates CYP2E1*2 R76H Less enzyme expressed ? CYP2E1*3 V389I No effects CYP2E1*4 V179I No effects Only very rare variants with no major functional effects L160H Gene deletion R144C 13 1 8 13 79 13 0 15 12 21 46 12 0 0 <1 <1 Orientals 0 15 0 23 23 32 6 10 02 1 6 50 (in Blacks) 1 0 nd

CYP2A6 CYP2C9

CYP2C19 CYP2D6

16 46

CYP2E1

CYP3A4
a

19

The number of variant alleles are given as shown at the (Human CYP allele Nomenclature Web site) (http://www.imm.ki.se/cypalleles) and includes only those with separate allelic numbers. b The allele frequencies are from many different studies provided, partly summarized in Ingelman-Sundberg et al. (1999).

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Fig. 1. Mechanisms for CYP gene modifications due to selection. In insecticide resistant Drosophila strains, a transposome (Accord) in the Cyp6g1 gene has been inserted, and selective mutations in the Cyp6a2 gene have taken place in resistant strains that change the CYP6A2 enzyme from being inactive to active in the metabolism of DDT. In humans, selection of CYP2D6 alleles carrying multiple copies of active CYP2D6 genes has most probably occurred as a pressure event for increasing the capability of alkaloid metabolism. Figure modified from Ingelman-Sundberg (2005).

enzyme from being inactive in the metabolism of insecticides to being very competent in this manner.

Functional effects of the interindividual variability in CYP genes The continuous generation of new variants of the cytochrome P 450 genes and the genetic drift and selection discussed above have created a global situation with large interethnic differences in the distribution of the polymorphic forms of P 450 genes. Based on the occurrence of mutations in these genes, gene deletions and gene duplications, the populations can be divided into poor (PM), intermediate (IM), efficient (EM) or ultrarapid (UM) drug metabolizers. The PMs lack the enzyme in question, the IMs are often heterozygous for a defect gene, the EMs have two functional alleles, whereas the UMs have multiple functional gene copies on a single allele (Ingelman-Sundberg, 2004). Among the human cytochromes P 450s, the CYP2D6 polymorphism has the highest impact on the pharmacokinetics of drugs. This enzyme metabolizes about 25% of all clinically used drugs, and a recent evaluation of Kirchheiner et al. (2004) focusing on psychoactive drugs reveals that dosing is an important issue for about 50% of drugs which are known to be metabolized by CYP2D6. This means that the CYP2D6 genotype is importantly determining the metabolism of 12% of all clinically used drugs. Adverse drug reactions are much more common at ordinary dosing among PMs for CYP2D6, cf. Ingelman-Sundberg, 2004. In addition, subjects being UMs are largely (5- to 10-fold) overrepresented among subjects treated with antidepressants showing no response to the treatment (Kawanishi et al., 2004; Rau et al., 2004). The incidence of PMs in the European population is about 7%.

However, the frequency of UMs is also very high, 5.5% taking into account the high number of subjects carrying duplicated CYP2D6 genes particularly in the Mediterranean area. This means that in total 50 million people living in the Western part of Europe are not properly dosed with drugs which are CYP2D6 substrates. Additional examples where the cytochrome P 450 polymorphism is of severe importance for drug treatment are, e.g. warfarin treatment in relation to the CYP2C9 genotype, the treatment with SSRIs and protein pump inhibitors in relation to the CYP2C19 polymorphism as well as the contribution of CYP2B6 polymorphism to the outcome of treatment with anti-HIV drugs and anti-cancer drugs, cf. Ingelman-Sundberg, 2004. In contrast to the enzymes mentioned, as yet no important functional polymorphism has been shown for CYP3A4, a very conserved enzyme. This is probably due to its important role in the metabolism of steroids and other endogenous compounds as well as its great impact on the metabolism of dietary constituents. By contrast to the rather extensive knowledge and proven importance of the polymorphism on the efficacy of drug treatment, much less has been shown regarding any importance of cytochrome P 450 polymorphism on the metabolic activation of procarcinogens. This is to a great extent dependent on the high conservation of the genes encoding cytochrome P 450 enzymes active in the metabolism of such compounds (cf. Table 1).

Conclusions In conclusion, the recent research in the cytochrome P 450 area has revealed the occurrence of many new extrahepatic enzymes in this enzyme family with some putative importance for the metabolic activation of potentially toxic and carcinogenic chemicals. Furthermore, it is believed that the most important and common forms of human cytochrome P 450 variant enzymes metabolizing drugs have been identified and characterized. We now know much about to what extent such polymorphism should be taken into account as to diminish the amount of adverse drug reactions and improve the therapeutic efficacy of drug treatment.

Acknowledgments The work in the authors laboratory is supported by grants from The Swedish Research Council and from NIH (NIGMS 1-R01 GM60548).

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