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Critical Reviews in Toxicology, 32(5):391–411 (2002)

1040-8444/02/$.50© 2002 by CRC Press LLC

Expression and Regulation of Xenobiotic-Metabolizing Cytochrome P450 (CYP) Enzymes inHuman Lung

Janne Hukkanen,1,3 Olavi Pelkonen,1 Jukka Hakkola,1 and HannuRaunio 2

Departments of Pharmacology and Toxicology, 1University of Oulu, Oulu and 2University of Kuopio, Kuopio,Finland, 3Department of Internal Medicine, Lapland Central Hospital, Rovaniemi, Finland

* Correspondence: Hannu Raunio, Department of Pharmacology and Toxicology, University of Kuopio, POB 1627, FIN-70211Kuopio, Finland. E-mail: hannu.raunio@uku.fi

ABSTRACT: Pathogenesis of lung diseases, such as lung cancer and chronic obstructive pulmonary disease, istightly linked to exposure to environmental chemicals, most notably tobacco smoke. Many of the compoundsassociated with these diseases require an enzymatic activation to exert their deleterious effects on pulmonary cells.These activation reactions are mostly catalyzed by cytochrome P450 (CYP) enzymes. Interindividual differencesin the in situ activation and inactivation of chemical toxicants may contribute to the risk of developing lungdiseases associated with these compounds. This review summarizes in detail the expression of individual CYPforms in human pulmonary tissue and gives a view on the significance of the pulmonary expression of CYPenzymes. The localization of individual CYP enzymes in various cell types of human lung and the emerging fieldof regulation of human pulmonary CYP enzymes are discussed. At least CYP1A1 (in smokers), CYP1B1,CYP2B6, CYP2E1, CYP2J2, and CYP3A5 proteins are expressed in human lung, and also other CYP forms arelikely to be expressed. Xenobiotic-metabolizing CYP enzymes are mostly expressed in bronchial and bronchiolarepithelium, Clara cells, type II pneumocytes, and alveolar macrophages in human lung, although individual CYPforms have different patterns of localization in pulmonary tissues. Problems in animal to human lung toxicityextrapolation and several specific aspects requiring more detailed assessment are identified.

I. INTRODUCTION

The lung is a major target organ for all in-haled toxicants. Many of these chemical com-pounds are not hazardous as such but arebiotransformed to reactive intermediates, oftenby enzymes in the cytochrome P450 (CYP) su-perfamily.1 The same chemicals can also be detoxi-fied by catalysis via these enzymes.2,3 Lung dis-eases most intimately linked to exogenouschemical substances include lung cancer andchronic obstructive pulmonary disease. Both ofthese diseases are caused primarily (about 90% ofcases) by tobacco smoke. Enzymes activating ordetoxifying environmental chemicals are not thesole factors in the etiology of chemical-inducedlung diseases. Prooxidant and antioxidant enzymesand chemicals, as well as various repair systems,play an important role in the etiology or modifi-

cation of pulmonary diseases. The role of theseenzymes in lung diseases induced by exogenousagents is reviewed in the references.4-6

This review gives a detailed picture of theCYP enzymes expressed in human lung and pre-sents studies on regulation of human pulmonaryCYP enzymes. Also, the localization of individualCYP enzymes in human lung is discussed. The roleof the genetic polymorphisms of CYPs in theinterindividual susceptibility to pulmonary diseasesis beyond the scope of this review. These aspectsare reviewed in recent articles.7-9 The lung containsalso other enzyme systems mediating biotransfor-mation, including cyclo-oxygenases and flavine-dependent monooxygenases, as well as phase IIconjugating enzymes such as glutathione S-trans-ferases, UDP-glucuronyltransferases, and DT-dia-phorases. All of these enzymes play a role in theearly cellular defense against acute and delayed

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pulmonary toxicity. The susceptibility of the lungto xenobiotics depends on the metabolic balancebetween toxication and detoxication pathways.6,10,11

The metabolism of endogenous substances,such as arachidonic acid, by pulmonary CYPenzymes is discussed here only superficially, andthe reader is advised to obtain a comprehensivepicture from recent reviews.12,13 Eicosanoids de-rived from CYP-catalyzed arachidonic acid me-tabolism are thought to play important roles inmaintaining lung homeostasis because they areinvolved in modulating airway smooth muscletone, lung inflammation, the composition of air-way lining fluid, and the tone of pulmonary vas-culature.13,14

II. GENERAL ASPECTS OF THEMETABOLIC CAPACITY OF HUMANLUNG

There is still great controversy regarding thesignificance of pulmonary metabolism ofxenobiotics due to the low levels of enzymespresent in human lung. It is possible that the liver,having by far the greatest metabolic capacity ofall organs, converts ingested and inhaled toxi-cants into proximate or ultimate reactive metabo-lites, which are then transported via the blood-stream into lung cells. In this case, intermediatescapable of interacting with cellular targets wouldhave to cross several cell membranes and escapenumerous scavenger systems. There is consider-able evidence from animal studies that reactivemetabolites generated in the liver are indeed trans-ported by red blood cells to specific cells in thelung.15,16 Considering the highly reactive natureof these intermediates, this is currently unknownwhether such transport occurs at the low concen-trations of toxicants that enter the human body. Incontrast, if the reactive intermediates were formedwithin (or in near proximity of) the actual targetcells, such as lung epithelial cells, they wouldneed to cross only intracellular membranes toreach their targets (DNA and protein).17

The majority of lung toxicants enter the bodythrough the respiratory tract, exposing pulmonaryepithelium to higher concentrations than liver cells,thus enabling even restricted local metabolism tocontribute significantly. It is also notable that

many inhaled, highly lipophilic compounds, suchas most polycyclic aromatic hydrocarbons (PAH),have longer retention times and higher local dosesin bronchial and bronchiolar epithelium than lesslipophilic compounds, indicating that at least theselipophilic substances are primarily site-of-entrytoxicants.18-21 On the other hand, the capability ofeven the most reactive ultimate carcinogen ofbenzo(a)pyrene, 7,8,9,10-tetrahydro-7,8-diol-9,10-epoxide, to cause pulmonary adenomas afterintraperitoneal injection in mice shows that evenhighly reactive intermediates may have carcino-genic effects outside the site-of-entry, althoughthe doses used were well above the relevant hu-man doses.22

It is of importance to emphasize the differentroles of alveolar and airway epithelia as picturedin Figure 1 (adapted from Gerde et al.).23 Theauthors propose a dosimetric model for inhaledPAHs in which a larger fraction of inhaled PAHsis deposited in the alveolar fraction. PAHs in thiscompartment are rapidly absorbed into the circu-lating blood with little influence of local metabo-lism. A smaller fraction of 10 to 20% of theinhaled PAHs is deposited, slowly absorbed, andextensively metabolized in the airway epitheliumat prolonged elevation of the local tissue concen-tration. Due to the retention of especially lipo-philic compounds in the airway epithelium, ampletime is given for local metabolism despite the lowabsolute capacity of lung cells for metabolic reac-tions. This is also reflected on the higher rate oftumor occurrence in airway epithelium comparedwith the alveolar epithelium.

Thus, a key question concerning organ-spe-cific chemical toxicity and carcinogenicity iswhether the actual target tissue has the capacity toactivate (or efficiently inactivate) chemicals. Ani-mal models show that, in the case of pulmonarytoxicity, several target cells in the lung have thecapacity to convert chemicals into reactive formsas well as to detoxify them.20 Finding evidence forthis in humans is more difficult, but various lines ofresearch have established that whole lung tissue aswell as several pulmonary cell types possess meta-bolic capacity toward numerous xenobiotics.24 Al-though the lung contains several enzymatic path-ways capable of xenobiotic metabolism, it isgenerally agreed that the CYP superfamily is themain system catalyzing the oxidative metabolism

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and metabolic activation of most toxicants. To-bacco smoke being the most widely studied pul-monary toxicant and carcinogen, it is of utmostimportance that 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its major me-tabolite NNAL as well as benzo(a)pyrene, the mostimportant tobacco carcinogens, are activated byCYPs,24,25 and that these activation reactions dotake place in human lung.26

Compared with the widespread interest in lo-cal pulmonary activation and inactivation of car-cinogens and toxicants, the role of pulmonarymetabolism in the systemic clearance of foreignchemicals has received less attention.27 Althoughthe pulmonary metabolic capacity is far lowerthan that in liver, there are some theoretical con-siderations that apply for the possible contribu-tion of the pulmonary enzymes in general:

1. The lungs receive 100% of the cardiac out-put, contrasting any other organ in human

body apart from the heart. This does notmean, however, that the whole quantity ofcirculating xenobiotics is exposed to pulmo-nary enzymes, which often reside severalcell boundaries away from the bloodstream.

2. Any xenobiotic given intravenously, intra-muscularly, subcutaneously, or topically iscirculated through the lung before reachingother organs, including liver. In these cases,pulmonary metabolism can be regarded asfirst-pass metabolism.

3. Due to its great metabolic capability, theliver converts many harmless compoundsinto more toxic and carcinogenic forms. Asmall portion of these is probably able toleave the liver for blood and on to the nextdownstream capillary bed of the lung. Forthese compounds the lung can be consideredthe second line of defense.

4. Pulmonary veins have very large vascularsurface area, exposing the circulating for-

FIGURE 1. A schematic dosimetric fate of an inhaled bioavailable fraction ofPAHs in the lungs. See the text for further details. (Reproduced with permissionof Oxford University Press and the author [Gerde et al. The rapid alveolarabsorption of diesel soot-absorbed benzo[α]pyrene: bioavailability, metabolismand dosimetry of an inhaled particle-borne carcinogen. Carcinogenesis 2001;vol. 22 no. 5; 741–749].)

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eign compounds efficiently to pulmonaryenzymes.

5. Certain basic amino compounds are accu-mulated in the lungs. These include suchdrugs as imipramine, amiodarone, and amidetype local anesthetics.4

These points make it a feasible assumptionthat pulmonary enzymes of the lung could alsohave some toxicological and clinical significancein the systemic clearance of chemical compounds.However, regarding the role of CYP enzymesspecifically, it must be taken into considerationthat the type I pneumocytes and endothelium,which are most directly exposed to blood flow,contain only small amounts of CYP enzymes.The only CYP enzyme with strong expression inthese cell types is CYP2J2, which is expressed inhuman pulmonary endothelium.28 Although be-ing primarily involved in arachidonic acid me-tabolism, CYP2J2 also has activity toward somexenobiotic drugs, such as diclofenac andbufuralol.29 The pulmonary cells with strongestCYP expression, that is, bronchial and bronchi-olar epithelium, are not in direct contact with thebulk of pulmonary circulation. Thus, there is nodirect evidence for a marked significance of thepulmonary CYP enzymes in the systemic clear-ance in humans.4,30

III. PNEUMOTOXIC AGENTS INANIMALS

Numerous studies with laboratory animalshave demonstrated the importance of respiratorycells as targets for both inhaled and ingested sub-stances. There is a large body of experimentalevidence from animal studies that some lung toxi-cants need metabolic activation by pulmonaryCYP enzymes to be able to cause toxicity. CYPenzymes in animals are preferentially localized inClara cells, alveolar type I and type II cells, en-dothelial cells, macrophages, and ciliated bron-chiolar cells.11 The nonuniform distribution ofxenobiotic bioactivation and detoxication enzymesin the lung is generally thought to be the basis forcell-specific toxicity associated with many pul-monary toxicants. In particular the nonciliatedbronchiolar epithelial (Clara) cells are very sensi-

tive to various toxicants due to the presence ofactivating enzymes, such as CYP2E1.6 The mouseis especially sensitive to a variety of metaboli-cally activated lung toxicants, and the rat is usu-ally resistant.

Table 1 summarizes some selected pneumotoxicchemicals requiring metabolic activation in animals.Naphthalene, a compound present in tobacco smokeand some industrial processes, is good example of aspecies-selective pulmonary toxicant. Naphthalenecauses pneumotoxicity especially in Clara cells ofmouse lung,31 possibly by activation by the pulmo-nary CYP2F2 enzyme.32 A baculovirus-expressedCYP2F2 enzyme was shown recently to effectivelymetabolize naphthalene and its 1-nitro and 2-methylderivatives to potentially cytotoxic intermediates.33

Human lungs express CYP2F1 mRNA, but evi-dence for the presence of catalytically active CYP2F1protein is still lacking. Expressed human CYP2F1metabolizes naphthalene only slowly but still at asubstantial level compared with other substratesmetabolized by human liver microsomes.34

1-nitronaphthalene is a mutagenic nitroaromatic de-tected in emissions from diesel engines. Recently,this compound was shown to be activated by thepulmonary CYP system in both rats and mice.35

3-Methylindole is a selective toxicant to pul-monary Clara cells in various species, and it isactivated through hydroxylation, epoxidation, anddehydrogenation pathways.36 Studies with ex-pressed CYP enzymes show that the humanCYP2F1 and goat CYP2F3 forms are uniquelycapable of dehydrogenation of 3-methylindole,whereas some other forms (CYP1A/1B andCYP2E1) preferentially oxygenate it.37 Mice andrats are also susceptible species for 3-methylindoleinduced pneumotoxicity.38 4-ipomeanol, anotherselective lung toxicant is activated locally byCYP4B1,39 but this is also a species-specific reac-tion since in humans 4-ipomeanol is hepatotoxicbut not pneumotoxic.40 An agent probably acti-vated by CYP2B enzymes is coumarin, whichcauses a selective Clara cell injury in the mouselung.41

Vinyl carbamate is derived from metabolismof ethyl carbamate, a byproduct formed duringthe fermentation process of various foods. Bothcarbamates induce a spectrum of tumors in ro-dents with the lung being the most susceptibleorgan.42 Neither vinyl nor ethyl carbamate is car-

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cinogenic in the parent form but requiresbioactivation to a metabolite to produce a carci-nogenic effect. The pathway of metabolism in-volves CYP-mediated oxidation of ethyl to vinylcarbamate, followed by an additional CYP-cata-lyzed oxidation to the electrophilic metabolitevinyl carbamate epoxide. Recent studies stronglyimplicate pulmonary CYP2E1 as being involvedin the two-step activation and carboxylesteraseenzymes mediating inactivation of ethyl carbam-ate in both experimental animals43 and humans.44

Because both enzymes systems metabolize ethylcarbamate in a similar way in mouse and humanlungs, these findings indicate that the murine modelis relevant for studying the mechanisms of ethylcarbamate toxicity in the human lung. However,direct comparisons of microsomes derived frommouse and human lung showed that CYP2E1enzyme activity is about sevenfold higher in mouselung, whereas levels of carboxylesterase are higherin human lung.44,45 Thus, quantitative risk assess-ment based on the activity of these enzymes couldlead to erroneous results.

Pulmonary toxicity follows after exposure to1,1-dichloroethylene, a prevalent environmental

contaminant.46 Dichloroethylene-induced Claracell damage in mouse lung was shown to be asso-ciated with in situ formation of the reactive ep-oxide metabolite.47 Mouse lung microsomesproduce higher levels of dichloroethylene epoxide-derived conjugates that human lung microsomes,45

and the production of the epoxide is predomi-nantly mediated by CYP2E1 in both liver andlung tissue.45-47

Trichloroethylene is both acutely toxic andcarcinogenic to the mouse lung following expo-sure by inhalation. It is not carcinogenic in the ratlung and markedly less toxic following acute ex-posure. The reactive metabolites are actively pro-duced in Clara cells in the mouse lung. There aremarked species differences in the number andmorphology of lung Clara cells. In mice they arenumerous and are distributed throughout the air-ways, whereas in rats they are significantly fewerin number. Clara cells are rare in human lung,residing in the distal bronchioles. The large quan-titative differences in the metabolic capacity ofmouse lung compared with rat and human lungsuggest that the risk of trichloroethylene carcino-genicity to humans is minimal.48

TABLE 1Examples of Pneumotoxic Chemicals Requiring Metabolic Activation in Animals

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IV. EXPRESSION AND SIGNIFICANCEOF SPECIFIC CYP FORMS IN HUMANLUNG

For the most recent update on CYP enzymes con-sult http://drnelson.utmem.edu/nelsonhomepage.html.

The detection of individual CYP forms inhuman lung has been difficult by conventionalmethods, such as protein purification, catalyticactivity studies, and Western immunoblotting, dueto the low abundance of CYPs in lung.49,50 De-pending on the procedure used, spectroscopicquantitation of CYPs in human lung has revealedthat the amount of microsomal CYP protein is ata level of about 2 to 10 pmol/mg protein.51,52 Also,the complex nature of the lung as an organ withmore than 40 different cell types with varyingamounts of CYP enzymes have complicated theresearch. CYP enzymes in human lung have beenstudied clearly less than CYPs in pulmonary tis-sue of animals, mainly due to difficulties in ob-taining sufficient amounts of human tissue. Therelative lack of CYP enzymes in human lungcompared with rodent lung has also hamperedthis research. Although human and baboon pul-monary microsomes contain essentially identicalconcentrations of CYP protein, they have only5% of the CYP protein levels measured in rat lungand only 1% of those found in rabbit lung.49,51

With the advent of the RT-PCR technology,it has become possible to detect minute amountsof mRNA in tissue samples. RT-PCR is extremelysensitive, and the results obtained with it cannotbe regarded as a direct indication of the existenceof corresponding proteins. Rather, RT-PCR isvaluable as a screening method, revealing mRNAthat can potentially be translated into functionalprotein in a given tissue. Conversely, the absenceof mRNA in RT-PCR analysis is a strong indica-tion of the lack of a corresponding protein prod-uct at biologically meaningful levels. The expres-sion patterns of individual CYP enzymes atmRNA, protein, and catalytic activity levels inhuman lung, based on available literature, is sum-marized in Table 2.

The biological functions of genes and therespective mRNAs are carried out by the proteinsthey encode. Thus, the final analysis on the role ofCYPs in pulmonary metabolism of foreign andendogenous agents must be made on the protein

level. At least three issues need to addressed whencarrying out such analysis: (1) differences in struc-tures and catalytic specificities among CYPs indifferent species, (2) highly uneven localizationof specific CYP forms in various lung cell types,and (3) because most toxic agents, includingprocarcinogens, are present at very low levels inlung cells, the affinities of these chemicals toCYP enzymes (Km) are important parameters tobe determined. Presently, information is insuffi-cient on each of these issues.

CYP1A1 is by far the most actively studiedhuman pulmonary CYP enzyme due to its impor-tance in PAH metabolism.53,54 The first report onthe expression of CYP1A1 mRNA in human lungwas published by Omiecinski et al. in 1990.55 Soonafter that the induction of CYP1A1 mRNA bytobacco smoking,56 the expression57 and purifica-tion52 of CYP1A1 protein in human lung, and thelocalization and induction of CYP1A1 protein bytobacco smoke58 were reported. CYP1A1 proteinis only detected in smokers,58 and CYP1A1 ex-pression correlates positively with the aryl hydro-carbon hydroxylation (AHH) and ethoxyresorufinO-deethylation (EROD) activities in human lungtissue.56-60 CYP1A1 mRNA expression in the lungmay be more abundant in female than male smok-ers.61 However, EROD activities are similar in fe-male and male smokers.9 Pulmonary EROD activ-ity is positively correlated with the number ofcigarettes smoked per day,62 but not with pack-year consumption.9 AHH activity and CYP1A1mRNA expression decrease to the basal levelswithin 2 months after the cessation of smoking.56,63

Recently, the expressions of pulmonary CYP1A1mRNA and protein were shown to correlate posi-tively with the aromatic/hydrophobic DNA adductlevels in human lung tissue.61,64

Because of the significance of CYP1A1 in theactivation of procarcinogens, there have been ac-tive efforts to link the polymorphisms of theCYP1A1 gene with the individual susceptibility tolung cancer. Nine variant alleles have been de-scribed, but none of them have been unequivocallyshown to correlate with altered catalytic activity ofthe CYP1A1 protein (CYP allele nomenclaturecommittee homepage: http://www.imm.ki.se/cypalleles/). The T3801C (MspI) and I462V poly-morphisms have been studied the most. It is con-cluded that these polymorphisms are associated

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TABLE 2Summary of Expression of CYPs in Human Lung

+

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with a higher risk of lung cancer in the Japanesepopulation, but not in Caucasians.65

Another PAH-metabolizing CYP, CYP1A2,was detected recently by RT-PCR and Westernblot in peripheral lung,66 but other reports do notcorroborate this finding.52,57,67-70 CYP1A2-relatedactivities acetanilide 4-hydroxylase and phenace-tin O-deethylation are not detected in human lungmicrosomes.52,57

There is still some controversy regardingCYP1B1 protein expression in lung tissue. Thereare three reports supporting the expression ofCYP1B1 protein and one report showing a nega-tive result, probably due to differences in the sen-sitivity of the antibodies used.71-74 Tang et al. esti-mated72 based on their results74 that CYP1B1 levelsare fivefold higher than CYP1A1 levels in lungmicrosomes. A portion of the AHH activity de-tected in human lung is probably contributed byCYP1B1, in addition to CYP1A1.57,59,60 CYP1B1mRNA is expressed in human lung,54,73,75,76 and itis inducible by smoking in bronchial epithelium.77

CYP1B1 is also induced by smoking in alveolarmacrophages78 and possibly also in lung tissue.73

CYP1B1 is highly active in the activation of PAHs,54

and it could play a significant role in the activationof PAHs, at least in such cell types as alveolarmacrophages where CYP1A1 is not expressed.78

Expression of CYP2A6 mRNA is detected inbronchial epithelium,66,79 but two reports on the ex-pression of the protein are contradictory.66,67 Ourgroup and others were unable to demonstrateCYP2A6 mRNA in whole lung tissue homogenate,perhaps due to dilution of bronchus-specific mRNAexpression with other cell types of the lung.70,80

CYP2A-catalyzed coumarin 7-hydroxylation has notbeen detected in human lung microsomes.67 Theexpression of pulmonary CYP2A6 protein would beof utmost interest because CYP2A6 has a crucialrole in the activation of NNK, the tobacco-specificprocarcinogen.81 It also activates other nitrosaminessuch as N-nitrosomethylphenylamine andN-nitrosopyrrolidine.82 Surprisingly, a novel findingindicates that CYP2A6 is also involved in the for-mation of NNK from nicotine, suggesting that NNKcould be formed endogenously during nicotine re-placement therapy.83 In parallel with studies onCYP1A1 polymorphisms, two Japanese studies haveshown protective effect of deletion of the CYP2A6gene against lung cancer,84,85 whereas studies with

other populations have yielded negative or opposingresults.86,87 Importantly, a recent study showed rela-tively high levels of CYP2A13 mRNA in humanlung and heterologously expressed CYP2A13 waseven more active in the activation of NNK thanCYP2A6.88

The CYP2B6 gene is expressed in humanlung as a splicing variant (previously calledCYP2B7),69,70,77,89-91 and the corresponding pro-tein is expressed as well.66,92,93 CYP2B6 mightplay a role in the activation of NNK.81,94 CYP2B6-related PROD (pentoxyresorufin O-deethylation)activity has been detected in human pulmonarymicrosomes.52,95

A recent immunohistochemistry study withunspecific CYP2C antibody suggested a cell-spe-cific expression of CYP2C protein(s) only in theserous cells of bronchial glands.96 Of the four ear-lier Western blot studies with unspecific CYP2Cantibodies, two support67,97 and two fail to supportthe expression of CYP2C proteins in humanlung.98,99 A study with CYP2C8-specific antibodyshowed no expression of CYP2C8 in human lungmicrosomes.100 CYP2C mRNAs (CYP2C8 andCYP2C18) have been detected in lung byRT-PCR,66,70 but not with Northern blot tech-nique.100 CYP2C8 has been implicated in theproduction of the endothelium-derived hyperpolariz-ing factor (EDHF), probably 11,12-epoxyeicosatrienoicacid, which affects vascular tone.101,102 If CYP2C8 isexpressed in certain cell types of lung, it could have arole in the regulation of pulmonary vascular and bron-chial tone.

There has been widespread interest in study-ing the expression of pulmonary CYP2D6 due toits alleged, but probably minor, role in the activa-tion of NNK.81 However, the findings on the ex-pression of CYP2D6 in human lung are inconsis-tent despite numerous studies.66,67,69,70,103-105 Animmunohistochemical study with CYP2D6 anti-body revealed neither uniform nor cell-specificCYP2D6 protein expression in lung,103 suggest-ing that the possible low-level expression ofCYP2D6 indicated in some studies is probablybiologically meaningless. CYP2D6-catalyzedbufuralol hydroxylation has not been detected inhuman lung.67,103 It has been speculated that theCYP2D6 polymorphism might affect the risk oflung cancer through modulating smoking behav-ior, because CYP2D6 might be involved in the

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signal transduction of the dopaminergic pathwayin brain.106 This is reflected in the result of arecent meta-analysis, which revealed that inactiveCYP2D6 alleles protect against lung cancer (OR= 0.69).107

The expression of pulmonary CYP2E1 mRNAand protein has been established in several stud-ies.66,67,69,70,79,91,98,108,109 CYP2E1 is interesting be-cause it is the CYP form that most actively gener-ates oxygen radicals causing tissue injury.110 Theseradicals are formed both in the absence and in thepresence of substrate.111 CYP2E1 is also highlyactive in the activation of tobacco-specific nitro-samines, such as N-nitrosodimethylamine (NDMA),N-nitrosodibutylamine, and N-nitrosodiethylamine,but contributes only slightly to the activation ofNNK, the most important nitrosamine in tobaccosmoke.82,112 CYP2E1-related catalytic activities,namely, oxidation of butadiene, demethylation ofNDMA, hydroxylation of p-nitrophenol, and6-hydroxylation of chloroxazone, are detected inlung microsomes.44,45,91,108,113,114

Expression of pulmonary CYP2F1 has beendetected at the mRNA level.69,70,75,115 but thereare no published results on the expression ofCYP2F1 protein. Recombinant CYP2F1 is ca-pable of activating the pulmonary toxicants3-methylindole and to a minor extent naphtha-lene.34

CYP2J2 mRNA and protein are expressed inhuman lung.28,116 CYP2J2 is active toward a verylimited number of xenobiotics, but it has beenshown to form epoxyeicosatrienoic acids (EETs)from arachidonic acid.29,116 EETs affect vascularand bronchial smooth muscle tone,12,117 and thusCYP2J2 might play an important physiologicalrole in the pulmonary regulation of both vascularand bronchial tone. Therefore, it is of interest thatCYP2J2 is strongly expressed in both bronchialand vascular smooth muscle cells and endothe-lium.28 Human lung is capable of forming EETsfrom arachidonic acid.28,118,119 Also, CYP2C hasbeen implicated in the production of EETs,101,102

but the pulmonary expression of CYP2C enzymesis in doubt and their localization is unknown.Importantly, the regiochemistry of human pulmo-nary EETs match that of EETs produced by re-combinant CYP2J2.28 Thus, CYP2J2 is a strongcandidate for catalyzing the synthesis of the pul-monary EETs.13

A novel enzyme, CYP2S1, was detected re-cently in human lung,120 where it was demon-strated at both the mRNA and protein levels. ThisCYP form is mainly extrahepatic. Nothing isknown about its catalytic properties yet. Interest-ingly, preliminary results show that CYP2S1 isinduced by AHR/ARNT in mouse lung and alsoin a human pulmonary cell line.121 Its inductionby dioxin could suggest that it may be able tometabolize chemical carcinogens.

Several studies demonstrate the expression ofCYP3A protein in human lung.66-68,98,122-124 Both thestudies with gene-specific RT-PCR and a study withisoform-specific antibodies demonstrate CYP3A5as the main pulmonary CYP3A form.66,69,123,124

CYP3A4 is expressed in about 20% of cases.124 It isinteresting to note that CYP3A5 also predominatesin the esophagus.125 CYP3A-catalyzed nifedipineoxidase and testosterone 6β-hydroxylation activitiesare detected in lung microsomes.67,68,126 There aretwo immunohistochemical studies showing noCYP3A protein in human lung.96,127 The antibodiesused were probably reactive with CYP3A4, but notwith CYP3A5. CYP3A5 could play some role in thepulmonary activation of both NNK and especiallybenzo(a)pyrene,24,81 at least when the CYP1A1 levelis low. In human pulmonary microsomes, the laststep of benzo(a)pyrene activation is stimulated byα-naphthoflavone, a CYP3A activator.52,126 PAH-DNA adduct levels correlate positively with theamount of CYP3A5 in alveolar macrophages ofsmokers.78 Our preliminary results suggest thatCYP3A5 is induced by low concentrations of gluco-corticoids in human lung,128 indicating that CYP3A5could have a physiological role in maintaining thesteroid hormone balance in lung, as CYP3A5 isactive in the metabolism of steroid hormones.129,130

Low levels of a novel CYP3A43 mRNA were de-tected recently by RT-PCR in human lung in one 131

but not in another study.132

There is only one preliminary report on theexpression of CYP4B1 protein in human lung,133

but at least human CYP4B1 mRNA is expressedin the lung.69,70,75,90,134 Heterologously expressedhuman CYP4B1 catalyzes 6β-hydroxylation oftestosterone, a typical CYP3A reaction, but not2-aminofluorene (2-AF) or the pulmonary toxin4-ipomeanol, which are typical CYP4B1-medi-ated reactions in animals.39,130,134 However, onestudy suggested that CYP4B1 is not functional

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due to its inability to incorporate heme, and it wasspeculated that the original human CYP4B1 ex-pression vector had been contaminated byCYP3A5, suggesting that the earlier results onexpressed CYP4B1 are erroneous.135 In contra-diction to this it was shown recently that humanbladder and kidney microsomes catalyzed 2-AFand this reaction was inhibited by an anti-CYP4B1antibody, suggesting that human CYP4B1 couldbe functional.136 Also, human CYP4B1 expressedin transgenic mouse liver was able to metabolize2-AF.137 Rabbit CYP4B1 has been suggested tobe involved in the synthesis of arachidonic acid-derived hydroxyeicosatetraenoic acids (HETEs).138

20-HETE is a vasodilator of human pulmonaryarteries associated with hypoxic pulmonary vaso-constriction,13,118,139 but it is probably producedby pulmonary CYP4A enzymes in humans.13,118

In conclusion, at least the CYP1A1 (in smok-ers), CYP2B6, CYP2E1, CYP2J2, and CYP3A5proteins are expressed in human lung, and recentresults indicate that also CYP1B1 protein is ex-pressed in lung tissue. Also, other CYP forms arelikely to be expressed, but their expression isprobably very low or restricted to specific celltypes or individuals. The studies on the expres-sion of certain CYP forms at protein level(CYP2F1 and CYP4B1) are still missing or pre-liminary. In alveolar macrophages, that is, themost easily accessible pulmonary cell type, atleast CYP1B1, CYP2E1, and CYP3A5 proteinsare expressed, while CYP1A1 is neither expressednor induced by tobacco smoke.78,108,124

V. LOCALIZATION OF INDIVIDUAL CYPFORMS IN HUMAN LUNG

Cell-specific localization of individual CYPenzymes in the lung is still poorly known, be-cause there are only a handful of good immuno-histochemical studies about CYP forms in humanlung. For a better understanding of cell-specificcarcinogenicity and toxicity, it would be of greatbenefit to have a comprehensive picture of thelocalization of different CYP forms. The overalldistribution of CYP enzymes can be estimatedfrom the immunohistochemical distribution ofNADPH-cytochrome P450 reductase, which isdetected in bronchial and bronchiolar epithelium,

Clara cells, alveolar lining cells (type I and IIpneumocytes), and alveolar macrophages.140

According to immunohistochemistry analy-ses, CYP1A1 is mainly expressed in the epithe-lium of the peripheral airways, that is, bronchi-olar, terminal bronchiolar, and alveolar epithelium.CYP1A1 expression is not present in the epithe-lium of bronchi larger than 1 mm in diameter, andit is only seen in the lung of smokers.58 Alveolarmacrophages do not express CYP1A1 regardlessof smoking status.58,78 In some cases, endotheliumexpresses CYP1A1.58 Induced CYP1A1 may be aprecondition for the development of peripherallung cancer in smokers, as not a single case of thisdisease with noninducible CYP1A1 in the lungwas found in two studies, and, furthermore,CYP1A1 was localized in the part of the airwayswhere peripheral cancers arise.58,141 A study of thelocalization of CYP1A1 mRNA by in situ hybrid-ization corresponded well to the protein findings.142

CYP1A1 mRNA has also been detected in bron-chial and peripheral samples by RT-PCR.66,77

Alveolar macrophages are the only pulmo-nary cell type in which the expression of CYP1B1protein has been established.78 CYP1B1 is in-duced at mRNA and protein levels by tobaccosmoking in these cells.

One immunohistochemical report localizesCYP2B6 to human Clara cells,92 and CYP2B6mRNA is expressed in bronchial and peripherallung.66,77 A recent study by Yokose et al. revealedCYP2C protein in serous cells of bronchial glands,but not in any other lung cell type.96 mRNAs ofCYP2C8 and CYP2C18 have also been detectedin both bronchial and peripheral tissue samples.66

CYP2E1 is localized to human bronchial, bron-chiolar and alveolar epithelium, and alveolarmacrophages.92,108,109 Endothelial cells have someexpression.108 The localization of maximum ex-pression cannot be determined, because none ofthese studies examined both bronchial and pe-ripheral lung. CYP2E1 mRNA has been detectedby RT-PCR in both bronchial and peripheralsamples.66,75,79

In an immunohistochemical study, CYP2J2was detected throughout the epithelium of thepulmonary airway from the trachea to alveoli andin alveolar macrophages.28 Surprisingly, bronchialand vascular smooth muscle cells and vascularendothelium also showed strong expression. In

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epithelium, this expression was most intense inciliated cells and less intense in alveoli.

Immunohistochemical analysis with CYP3A5-specific antibody shows that CYP3A5 protein ispresent in all lung samples studied and is local-ized to the bronchial, bronchiolar, and alveolarepithelium as well as endothelium and alveolarmacrophages.124 Compared with CYP1A1 local-ization, CYP3A5 expression extends up to largerbronchi. The highest CYP3A5 level is detected inthe bronchial lung. Also, CYP3A4 protein is foundin some cell types in a minority (about 20%) oflung samples with CYP3A4-specific antibody.124

The immunohistochemical studies with nonspe-cific CYP3A antibody also show the same patternof expression as CYP3A5 antibody.122,123 The re-sults on CYP3A mRNA expression in peripheraland bronchial samples establish CYP3A5 as themain CYP3A form.66,123

In general, it can be concluded that the ex-pression of CYPs in human lung is more widelydistributed than in pulmonary tissues of labora-tory animals, where the expression is morepronouncedly concentrated in Clara cells, type IIalveolar cells, and alveolar macrophages.49,143

Perhaps the most obvious difference between thelocalization of CYP enzymes in man and rodentsis the weaker expression of CYPs in human Claracells. These cells are also less numerous in humanthan in rodent lung, and their numbers are dimin-ished by smoking.144,145

VI. REGULATION OF HUMANPULMONARY CYP EXPRESSION

A. Regulation In Vivo

Knowledge about the regulation of CYPs inhuman lung in vivo is rather limited. By far thegreatest amount of information has been gatheredconcerning the induction of CYP1A1 andCYP1A1-related enzyme activities AHH andEROD.49 Both aryl hydrocarbon receptor (AHR)and AHR nuclear translocator (ARNT), whichregulate the induction of CYP1, are expressedabundantly in human lung.146,147 CYP1A1 proteinis only detected in smokers,58 and CYP1A1 ex-pression correlates positively with the AHH andEROD activities in human lung tissue.56-60 AHH

activity decreases to the basal level within 2months and CYP1A1 mRNA expre. 56,63 CYP1A1mRNA expression in the lung may be more thantwo-fold higher in female smokers compared withmale smokers.61 It has been speculated that thecomplex interactions between the estrogen recep-tor and AHR pathways could explain this differ-ence.61 However, EROD activity is similar in maleand female smokers.9 A 10 to 20 % subset ofsmokers does not have measurable CYP1A1mRNA, protein, or activity in lung.9,58,142,148 Thispoor inducibility phenotype is not explained byAHR, ARNT, or CYP1A1 gene polymorphisms.148

CYP1B1, which is also controlled by AHR/ARNT, is induced by tobacco smoking in bron-chial epithelium and alveolar macrophages.77,78

However, the induction of CYP1B1 protein bytobacco smoke is probably modest at best.73,78

AHH activity in alveolar macrophages is increasedby smoking,149 and because CYP1A1 is neitherexpressed nor induced in these cells,58,78 the in-duced CYP form with AHH activity is likelyCYP1B1. Early research by some authors sug-gested that CYP1B1 protein expression could beused as a tumor-marker because they detected noexpression of CYP1B1 protein in normal humantissues.71,150 This is probably more likely to be amisjudgment caused by a nonsensitive antibodyas CYP1B1 protein has been detected in numer-ous normal tissues, including lung, using otherCYP1B1-antibodies.72-74,78 However, it is possiblethat CYP1B1 is slightly up-regulated in sometumors, including lung cancers.71,73,151

Pulmonary CYP2B6 and CYP4B1 mRNAsare not affected by smoking.77,79,90 Tobacco smok-ing has been shown to repress CYP2A6 mRNA inbronchial epithelial cells79 and CYP3A5 proteinand mRNA in alveolar macrophages 78 (Hukkanenet al., unpublished results). The mechanisms ofthese repressions are unknown.

No expression of pregnane X receptor (PXR),the receptor participating in the regulation ofCYP2B6, CYP2C, CYP3A4, and CYP3A7,152-156

has been detected in human lung tissue by North-ern blot analysis.157-159 This does not exclude thepossibility of expression in some specific pulmo-nary cell types. Extremely low levels of constitu-tively active receptor (CAR) mRNA are detectedin pulmonary tissue by Northern blot after longexposure times.160 It would be of interest to clarify

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the possible cell specificity. CAR is involved inthe induction of CYP2B6, CYP2C, CYP3A4, andCYP3A7.156,161,162 The CYP inducer and PXRligand rifampicin induces 7-ethoxycoumarinO-deethylation (ECOD) activity in human lungmicrosomes of patients with tuberculosis morethan two-fold.163 ECOD is catalyzed by manyCYP enzymes, including CYP1A1, CYP2A6,CYP2B6, and CYP2E1, and CYP3A.130,164,165 It isnot known which, if any, of these enzymes isinduced in human lung by rifampicin. It is alsopossible that the inducer is not rifampicin, but thechronic inflammation caused by tuberculosis orother microbes because very high EROD activityhas also been detected from a current smoker withaspergillosis.9 CYP2E1 and CYP3A are regulateddifferentially in human lung, because there is noassociation between their expression in bronchialtissue as assessed by immunohistochemistry.109

In conclusion, CYP1A1 induction in lung tis-sue and CYP1B1 induction in alveolar macroph-ages are probably mediated by the AHR/ARNTpathway, and these inductions are seen at themRNA, protein, and catalytic activity levels.CYP1B1 is probably also induced modestly inlung tissue. The regulation of other CYP enzymesin human lung tissue remains to be characterized.

B. Regulation In Vitro

In vitro induction of CYP1A1 has been studiedquite extensively in various human pulmonary celllines, the most popular ones being the adenocarci-noma line A549 and the bronchioloalveolar carci-noma line NCI-H322. In general, CYP1A1 is in-duced by AHR agonists in various pulmonarycancer cell lines166-174 and in the human immortal-ized bronchial cell lines BEAS-2B, BEP2D and16HBE.75,175-177 McLemore et al. tested 24 humanlung cancer cell lines for the benz(a)anthraceneinduction of CYP1A1 mRNA.166 Eleven cell linesshowed low-level basal CYP1A1 mRNA expres-sion, and 16 were induced after benz(a)anthracenetreatment. AHH activity correlated positively withthe CYP1A1 mRNA levels. In A549 cells, trans-forming growth factor-β1 represses both basal andinduced expression of CYP1A1 and CYP1B1 andbasal expression of AHR, while ARNT expressionis not affected.168,169 In NCI-H322 cells, interferon γ

decreases both basal and induced CYP1A1-relatedEROD activity, while interleukin-1β, tumor necro-sis factor, interferon α, and interferon β are with-out effect.167 We have studied the regulation ofAHR-dependent induction of CYPs by phosphory-lation in A549 cells and shown that the proteinkinase C and the tyrosine kinase are involved inCYP1A1 induction, but not in CYP1B1 induc-tion.173 Our study173 and a study by Mollerup etal.176 show that constitutive CYP1A1 mRNA ex-pression is lower than constitutive CYP1B1 mRNAexpression, but CYP1A1 is induced 20 to 60-foldby AHR agonists, whereas CYP1B1 is inducedonly 2- to 6-fold. Resveratrol, an antioxidant foundin plants and red wine, is able to inhibit both basaland induced CYP1A1 and CYP1B1 mRNA ex-pressions in BEP2D cells.176

In A549 cells, CYP2B6 protein and the re-lated PROD (pentoxyresorufin O-deethylation)activity were detected, but PROD activity was notinduced by phenobarbital,170 CYP2B6 mRNA wasnot induced by AHR agonists, phenobarbital, orethanol in CL5 cells.174 In a study with NCI-H322cells,167 CYP1-, CYP2B-, CYP2C-, and CYP3A-related 7-benzyloxyresorufin O-debenzylation(BROD) activity93,130,165 was increased by Aroclor1254, a mixture of polychlorinated biphenyls con-taining both AHR ligands and phenobarbital-likeinducers.178 BROD activity was also induced bybeta-naphthoflavone and phenobarbital in humancultured lung slices and bronchial epithelial cells,respectively.179,180 CYP3A5 mRNA is induced bydexamethasone and phenobarbital in A549 cells.173

Work in our laboratory has given further evidencefor the induction of CYP3A5 by low concentra-tions of glucocorticoids in A549 cells.128 Thereare also novel results indicating the induction ofCYP2E1 by ethanol in primary culture of humanbronchial epithelium.91 Recent preliminary resultsshow that CYP2S1 mRNA is regulated by AHR/ARNT and it is induced by AHR agonist in A549cells.121

In conclusion, in vitro results are in agree-ment with the in vivo studies on CYP1A1 induc-tion. Also, in vitro results on CYP1B1 inductionseem to correlate with in vivo results, althoughCYP1B1 is relatively weakly induced in cellmodels and humans alike, making it more diffi-cult to study. Most of the in vitro studies oninduction of other CYP enzymes are difficult to

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interpret due to the use of unspecific catalyticactivities, but there are promising results acquiredwith gene-specific methods.

VII. CONCLUSIONS AND AREAS FORFURTHER STUDY

The expression of CYP forms in human lungis being unraveled with increasing precision. TheCYP proteins demonstrated most convincingly inhuman lung include CYP1A1 in smokers,CYP1B1, CYP2B6, CYP2E1, CYP2J2, andCYP3A5. The catalytic activities associated withthese enzymes are also detected in human lung.However, more detailed analysis is needed oncatalytic specificities and kinetic properties ofthese enzymes toward key exogenous and endog-enous substrates. In addition, knowledge of thecell-specific localization of the pulmonary CYPforms is still incomplete due to the relative lack ofgood-quality immunohistochemical studies onCYP enzymes. Only the localizations of CYP1A1,CYP2J2, and CYP3A5 proteins are adequatelycharacterized. Also, the use of unspecific anti-bodies and catalytic activities adds uncertainty tothe results on both the expression and the regula-tion of pulmonary CYP enzymes. The regulationof pulmonary CYP forms is still quite poorlycharacterized, with the exception of CYP1A1 in-duction in smokers. In addition, very little is knownabout the consequences of interindividual varia-tions in both basal and induced pulmonary CYPexpression.

To what extent does lung metabolism in ro-dents and other laboratory animals reflect the situ-ation in the human lung? Although only limitedcomparison is possible based on current knowl-edge, it is still an interesting exercise. Pulmonarydosimetry studies in experimental animals in vivohave added important information on the behav-ior of xenobiotics in different lung compartments.Due to their extensive retention in airway mu-cosa, many inhaled procarcinogens such as PAHsshow nonlinear relations between exposure leveland dose to airway target cells. Thus, the airwayepithelium attains comparatively high concentra-tions at low exposure levels. Such effects compli-cate high- to low-dose extrapolations commonlyemployed in carcinogenesis risk assessment. Fur-

ther problems arise when animal data are used forhuman risk assessment. It is fair to conclude thatwe still do not know enough about quantitativemetabolism pathways in either human or animallung tissue, and that much further work is neededbefore the role of local metabolism in target lungcells is fully understood.

At least the following areas should be cov-ered by future research:

1. There are several CYP enzymes that havestrong indications for pulmonary expression,but that have not been studied in detail.These CYP forms include CYP2A6,CYP2A13, CYP2F1, and CYP4B1.

2. More immunohistochemical studies withspecific antibodies are needed to clarify thecell-specific localization of individual CYPforms. An alternative technique would be insitu RT-PCR. This knowledge would be vitalto understand the cell-specific carcinoge-nicity and toxicity of inhaled and blood-borne compounds.

3. Is there a common transcriptional factor in-volved in the basal expression of pulmonaryCYP enzymes? CYP forms, such asCYP2A13, CYP2F1, CYP2S1, CYP3A5,and CYP4B1, which are relatively selec-tively expressed in lung (or at least in extra-hepatic organs), could be investigated in thisregard. This task is a major undertaking,because the mechanisms of tissue-specificCYP expression outside liver have beenlargely overlooked.

4. It should be established which of the pulmo-nary CYP forms are inducible in humanlung, and the regulation of CYP inductionshould be elucidated. These goals are mostconveniently achieved with the use of pul-monary cell models, such as cancer cell linesand immortalized cell lines, and gene-spe-cific methods such as quantitative RT-PCR.Naturally, the human in vivo studies are “thegolden standard”.

5. A systematic and quantitative comparisonof pulmonary xenobiotic metabolism in hu-mans and common experimental animals isneeded to refine the current risk assessmentprocedures. The commonness of the pulmo-nary diseases caused by xenobiotics war-

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rants more research on the enzymes metabo-lizing these compounds. In addition to thesignificance in the xenobiotic metabolismand chemical carcinogenesis, the role of CYPenzymes in the physiological regulation ofvascular and bronchial tone also justifiesmore studies. Thus, pulmonary CYP en-zymes are still an intriguing subject for fu-ture studies.

ACKNOWLEDGMENTS

The studies in the authors’ laboratories havebeen financially supported by the Academy ofFinland, the Biomed2 program (EUROCYPProject), and TEKES (Technology DevelopmentCenter, Finland).

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