Olavi Pelkonen and Hannu Raunio
Department of Pharmacology and Toxicology, University of Oulu, Oulu, Finland
Key words: xenobiotic metabolism, metabolic activation, cytochrome P450, CYP, tissue-specific expression, polymorphism
This paper was prepared as background for the Workshop on Susceptibility to Environmental Hazards convened by the Scientific Group on Methodologies for the Safety Evaluation of Chemicals (SGOMSEC) held 17-22 March 1996 in Espoo, Finland. Manuscript received at EHP 5 November 1996; accepted at EHP 18 November 1996.
The research in the authors' laboratory has been supported by The Academy of Finland (contracts 1051029 and 29456). This article was written within the framework of the Action COST B1.
Address correspondence to Dr. O. Pelkonen, Department of Pharmacology and Toxicology, University of Oulu, FIN-90220 Oulu, Finland. Telephone: (358) 81-5375011. Fax: (358) 81-5375247. E-mail: olavi.pelkonen@oulu.fi
Abbreviations used: AHH, aryl hydrocarbon (benzo[a]pyrene) hydroxylase; CYP, cytochrome P450; MAO-B, monoamine oxidase B; MTPT, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MPP+, 1-methyl-4-phenyl-pyridinium ion; PAH, polycyclic aromatic hydrocarbon; RFLP, restriction fragment length polymorphism; RT-PCR, reverse transcriptase-polymerase chain reaction.
Formation of reactive metabolites is catalyzed principally by the so-called drug- or xenobiotic-metabolizing enzymes, but some activating reactions are catalyzed also by enzymes principally involved in endogenous metabolism. The most important group of activating enzymes is the large P450 (CYP) "superfamily" (1) catalyzing oxidative (and sometimes reductive) reactions; but practically all conjugating enzymes are also capable of forming reactive metabolites from a few selected substrates, e.g., reactive glutathione conjugates are generated of some halogenated compounds.
The prevailing opinion thus far has been that for a given compound, metabolic activation is rather difficult to predict because it depends on both the structure of the substrate and the specific enzymes involved. Another hindrance in the prediction of consequences of metabolic activation has been the lack of exact knowledge about endogenous and exogenous factors affecting the level and balance of enzymes. However, recent studies on the activation of numerous potent genotoxins and potential carcinogens by human liver enzymes demonstrate that a relatively small number of CYP forms primarily activate genotoxins and other toxicants. Such studies show promise in that it may be possible in the future to predict an individual's ability (even the inherited ability) to activate specific toxins.
One of the crucial endogenous factors is polymorphic variability of various CYP forms (Table 1), which is of considerable clinical importance but which may also be significant in individual susceptibility to chemical carcinogens and other toxic substances. However, the significance of CYP polymorphisms in the expression and metabolic activation of toxic substances in target tissues other than liver has not been elucidated to any great degree although, hypothetically, variant alleles of structural genes should be similarly expressed in all expression-competent tissues.
This article surveys the present status of the field in terms of tissue-specific expression, metabolism of some toxicants, and especially the methodologies that measure their parameters while focusing upon CYP enzymes.
Figure 1. Possible pathways of a xenobiotic and its toxic metabolites in the body. After absorption, the xenobiotic is distributed throughout the body through the bloodstream. The parent compound, if hydrophobic enough, can travel freely in different tissue compartments. Xenobiotic-metabolizing enzymes act in concert to convert the parent compound to a water-soluble metabolite. Toxic metabolites can be formed in virtually any tissue, but the main bulk of metabolites is usually generated in liver. Tissue-specific toxicity depends partially on the amount of metabolizing enzymes present in target tissues. A reactive metabolite can migrate to sites distant from the tissue of origin. For example, reactive metabolites formed in liver can cause toxic effects in lungs.
A heavily studied aspect of tissue distribution of xenobiotic metabolizing enzymes relates to target organ selectivity of chemical carcinogens. A large proportion of the known or suspected carcinogens require metabolic activation; many carcinogens exhibit marked tissue specificity, with a given compound producing cancers at some sites and no effects at others. There is thus a clear urgency in understanding tissue-specific activation mechanisms. Many human CYP forms are known to be able to activate numerous procarcinogens. The inactivation pathways by CYP and conjugating enzymes are also being unraveled (3-6). The genetic variations in xenobiotic-metabolizing enzymes in relation to cancer have been reviewed in several articles (7-10).
Furthermore, after the demonstration of a specific CYP enzyme in a given tissue, we need a variety of approaches to link the activity with the toxic outcome.
There are basically five groups of methods to study the expression of individual CYP forms: determination of enzyme catalytic activities in tissue samples using form-selective diagnostic substrates and inhibitors, methods that can also be applied to the in vivo situation under certain constraints; purification and reconstitution of CYP proteins from different tissues; utilization of antibodies in various immunodetection protocols; cloning and sequencing of CYP cDNAs and their use as probes in various types of mRNA analysis; and RT-PCR, often combined with the use of specific restriction enzymes, RFLP.
A new and exciting approach is to generate knock-out mice in which specific CYP structural genes or genes encoding regulatory receptor proteins are disrupted by targeted mutations (11). These mice, lacking the proper function of the disrupted gene, serve as excellent models in the study of the role of these genes in organ development and, if the mutations are compatible with life, also in the various toxicities caused by metabolized xenobiotics (12).
Diagnostic Substrates and Inhibitors
Numerous chemicals have been tested as substrates or inhibitors of CYP forms in experimental animals and humans (13-16). Table 3 lists some of the best established form-specific substrates and inhibitors of human CYPs. Substrates or inhibitors are very seldom, if ever, completely enzyme specific; usually a single substrate can be metabolized by several CYP forms, and a single form can accept several substrates. On the other hand, at low concentrations of several substrates, enzyme kinetics often favor a single form being the primary catalyst of metabolism.
A number of compounds have been found to inhibit P450-mediated metabolism of other chemicals (14,17). Inhibition can be either reversible or irreversible depending upon the chemical. Irreversible inhibitors binding covalently to P450 enzymes are often called mechanism-based or suicide inhibitors. These are often more form specific than reversible ones because the enzyme must initially accept the inhibitor as a substrate, which is then metabolized to a reactive species (13,14).
Currently, with the aid of known enzyme-specific reactions and purified and heterologously expressed human enzymes, it is possible to unequivocally assign relative inhibitor affinities to a series of related enzymes. A selection of inhibitors listed in Table 3 is claimed to express at least a degree of enzyme specificity, which might be useful in the assignment of metabolism of any new substance to each specific enzyme. Specificity is seldom, if ever, absolute; rather, an inhibitor shows variable affinities to different enzymes. However, for practical purposes, one or two orders of magnitude difference is probably enough for a compound to be useful as a specific or diagnostic inhibitor.
In human studies, for obvious reasons, it would be very useful to have a substance that could be used to measure the activity of an enzyme in vivo (a probe drug). However, such an in vivo probe has to fulfill more stringent conditions than an in vitro substrate (18). Also the validation of an in vivo probe, which has to be performed in human subjects--healthy volunteers or patients--is a demanding and cumbersome task and has been accomplished only for a handful of substances. The most thoroughly validated probes are those for polymorphic CYP enzymes (such as debrisoquine or mephenytoin) because they display phenotypically separable, pharmacokinetically defined groups and the correspondence between genotype and phenotype is often relatively direct, e.g., lack of an enzyme shows up as a poor metabolizer.
An example of an in vivo probe drug is caffeine; although several distal metabolites are produced, these can be grouped under three enzymes (CYP1A2, NAT2, and xanthine oxidase) and consequently can be used as probes for the respective enzyme. However, despite the wide use of caffeine as an in vitro and in vivo probe, concern still exists about the uncritical use of caffeine metabolites as probes (19).
P450 Protein Purification
Comprehensive reviews are available about this issue (20,21). Approximately 10 human hepatic CYP proteins in families 1 to 4 have been purified to a degree at which unequivocal form assignment is possible. It is obvious that these forms represent the most abundant ones present in human liver and that forms expressed at low levels in the liver or other tissues cannot be readily purified to homogeneity.
Antibodies and Immunochemical Methods
A large number of polyclonal and monoclonal antibodies have been developed against purified animal and human CYP forms, and many of these are commercially available (21,22). A novel and promising technology is the production of monospecific antibodies against synthetic CYP peptides (23,24). In general, monoclonal antibodies are epitope specific and thus yield more precise information than polyclonal antibodies. Use of monoclonal anti-CYP antibodies in qualitative and quantitative detection of individual CYP forms has recently been reviewed (21). The advantage of using polyclonal antibodies is that they are usually inhibitory to the target enzyme. Anti-CYP antibodies are used in several techniques as probes to detect the presence of individual CYP forms. The basic methods are catalytic activity inhibition, immunoblotting (Western blotting) and immunohistochemistry. The main advantage of immunohistochemistry is that with this technique the distribution of the detected antigen in tissues can be elucidated. This is an important feature in extrahepatic tissues, which often consist of several parenchymal cell types mixed with supporting cells.
Current immunohistochemical techniques have the sensitivity and resolution to locate specific cell types containing identifiable CYP forms, even in tissues with a low overall P450 content (25). The lung is the best example of a complex tissue in which substantial progress has been made with immunohistochemical approaches in the localization of several distinct CYP forms in various cell types. No other method, excluding in situ hybridization, could have yielded such precise information. A comprehensive review on the use of immunohistochemical methods in pulmonary P450 research is available (26).
cDNA Cloning
To characterize the amino acid sequence of almost any protein, the best approach is to determine the nucleotide sequence of its cDNA. cDNA and genomic cloning of CYP forms have made it possible to construct the CYP superfamily (1). The molecular biology of CYPs has been reviewed by Gonzalez (27,28). The cDNAs produced can be employed for various types of mRNA detection protocols, such as the Northern blot and dot-blot methods. In situ hybridization, a method that combines the virtues of histochemistry and RNA hybridization techniques, has much potential for studying expression patterns in extrahepatic tissues. The basic steps of this technique are preparation and fixation of tissue slices, treatment to render them permeable to nucleic acid probes, hybridization with radioactive DNA or RNA probes, and autoradiography.
As in immunohistochemistry, the main advantage of in situ hybridization is that the distribution of the target molecule in the tissue under study can be evaluated. This is especially helpful in tissues that are composed of several types of cells. This technique is rather laborious and sensitive, and there is no consensus on the optimal handling protocols for human tissues. However, some groups have recently obtained reproducible results with human liver and extrahepatic tissues (29,30).
RT-PCR
The use of PCR-based methods is rapidly increasing in the study of gene expression, and several novel PCR-based methods are now available to measure the amount of specific mRNAs in a quantitative manner (31). The basic protocol to measure gene expression by PCR involves synthesis of cellular mRNA to cDNA with reverse transcriptase, amplification of the cDNA template using gene-specific primers, and detection of the amplification product.
Several groups have used this method to assess the expression of CYP genes in a qualitative manner, i.e., to screen whether a given CYP form is expressed or not in the tissue under study. In the first report in which RT-PCR was used for detection of CYP forms in humans, CYP1A1 message was found in several tissues, including liver and lung (32). Hakkola et al. (33) assessed by qualitative RT-PCR the expression of all known CYP forms in families CYP1 to CYP4 in human liver. The results correlated well with known features of CYP expression (Table 2). The RT-PCR approach is being increasingly used to detect CYP form mRNAs in several extrahepatic tissues (34).
Quantitative RT-PCR technique for measuring CYP1A1 expression in human lymphocytes was applied first by Vanden Heuvel et al. (35). An artificial recombinant mRNA (rcRNA) containing the same primer annealing sites as CYP1A1 was used as an internal standard. The rcRNA is added to the cDNA synthesis reaction together with the test RNA, and the amplified products are distinguished by their different sizes in gel electrophoresis. After optimization, this approach yields an absolute quantitation of the amount of CYP1A1 mRNA present in the sample. Treatment of lymphocytes with 10 nM 2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD) resulted in a 20-fold increase in the amount of CYP1A1 mRNA, which is very low in uninduced lymphocytes (35).
The PCR methodology is also central in the development of genotyping assays to detect variant alleles of CYP genes. The best validated methods are those that detect the most common variant alleles of CYP2D6 (36,37) and CYP2C19 (38). The latest addition to the genotyping assays is the PCR-based method to detect variant CYP2A6 alleles (39).
Naturally, when we are dealing with humans, it is seldom possible to establish an unequivocal sequence of events; but we should try to demonstrate at least some of the necessary factors, e.g., the expression of activating enzyme(s) in the target tissue. A compilation of CYP expression in various tissues is presented in Table 2. Furthermore, some better known examples of chemical-induced tissue toxicities are shown in Table 4. In the next few paragraphs, we summarize examples of CYP expression in potential target tissues.
Liver
Perhaps the most compelling case is provided by paracetamol-induced liver toxicity in which it is possible to construct the logical and plausible sequence of events and also to delineate factors enhancing and opposing toxicity. With most other toxicants, and especially with those causing toxicity in extrahepatic tissues, it is not possible to construct such a logical and well-researched scheme, but with paracetamol-induced toxicity, the following discrete steps can be traced:
Another established case is the association between activation and covalent binding of numerous hepatocarcinogens (e.g., aflatoxin B1) and the development of liver cancer. Relatively good correlation exists between DNA adducts and carcinogenic potency of several hepatocarcinogens. P450 enzyme-specific activation reactions have been elucidated for many of these compounds, making it possible, for example, to predict the influence of appropriate P450 polymorphisms on cancer risk (40,41).
Lung
Pulmonary tissue is the first to encounter all inhaled xenobiotics. Several mechanisms have developed in the lung to minimize the entry of foreign compounds into the systemic circulation, including the presence of P450 and conjugating enzymes in the various pulmonary cell types. The lung is a target for tobacco smoke-induced lung cancer, one of the best characterized chemical toxicities in humans. Several procarcinogens present in tobacco smoke are, after metabolic activation, capable of inducing and promoting neoplastic transformation in the lung cells (42).
The human lung has an extensive complement of xenobiotic-metabolizing enzymes, the main features of which are the following:
g pellet (microsomal fraction) contains little endoplasmic reticulum
Benzo[a]pyrene and other polycyclic aromatic hydrocarbons are activated to DNA-binding metabolites by human lung tissue and catalytic activities mediated by members in the CYP1A subfamily are measurable and inducible in the lungs.
More direct evidence for the presence of cigarette smoke-inducible CYP1A1 in lung was provided by several studies in which CYP1A1 was assessed at the mRNA, protein, and catalytic activity levels (43,44). These studies have also demonstrated that CYP1A1 inducibility is higher in patients with lung cancer than in control subjects. The observed induction is likely to be Ah receptor mediated, since the receptor is present in lung (45,46). The CYP1A1 protein capable of oxidizing 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene to a genotoxic product has recently been purified from human lung (6).
An example of selective toxicity based on metabolic activation in the target tissue is lung cell death caused by 4-ipomeanol. This toxin is activated by P450 enzymes in several cell types in the lung, especially the Clara cells, whereas only marginal activation occurs in other tissues (47). Studies with a panel of heterologously expressed human CYP forms have shown that metabolic activation of 4-ipomeanol is mediated by CYP1A2 and CYP3A4 (48), a finding that fits well with the presence of CYP3A members in the lung (49,50). Another lung toxin, skatole (3-methylindole), is activated by a lung-specific form, CYP2F1 (51).
Brain
Considerable attention has been paid to the role of environmental toxins in the etiology of central nervous system disorders. In particular, exogenous neurotoxins have been suggested to cause Alzheimer's and Parkinson's disease (52). Xenobiotics entering the central nervous system have to be very lipophilic to be able to cross the specialized endothelium of cerebral blood vessels (blood-brain barrier). It is thus conceivable that metabolism in blood vessel endothelial cells, as well as in neuronal and adjacent support cells, would greatly affect the toxicity elicited by the xenobiotics entering the brain.
The role of CYPs in brain may include such diverse functions as metabolism of xenobiotics, aromatization of androgens to estrogens, and formation of catechols; CYPs may also participate in metabolism of neurotransmitters (53). In rodents, brain P450 levels are low, but there exist some hot spots such as olfactory areas, cerebellum, and brainstem where P450 activities are exceptionally high. The same type of localization is found in human brain (54). Demonstration of the presence of P450 in the human brain microvessels and choroid plexuses (55) opens the question of whether metabolism in endothelial cells may regulate the penetration of the xenobiotics to the brain compartment.
Multiple CYP forms are found in the brain of rats (56). Several CYP forms are inducible in the rat brain during pregnancy, lactation, and treatment with P450 inducers, such as ethanol, chlorpromazine, phenobarbital, and nicotine (53). Information about human brain P450 is limited and is mainly based on autopsy material that may not reflect the true in vivo situation for a number of reasons. For example, there may be considerable discrepancy in overall P450 levels among different studies. Localization is probably extremely nonhomogeneous, and there may be xenobiotic-metabolizing CYP forms in subfamilies 1A, 2D and 3A. We know that steroid-metabolizing CYP forms are definitively present in subfamilies 11A and 19. There may also be a possible role for CYPs in neurotoxin metabolism, either in activation or inactivation. Evidence thus far has been fragmentary and not always convincing (34).
An especially intriguing case is cerebral CYP2D6. There are indications for its presence in distinct regions of human brain and involvement in detoxification of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a potent neurotoxin that causes Parkinson's disease-like symptoms. MPTP is oxidized to the 1-methyl-4-phenol-pyridinium (MPP+) ion by monoamine oxidase B (MAO-B) in astrocytes and then taken into dopaminergic neurons via the dopamine transporter. CYP2D6, in turn, oxidizes MPTP into a less active metabolite (52,57). Several studies have demonstrated that a CYP2D6-poor metabolizer genotype is connected with a 2- to 3-fold increased risk of developing Parkinson's disease, whereas other studies have not come to the same conclusion (57). The overrepresentation of CYP2D6-poor metabolizers in patients with Parkinson's disease might reflect a reduced capacity to detoxify ingested MPTP-like neurotoxins.
CYP2D6 is also known to metabolize several centrally acting drugs (58,59). It is thus likely that activation and inactivation of several CNS-acting drugs may take place at or close to the target neuronal tissues, adding a new dimension to the kinetics of these drugs. It is possible that CYP2D6 and other forms may participate in the synthesis and degradation of exogenous and endogenous neuroactive compounds (53). Thus, generation of reactive metabolites or metabolic disorders in situ may lead to neurotoxicity or neoplasia.
Leukocytes
Because of the abundance and relatively easy availability of human peripheral leukocytes, much effort has been devoted to using them as surrogates for P450 activities in other tissues. Analogous with several other extrahepatic tissues, aryl hydrocarbon hydroxylase (AHH) was the first monooxygenase activity demonstrated in human leukocytes and pulmonary alveolar macrophages (34).
Beginning with the pioneering studies of Kellerman et al. (60), there was a flurry of reports on the association between lymphocyte AHH activity and cigarette smoke-induced lung cancer. Many reports suggested that a relationship exists between higher AHH inducibility in lymphocytes and the occurrence of carcinoma of the lung and other tissues, while other studies did not find such an association (3,7). Because of differences between monocytes and lymphocytes (61), there is concern about the validity of studies done with unselected leukocyte fractions. Recent studies have also demonstrated that the metabolic activation of benzo[a]pyrene in peripheral leukocytes correlates poorly with that in lung tissue (62). Recent studies using both conventional Northern blotting technique (63) and quantitative RT-PCR approach (35) for lymphocyte CYP1A1 and Ah receptor expression (45) have not resolved these discrepancies. It seems that the expression of CYP1A1 may be controlled distinctly in the lung and lymphocytes, which consequently may not be suitable as a surrogate for lung microsomes concerning the pulmonary activation of PAHs (62,64).
Mammary Gland
As in most other extrahepatic tissues, oxidative metabolism and activation of benzo[a]pyrene were first indications of the presence of P450 in mammary gland. AHH activities in human mammary epithelial cells and mammary tumors range from nondetectable to relatively high levels and are inducible by some aromatic substances (34). In a study involving 188 patients with malignant or benign breast tumors, AHH activity was found to be higher in malignant than benign breast tumors (65). In addition, the survival and the disease-free interval of the cancer patients who had low mammary AHH activity was significantly longer than with patients having high AHH activity, suggesting that AHH activity may reflect the overall malignant potential of breast cancer.
Using a monoclonal antibody against rat CYP1A in an immunohistochemical study, Murray and Burke (25) detected positive staining in the cytoplasm of breast tumor cells, while no staining occurred in adjacent normal breast epithelium. Thus, studies in which either AHH activity or CYP1A protein were used as indicators show that the expression of CYP1A member(s), most probably CYP1A1, is increased in cancerous breast cells.
First, some of the hepatic P450s seem to be liver specific, i.e., they are not found in extrahepatic tissues. One such form is CYP1A2, which is the predominant form of the two CYP1A members in the liver, although it is quite variably expressed. CYP1A2 is capable of activating various carcinogens, which give rise to extrahepatic tumors. It is possible that CYP1A2 as an activating enzyme represents a case in which activation occurs in the nontarget tissue and reactive products are transferred to the target tissue.
Another problem is concerned with the significance of low-level expression of mRNA in various tissues, i.e., whether it has any functional or toxicological consequences. Basically here we are faced with an exceedingly sensitive new methodology, RT-PCR. In addition, advances in immunological detection methods have made it possible to show low levels of specific proteins in tissues and here we have the same problem as with RT-PCR: what is the role of these low-level proteins?
Use of isolated hepatocytes has been increasing for mechanistic (and even for toxicological screening) studies. Although these cells tend to lose (or sometimes acquire) properties of importance for the activation of toxicants, nevertheless it is expected that hepatocytes will serve as a useful model in studies of CYP-expression, metabolic activation, and induction in the near future. However, the development of similar approaches for extrahepatic tissues faces tougher challenges.
Finally, we have to consider the future requirements and potential of molecular biological techniques. In vivo drug probes, however scientifically appropriate and validated, are cumbersome and expensive, and they carry at least some--however small-- risk to the subject. For these reasons it would be useful to have methods that would give the same crucial information with minimal invasiveness. For well characterized and properly validated genetic polymorphisms (a prime example is CYP2D6) this has been possible in the form of DNA-based RFLP and PCR methods (10,37). It would be useful to have similar methods to measure, for instance, the potential inducibility, although necessarily in this case also the exposure should be quantitated. The advances in the understanding of regulation of drug metabolizing enzymes should make it possible to develop such assays in the not too distant future, or at least to answer the question of whether there are people who for genetic reasons have varying responses to inducers or other factors that affect drug metabolism.
2. Cohen GM, Moss EJ. Tissue distribution of drug metabolizing enzymes in relation to toxicity. In: Drug Metabolism--from Molecules to Man (Benford DJ, Bridges JW, Gibson GG, eds). London:Taylor & Francis, 1978;90-707.
3. Pelkonen O, Nebert DW. Metabolism of polycyclic aromatic hydrocarbons: etiologic role in carcinogenesis. Pharmacol Rev 34:189-222 (1982).
4. Harris CC. Human tissues and cells in carcinogenesis research. Cancer Res 47:1-10 (1987).
5. Autrup H. Carcinogen metabolism in cultured human tissues and cells. Carcinogenesis 11:707-712 (1990).
6. Guengerich FP. Bioactivation and detoxication of toxic and carcinogenic chemicals. Drug Metab Dispos 21:1-6 (1993).
7. Law MR. Genetic predisposition to cancer. Br J Cancer 61:195-206 (1990).
8. Idle JR. Is environmental carcinogenesis modulated by host polymorphism? Mutat Res 247:259-266 (1991).
9. Kawajiri K, Nakachi K, Imai K, Watanabe J, Hayashi S. The CYP1A1 gene and cancer susceptibility. Crit Rev Oncol Hematol 14:77-87 (1993).
10. Raunio H, Husgafvel-Pursiainen K, Anttila S, Hietanen E, Hirvonen A, Pelkonen O. Diagnosis of polymorphisms in carcinogen-activating and inactivating enzymes and cancer susceptibility. Gene 159:113-121 (1995).
11. Pineau T, Fernandez-Salguero P, Lee SST, McPhail T, Ward JM, Gonzalez FJ. Neonatal lethality associated with respiratory distress in mice lacking cytochrome P450 IA2. Proc Natl Acad Sci USA 92:5134-5138 (1995).
12. Gonzalez FJ, Fernandez-Salguero P, Lee SST, Pineau T, Ward JM. Xenobiotic receptor knockout mice. Toxicol Lett 82/83:117-121 (1995).
13. Testa B, Jenner P. Inhibitors of cytochrome P450s and their mechanism of action. Drug Metab Rev 12:1-118 (1981).
14. Murray M. P450 enzymes: inhibition mechanisms, genetic regulation and effects of liver disease. Clin Pharmacokinet 23:123-146 (1992).
15. Birkett DJ, Mackenzie PI, Veronese ME, Miners JO. In vitro approaches can predict human drug metabolism. Trends Pharmacol Sci 14:292-294 (1993).
16. Pelkonen O, Breimer DD. Role of environmental factors in the pharmacokinetics of drugs--considerations with respect to animal models. In: Handbook of Experimental Pharmacology (Welling PG, Balant LP, eds). Basel:Karger, 1994;289-332.
17. Guengerich FP, Shimada T. Oxidation of toxic and carcinogenic chemicals by human cytochrome P-450 enzymes. Chem Res Toxicol 4:391-407 (1991).
18. Pelkonen O, Rautio A, Raunio H. Specificity and applicability of probes for drug metabolizing enzymes. In: European Cooperation in the Field of Scientific and Technical Research--COST B1 Conference on the Variability and Specificity in Drug Metabolism (Alvan G, Balant LP, Bechtel PR, Boobis AR, Gram LF, Paintaud G, Pithan K, eds). Luxembourg:European Commission, 1995;147-158.
19. Kalow W, Tang B-K. The use of caffeine for enzyme assays: a critical appraisal. Clin Pharmacol Ther 53:503-514 (1993).
20. Guengerich FP. Characterization of human microsomal cytochrome P-450 enzymes. Annu Rev Pharmacol Toxicol 29:241-264 (1989).
21. Ryan DE, Levin W. Purification and characterization of hepatic microsomal cytochrome P-450. Pharmacol Ther 45:153-239 (1990).
22. Gelboin HV. Cytochrome P-450 and monoclonal antibodies. Biochem Pharmacol 45:413-453 (1993).
23. Murray BP, Edwards RJ, Murray S, Singleton AM, Davies DS, Boobis AR. Human hepatic CYP1A1 and CYP1A2 content, determined with specific anti-peptide antibodies, correlates with the mutagenic acivation of PhIP. Carcinogenesis 14:585-592 (1993).
24. Edwards RJ, Singleton AM, Murray BP, Davies DS, Boobis AR. Short synthetic peptides exloited for reliable and specific targeting of antibodies to the C-termini of cytochrome P450 enzymes. Biochem Pharmacol 49:39-47 (1995).
25. Murray GI, Burke MD. Immunohistochemistry of drug-metabolizing enzymes. Biochem Pharmacol 50:895-903 (1995).
26. Baron J, Voigt JM. Localization, distribution, and induction xenobiotic-metabolizing enzymes and aryl hydrocarbon hydroxylase activity within lung. Pharmacol Ther 47:419-445 (1990).
27. Gonzalez FJ. Molecular genetics of the P-450 superfamily. Pharmacol Ther 45:1-38 (1990).
28. Gonzalez FJ. Human cytochromes P450: problems and prospects. Trends Pharmacol Sci 13:346-352 (1992).
29. McKinnon RA, Hall PM, Quattrochi LC, Tukey RH, McManus ME. Localization of CYP1A1 and CYP1A2 messenger RNA in normal human liver and in hepatocellular carcinoma by in situ hybridization. Hepatology 14:848-856 (1991).
30. Palmer CNA, Coates PJ, Davies SE, Shephard EA, Phillips IR. Localization of cytochrome P-450 gene expression in normal and diseased human liver by in situ hybridization of wax-embedded archival material. Hepatology 16:682-687 (1992).
31. Foley KP, Leonard MW, Engel JD. Quantitation of RNA using the polymerase chain reaction. Trends Genet 9:380-385 (1993).
32. Omiecinski CJ, Redlich CA, Costa P. Induction and developmental expression of cytochrome P450IA1 messenger RNA in rat and human tissues. Cancer Res 50:4315-4321 (1990).
33. Hakkola J, Pasanen M, Purkunen R, Saarikoski S, Pelkonen O, Mäenpää J, Rane A, Raunio H. Expression of xenobiotic-metabolizing cytochrome P450 forms in human adult and fetal liver. Biochem Pharmacol 48:59-64 (1994).
34. Raunio H, Pasanen M, Mäenpää J, Hakkola J, Pelkonen O. Expression of extrahepatic cytochrome P450 in humans. In: Advances in Drug Metabolism in Man (Pacifici GM, Fracchia GN, eds). Luxembourg:European Commission, Office for Official Publications of the European Communities, 1995;234-287.
35. Vanden Heuvel JP, Clark GC, Thompson CL, McCoy Z, Miller CR, Lucier GW, Bell DA. CYP1A1 mRNA levels as a human exposure marker: use of quantitative polymerase chain reaction to measure CYP1A1 expression in human peripheral blood lymphocytes. Carcinogenesis 14:2003-2006 (1993).
36. Kroemer HK, Eichelbaum M. Molecular bases and clinical consequences of genetic cytochrome P450 2D6 polymorphism. Life Sci 56:2285-2298 (1995).
37. Meyer UA, Skoda RC, Zanger UM. The general polymorphism of debrisoquine/sparteine metabolism-molecular mechanisms. Pharmacol Ther 46:297-308 (1990).
38. de Morais SMF, Wilkinson GR, Blaisdell J, Nakamura K, Meyer UA, Goldstein JA. The major genetic defect responsible for the polymorphism of S-mephenytoin metabolism in humans. J Biol Chem 269:15419-15422 (1994).
39. Fernandez-Salguero P, Hoffman SMG, Cholerton S, Mohrenweiser H, Raunio H, Rautio A, Pelkonen O, Huang J, Evans WE, Idle JR et al. A genetic polymorphism in coumarin 7-hydroxylation: sequence of the human CYP2A genes and identification of variant CYP2A6 alleles. Am J Hum Genet 57:651-660 (1995).
40. Caporaso N, Goldstein A. Cancer genes: single and susceptibility: exposing the difference. Pharmacogenetics 5:59-63 (1995).
41. Wolf CR, Smith CAD, Forman D. Metabolic polymorphisms in carcinogen metabolizing enzymes and cancer susceptibility. Br Med Bull 50:718-731 (1994).
42. IARC. Tobacco smoking. In: IARC Monographs on Evaluation of Carcinogenic Risk of Chemicals to Humans. Vol 38. Lyon:International Agency for Research on Cancer, 1990;83-139.
43. McLemore TL, Adelberg S, Liu MC, McMahon NA, Yu SJ, Hubbard WC, Czerwinski M, Wood TG, Storeng R, Lubet RA, Eggleston JC, Boyd MR, Hines RN. Expression of CYP1A1 gene in patients with lung cancer: evidence for cigarette smoke-induced gene expression in normal lung tissue and for altered gene regulation in primary pulmonary carcinomas. J Natl Cancer Inst 83:1333-1339 (1990).
44. Anttila S, Hietanen E, Vainio H, Camus A-M, Gelboin HV, Park SS, Heikkilä L, Karjalainen A, Bartsch H. Smoking and peripheral type of cancer are related to high levels of pulmonary cytochrome P450IA in lung cancer patients. Int J Cancer 47:681-685 (1991).
45. Landers JP, Bunce NJ. The Ah receptor and the mechanism of dioxin toxicity. Biochem J 276:273-287 (1991).
46. Dolwick KM, Schmidt JV, Carver LA, Swanson HI, Bradfield CA. Cloning and expression of a human Ah receptor cDNA. Mol Pharmacol 44:911-917 (1993).
47. Gram TE. Pulmonary toxicity of 4-ipomeanol. Pharmacol Ther 43:291-297 (1989).
48. Czerwinski M, McLemore TL, Philpot RM, Nhamburo PT, Korzekwa K, Gelboin HV, Gonzalez FJ. Metabolic activation of 4-ipomeanol by complementary DNA-expressed human cytochromes P-450. Cancer Res 51:4636-4638 (1991).
49. Kivistö KT, Kroemer HK, Eichelbaum M. The role of human cytochrome P450 enzymes in the metabolism of anticancer agents: implications for drug interactions. Br J Clin Pharmacol 40:523-530 (1995).
50. Kivistö KT, Griese E-U, Fritz P, Linder A, Hakkola J, Raunio H, Beaune P, Kroemer HK. Expression of cytochrome P4503A enzymes in human lung: a combined RT-PCR and immunohistochemical analysis of normal tissue and lung tumors. Naunyn-Schmiedeberg Arch Pharmacol 353:207-212 (1996).
51. Thornton-Manning JR, Ruangyuttikarn W, Gonzalez FJ, Yost GS. Metabolic activation of the pneumotoxin, 3-methylindole, by vaccinia-expressed cytochrome P450s. Biochem Biophys Res Commun 181:100-107 (1991).
52. Mizuno Y, Mori H, Kondo T. Potential of neuroprotective therapy in Parkinson's disease. CNS Drugs 1:45-56 (1994).
53. Warner M, Gustaffson J-Å. Cytochrome P450 in the brain: neuroendocrine functions. Front Neuroendocrinol 16:224-236 (1995).
54. Ravindranath V, Bhamre S, Bhagwat SV, Anandatheerthavarada HK, Shankar SK, Tirumalai PS. Xenobiotic metabolism in brain. Toxicol Lett 82/83:633-638 (1995).
55. Ghersi-Egea J-F, Perrin R, Leininger-Muller B, Grassiot M-C, Jeandel C, Floquet J, Cuny G, Siest G, Minn A. Subcellular localization of cytochrome P450, and activities of several enzymes responsible for drug metabolism in the human brain. Biochem Pharmacol 45:647-658 (1993).
56. Strobel HW, Kawashima H, Geng J, Sequeira D, Bergh A, Hodgson AV, Wang H, Shen S. Expression of multiple forms of brain cytocrome P450. Toxicol Lett 639-643 (1995).
57. Sim E, Stanley LA, Risch A, Thygesen P. Xenogenetics in multifactorial disease susceptibility. Trends Genet 11:509-512 (1995).
58. Cholerton S, Daly AK, Idle JR. The role of individual human cytochromes P450 in drug metabolism and clinical response. Trends Pharmacol Sci 13:434-439 (1992).
59. von Moltke LL, Greenblatt DJ, Harmatz JS, Shader RI. Cytochromes in psychopharmacology. J Clin Psychopharmacol 14:1-14 (1994).
60. Kellerman G, Shaw CR, Luyten-Kellerman M. Aryl hydrocarbon hydroxylase inducibility and bronchogenic carcinoma. N Engl J Med 289:934-937 (1973).
61. Holz O, Krause T, Rudiger HW. Differences in DNA adduct formation between monocytes and lymphocytes after in vivo incubation with benzo[a]pyrene. Carcinogenesis 12:2181-2183 (1991).
62. Rojas M, Camus A-M, Alexandrov K, Husgafvel-Pursiainen K, Anttila S, Vainio H, Bartsch H. Stereoselective metabolism of (-)-benzo[a]pyrene-7,8-diol by human lung microsomes and peripheral blood lymphocytes: effects of smoking. Carcinogenesis 13:929-933 (1992).
63. Cosma GN, Toniolo P, Currie D, Pasternak BS, Garte SJ. Expression of CYP1A1 gene in peripheral lymphocytes as a marker of exposure in railroad workers. Cancer Epidemiol Biomarkers Prev 1:137-142 (1992).
64. Okano P, Miller HN, Robinson RC, Gelboin HV. Comparison of benzo[a]pyrene and (-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene metabolism in human blood monocytes and lymphocytes. Cancer Res 39:3184-3193 (1979).
65. Pyykkö K, Tuimala R, Aalto L, Perkiö T. Is aryl hydrocarbon hydroxylase activity a new prognostic indicator for breast cancer? Br J Cancer 63:596-600 (1991).
66. Chang TKH, Gonzalez FJ, Waxman DJ. Evaluation of triacetyloleandomycin, *-naphthoflavone and diethyldithiocarbamate as selective chemical probes for inhibition of human cytochrome P450. Arch Biochem Biophys 311:437-442 (1994).
67. Carriere V, Goasduff T, Ratanasavanh D, Morel F, Gautier JC, Guillouzo A, Beaune P, Berthou F. Both cytochromes P450 2E1 and 1A1 are involved in the metabolism of chlorzoxazone. Chem Res Toxicol 6:852-857 (1993).
Last Update: June 13, 1997