Expression of Xenobiotic-Metabolizing Enzymes in Human Pulmonary Tissue: Possible Role in Susceptibility for ILD

SELECTED REPORT

Expression of xenobiotic-metabolizing enzymes in human pulmonary tissue: possible role in susceptibility for ILD

J. Hukkanen*, O. Pelkonen*, H. Raunio*,#

Expression of xenobiotic-metabolizing enzymes in human pulmonary tissue: possible *Dept of Pharmacology and Toxico- # logy, University of Oulu, Oulu, and role in susceptibility for ILD. J. Hukkanen, O. Pelkonen, H. Raunio. ERS Journals # Ltd 2001. UniversityofKuopio,Kuopio,Finland. ABSTRACT: The lung is a major target for all inhaled toxicants. Many inhaled Correspondence: O. Pelkonen, Dept of chemicals are not hazardous as such, but are biotransformed to reactive intermediates. Pharmacology and Toxicology, Uni- Therefore, the pathogenesis of interstitial and other lung diseases is intimately linked to versity of Oulu, POB 5000, FIN-90014 exposure to environmental and other chemicals, which may be causative or modifying Oulu, Finland. factors in the cellular pathways and mechanisms mediating oxidative stress and cell Fax: 358 85375247 protection in the pulmonary tissue. Several different xenobiotic-metabolizing cytochrome P450 (CYP) and phase II Keywords: Cytochrome P450 enzymes (i.e. conjugation enzymes including several transferases) are present in the cytochrome P450 enzymes human lung and lung-derived cell lines, possibly contributing to in situ activation and xenobiotic-metabolizing enzymes inactivation of chemical toxicants. This paper describes the expression and localization of individual CYP-forms in the lung. The studies in the authors9 laboratories Interindividual differences in the expression of these enzymes may contribute to the have been ﬁnancially supported by the Academy of Finland and the Biomed2 risk of developing interstitial and other lung diseases initiated by agents that require programme (EUROCYP Project). metabolic activation. Eur Respir J 2001; 18: Suppl. 32, 122s–126s.

The lung is a major target for all inhaled toxicants. forms as well as to detoxify them [6]. Finding evidence Many chemicals are not hazardous as such, but are for this in humans is more difficult, but various lines biotransformed to reactive intermediates, often by of research have established that whole lung tissue, as enzymes in the cytochrome P450 (CYP) superfamily well as several cell types in the lung, possess metabolic [1]. The same chemicals are also detoxified by cataly- capacity towards numerous xenobiotics [7]. Although sis via these enzymes. For example, of the several the lung contains several enzymatic pathways capable thousands of compounds present in tobacco smoke, of xenobiotic metabolism, it is generally agreed that several have been found to undergo metabolic acti- the CYP superfamily of enzymes is the main system vation through xenobiotic-metabolizing CYP enzymes catalyzing the oxidative metabolism and metabolic [2, 3]. activation of most toxicants. However, from the Enzymes activating or detoxifying environmental susceptibility point of view, it is equally important chemicals are not the sole factors in the aetiology of to measure variations in phase II detoxifying enzymes chemical-induced lung diseases. Pro-oxidant and anti- (i.e. conjugation enzymes, such as uridine diphos- oxidant enzymes and chemicals (many xenobiotic- phate (UDP)-glucuronyl transferases and glutathione metabolizing enzymes have these same properties), as S-transferases) and pro-oxidant and antioxidant well as various repair systems, also play an impor- systems, although these areas are less well character- tant role in the aetiology or modification of diseases. ized. It is also notable that, like most of the polycyclic Their possible role in interstitial lung disease (ILD) aromatic hydrocarbons (PAH), such as benzo(a)pyr- induced by exogenous agents is outlined by NEMERY ene, the inhaled, highly lipophilic compounds have et al. [4] in this Supplement. longer retention times and higher local doses in pulmonary epithelium than less lipophilic compounds, indicating that at least these lipophilic substances are Metabolic capacity of pulmonary cells primarily site-of-entry toxicants [6].

A key question concerning organ-specific chemical toxicity is whether the actual target tissue has the Expression of cytochrome P450 forms in human lung capacity to activate (or efficiently inactivate) chemicals [5]. Animal models show that in the case of For the most recent update on CYP enzymes, pulmonary toxicity, several target cells in the lung consult [8]. have the capacity to convert chemicals to reactive The detection of individual CYP forms in human XENOBIOTIC-METABOLIZING ENZYMES IN ILD 123s lung by conventional methods, such as protein and it is inducible by smoking [27]. CYP1B1 is also purification, catalytic activity studies, and Western induced by smoking in alveolar macrophages [28]. immunoblotting, has been difficult due to the low CYP2A6 mRNA was detected in bronchial epithe- abundance of CYPs in lungs [9]. With the advent of lium [17, 29], but two reports on the expression of the the reverse transcriptase-polymerase chain reaction CYP2A6 protein are contradictory [17, 18]. Studies by (RT-PCR) technology it has become possible to detect the author did not demonstrate CYP2A6 mRNA in minute amounts of messenger ribonucleic acid whole lung tissue homogenate, probably due to (mRNA) in tissue samples. RT-PCR is extremely dilution of bronchus-specific mRNA expression with sensitive and results obtained with it cannot be other cell types of the lung [30]. The expression of regarded as a direct indication of the existence of pulmonary CYP2A6 protein would be of utmost corresponding proteins. Rather, RT-PCR is valuable interest because CYP2A6 has a crucial role in the acti- as a screening method, revealing mRNA that can vation of 4-methylnitrosamino-1,3-pyridyl-1-butanone potentially be translated to functional protein in a (NNK), the tobacco-specific procarcinogen [31]. given tissue. Conversely, absence of mRNA in RT- The CYP2B6 gene is expressed in human lung as a PCR analysis is a strong indication of the lack of a splicing variant called CYP2B7 [21, 27, 32] and the corresponding protein product at biologically mean- corresponding protein is also expressed [17, 33, 34]. A ingful levels. Expression of various CYP enzymes in recent immunohistochemistry study suggests a cell- human lung at mRNA and protein level is summa- specific expression of CYP2C proteins only in the rized in table 1. serous cells of bronchial glands [35]. Out of four CYP1A1 is by far the most studied human previous Western blot studies, two support [19, 36] pulmonary CYP [10]. The first report on the expres- and two oppose the expression of CYP2C proteins in human lung [37, 38]. CYP2C8 and CYP2C18 mRNAs sion of CYP1A1 mRNA in human lung was by have also been detected in the lung [17]. OMIECINSKI et al. [11] in 1990. Soon after, the There has been great interest in studying expression induction of CYP1A1 mRNA by tobacco smoking of pulmonary CYP2D6 due to its alleged role in the [12], the expression of CYP1A1 protein in human lung activation of the tobacco-specific procarcinogen NNK [13] and the localization and induction of CYP1A1 [30]. However, although it is the focus of numerous protein by tobacco smoke [14] were reported. studies [17, 19, 21, 39–41], the data on the expression CYP1A1 protein is only detected in smokers [14] of CYP2D6 in human lung is inconsistent. The and CYP1A1 expression is positively correlated with expression of pulmonary CYP2E1 mRNA and pro- aryl hydrocarbon hydroxylase (AHH) activity in tein have been established in several studies [17, 19, human lung tissue [15, 16]. 21, 29, 37, 42, 43]. CYP2E1 is an interesting CYP Another PAH-metabolizing CYP, CYP1A2, was form because it is the most active CYP enzyme in detected recently by RT-PCR and Western blot in forming oxygen radicals, causing tissue injury [44]. peripheral lung [17], but earlier reports do not corro- The expression of CYP2F1 has been detected at the borate this finding [13, 18–21]. Also, the presence of mRNA level [21, 24, 45], but there are no published CYP1B1 protein in the lung is controversial [22, 23]. results on the expression of CYP2F1 protein. Recom- CYP1B1 mRNA is expressed in human lung [24–26] binant CYP2F1 is capable of activating the pulmonary toxicant 3-methylindole [46]. Several studies have demonstrated the expression of Table 1. – Summary of expression of cytochrome P450s in human lung CYP3A protein in human lung [17, 19, 20, 37, 47, 48]. The main pulmonary CYP3A form is CYP3A5 CYP mRNA Protein Comments [17, 21, 48, 49]. There are no published results on the expression of CYP4B1 protein, but mRNA is 1A1 zzzzzz Smokers expressed in human lung [21, 24, 32, 50]. It has been z/- -- Nonsmokers speculated that native human CYP4B1 enzyme is not 1A2 z/- z/- functional due to inability to incorporate haeme [51]. 1B1 zzz/- Protein in alveolar In conclusion, at least CYPs 1A1 (in smokers), 2B7, macrophages 2E1 and 3A5 proteins are expressed in human lung. 2A6 zzz/- Other CYP forms are also likely to be expressed, but 2B6/7 zzzzzz z z their expression is probably very low or restricted 2C /- /- Protein in serous cells to specific cell types or individuals. Studies on the of bronchial glands 2D6 z/- z/- expression of certain CYP forms (CYP2F1 and 2E1 zzzzzz CYP4B1) are still required. 2F1 zzz 3A4 z/- z Protein in y20% of cases 3A5 zzzzz Localization of individual cytochrome P450 forms in 3A7 z/- the lung 4B1 zzz CYP: cytochrome P450; mRNA: messenger ribonucleic The cell-specific localization of individual CYP acid. --: moderate negative evidence; -: weak negative enzymes in the lung is still largely unknown, since evidence;z/-: conflicting evidence;z: weak positive evidence; there are only a handful of good immunohisto- zz: moderate positive evidence; zzz: strong positive chemical studies concerning CYP forms in human evidence. lung. It would be of great benefit to the understanding 124s J. HUKKANEN ET AL. of cell-specific toxicity to have a comprehensive alveolar macrophages may provide a model for picture of localization of different CYP forms. The investigating the relationship between the capacity to overall distribution of all CYP enzymes can be esti- activate and detoxify chemicals in an easily accessible mated from the immunohistochemical distribution of lung cell type [28]. In these cells, at least CYP1B1, reduced nicotinamide adenine dinucleotide phosphate CYP2E1 and CYP3A5 proteins are expressed, while (NADPH)-cytochrome P450 reductase, which is CYP1A1 is not [15, 28, 42, 49]. detected in bronchial and bronchiolar epithelium, Clara cells, alveolar lining cells and alveolar macrophages [52]. Inducibility of cytochrome P450 forms in the lung According to immunohistochemistry, CYP1A1 is mainly expressed at the epithelium of the peripheral CYP1A1 is induced by tobacco smoking in human airways. CYP1A1 expression does not extend to the lung, and there is also evidence for the induction of epithelium of bronchi w1 mm in diameter and is CYP1B1 in smokers [14, 27, 28]. Information about expressed only in the lungs of smokers [14]. Alveolar induction of other pulmonary CYP forms in vivo does macrophages do not express CYP1A1 [14, 28]. not exist. However, studies in a continuously growing Induced CYP1A1 may be a precondition for the pulmonary adenocarcinoma cell line (A549) indicate development of peripheral lung cancer in smokers, as that CYP3A5 is inducible by glucocorticoids [56]. not a single case of this disease with noninducible CYP1A1, CYP1B1 and CYP3A5 participate in the CYP1A1 in the lung has been found, and, further- activation of PAHs in the tobacco smoke [10, 57, 58] more, CYP1A1 is localized in the part of the airways and thus, their induction could have practical in which peripheral cancers arise [14, 53]. A study on significance. the localization of CYP1A1 mRNA by in situ hybri- dization corresponded well with the protein data [54]. Immunohistochemical analysis with CYP3A5- Genetic differences in the pulmonary expression of specific antibody shows that CYP3A5 protein is cytochrome P450s present in all lung samples studied and is localized to the bronchial, bronchiolar and alveolar epithelium, The expression of CYP1A1 is regulated by genetic as well as endothelium and alveolar macrophages [49]. polymorphism and it has been claimed that this Compared with CYP1A1 localization, CYP3A5 hereditary variability contributes to individual sus- expression extends up to larger bronchi. The highest ceptibility to environmental chemicals [59]. Although CYP3A5 level is detected in the bronchial lung. Also, the interindividual variability of CYP3A5 expression CYP3A4 protein is found in some cell types in a in the liver and lung is rather large, several 10-fold, minority (y20%) of lung samples with CYP3A4 relative contributions of environmental and genetic specific antibody [49]. A few studies with nonspecific factors have not been elucidated. "Classical" poly- CYP3A antibody also show the same pattern of morphic enzymes CYP2D6 and CYP2C19 have not expression as CYP3A5 antibody [47, 48]. been demonstrated in the human lung. Among phase CYP2E1 is also localized to human bronchial, II enzymes, at least glutathione S-transferase M1 bronchiolar and alveolar epithelium, and alveolar (GSTM1) has been shown to be polymorphic in macrophages [33, 42, 43]. The localization of the human lungs and may be associated with differential highest expression cannot be evaluated since none of susceptibility to lung cancer [53, 60]. these studies examined both bronchial and peripheral lung. One immunohistochemical report localizes CYP2B7 to human Clara cells [33] and a recent Conclusions study by YOKOSE et al. [35] observed CYP2C protein in serous cells of bronchial glands, but not in any The patterns of constitutive and induced expression other cell type of the lung. of cytochrome P450 forms in several relevant human pulmonary cell types are being unravelled with increasing precision. However, knowledge of the Surrogate tissues functional activities and cell-specific localization of the pulmonary cytochrome P450 forms is still Various surrogate tissues have been used in human incomplete, mostly due to sensitivity problems asso- biomonitoring studies, the most popular being ciated with measurements in tissues with a low peripheral blood lymphocytes [55]. Mitogen-treated abundance of cytochrome P450 enzymes. In addition, lymphocytes display inducible CYP1A1 and forma- very little is known about the consequences of tion of benzo(a) pyrene-deoxyribonucleic acid (DNA) interindividual variations in both basal and induced adducts, and it is suggested that these parameters may pulmonary cytochrome P450 expression. Reactions correspond with those of the lung tissue in the same catalyzed by cytochrome P450 enzymes are often individual, although this issue is controversial and still "leaky", resulting in the transfer of electrons to not definitively settled [5, 9]. Alveolar macrophages, produce oxygen radicals. Cytochrome P450-catalyzed which presumably originate from peripheral blood reactions can thus produce xenobiotic metabolites and monocytes, have an important role as defence cells oxygen species capable of interfering with cell home- against inhaled particles. The expression pattern of ostasis. 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