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Metabolism and covalent binding of benzo[a]pyrene in human peripheral lung
Chem.-Biol. Interactions, 28 (1979) 345--358 © Elsevier/North-Holland Scientific Publishers Ltd.
345
METABOLISM AND COVALENT BINDING OF BENZO[a]PYRENE IN HUMAN PERIPHERAL LUNG
REKHA MEHTA, M. MEREDITH-BROWN a and G E R A L D M. COHEN
Department of Biochemistry, University of Surrey, Guildford, Surrey, GU2 5XH and aMilford Chest Hospital, Milford, Surrey (United Kingdom) (Received July 21st, 1979) (Accepted September 15th, 1979)
SUMMARY
Short-term organ cultures of peripheral lung from lung cancer patients metabolise benzo[a]pyrene to ethylacetate-soluble metabolites, which covalently bind to tissue macromolecules. The nature and quantities of metabolites formed and the extent of covalent binding are dependent upon the time of incubation, the substrate concentration and interindividual variability in the metabolic activity of the lung. Individuals whose lungs rapidly metabolise the carcinogen exhibit more extensive further metabolism of primary metabolites and higher levels of covalent binding. Certain striking differences in the relative retention iIl the tissue or release into the extracellular medium of different metabolites have also been found as illustrated by the observation that the ratio of 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene to 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene was always significantly higher in the tissue than in the extracellular medium. INTRODUCTION
As environmental contaminants and constituents of cigarette smoke, polycyclic aromatic hydrocarbons such as benzo[a]pyrene have been implicated as major factors in the aetiology of human lung carcinoma [1,2]. Many chemical carcinogens, including polycyclic aromatic hydrocarbons, require metabolic activation to reactive electrophiles to elicit cell transformation, mutagenicity and cytotoxicity [3--6]. Benzo[a]pyrene and related chemicals are metabolised to various hydroxylated derivatives including epoxides, phenols, dihydrodiols, quinones and diol-epoxides [4,5,7--10]. Certain of these metabolites, namely epoxides, diol-epoxides and some phenols have been shown to be mutagenic, carcinogenic and capable of reacting with nucleic acids and proteins [9,11--16]. Inter-individual variation in the ability to activate and deactivate chemical
346 carcinogens, particularly in target tissues, may influence the susceptibility of an individual to hydrocarbon-induced carcinogenesis. Organ cultures of human bronchus [17], peripheral lung [18,19] and cultured alveolar macrophages [20] can metabolise b e n z o [ a ] p y r e n e to intermediates that are bound to cellular macromolecules including DNA. A 75-fold and 9-fold variation between individuals has been found in the binding of b e n z o [ a ] p y r e n e to bronchial cell DNA and alveolar macrophage DNA, respectively [17,18]. Whilst most primary malignancies of the human lung are believed to originate from the bronchial epithelium, some recent reports suggest that a considerable fraction possibly up to 50% may arise in peripheral subpleural regions [1]. Recently, we reported a large interindividual variation in the metabolism of b e n z o [ a ] p y r e n e by short-term organ culture of human peripheral lung from six lung cancer patients [21]. Besides the nature of metabolites formed, other factors such as uptake of benzo[a]pyrene, inter-relationships between activating and deactivating metabolism and the rates of formation and release of free and conjugated metabolites from the cell into the extracellular medium may be crucial in determinmg the levels of reactive intermediates available to interact with cellular macromolecules, and hence, the susceptibility of a particular tissue to polycyclic aromatic hydrocarboninduced carcinogenesis. We have, therefore, investigated the kinetics of formation and covalent binding of b e n z o [ a ] p y r e n e metabolites in short-term organ cultures of human peripheral lung in the presence of different concentrations of benzo[a]pyrene, with particular reference to the distribution of metabolites between the tissue and extracellular medium. MATERIALS AND METHODS
Organ cultures Peripheral lung specimens from lung cancer patients were obtained at the time of surgery. These sepcimens were transported to the laboratory in phosphate buffered saline (lacking calcium and magnesium salts) at 4°C and cultured within 3 h of removal at surgery. Samples of lung (100 -+ 10 mg) that appeared macroscopically normal were cultured for 18 h at 37°C as described previously [21]. The full clinical details of these patients and the ethylacetate-soluble metabolites formed after short-term organ culture of peripheral lung with b e n z o [ a ] p y r e n e (2 uM, final concentration) have been published recently [ 21].
Benzo[a]pyrene metabolism After culture for 18 h, the lung samples were transferred to media containing [3H]benzo[a]pyrene (10--20 ttCi/ml; specific activity, 24 Ci/mmol; Radiochemical Centre, Amersham, U.K.) and unlabelled benzo[a]pyrene (Koch-Light Laboratories Limited, Bucks. U.K.) at final concentrations in the range 1 - 1 0 pM, and incubated in a shaking water-bath at 37°C for periods of up to 24 h. Control incubations containing tissue and similar
347 concentrations of b e n z o [ a ] p y r e n e as the test incubations, were incubated for the same periods at 4°C.
Analysis of ethylacetate-soluble metabolites After culture, the tissue and medium were separated and the tissue homogenised in 2 ml of 1.15% KC1. The tissue homogenate and medium were extracted with 3 × 1 vol. of ethylacetate. The pooled ethylacetate extracts were dried with anhydrous sodium sulphate and concentrated to dryness on a rotary evaporator. The metabolites were redissolved in ethylacetate and separated by high pressure liquid chromatography using a linear methanol/water gradient (1 : 1 to 4 : 1) as previously described [22].
Covalent binding The tissue homogenates, after extraction with ethyl acetate, were stored at - 2 0 ° C until the amount of covalent binding was determined essentially by the m e t h o d of Siekevitz [23] as described previously [24]. RESULTS
Uptake and distribution of benzo[a]pyrene Organ cultures of peripheral lung from patient I were used to study the time course of b e n z o [ a ] p y r e n e metabolism over a period of 24 h. Within 3 h, 56% of the initial radioactivity had been taken up from the medium into lung tissue {Fig. lb). The total activity in the tissue then slowly declined as metabolism of the substrate continued. Examination of the radioactivity in the medium with respect to its association with either ethylacetatesoluble or water-soluble metabolites, or unchanged benzo[a]pyrene, indicated that after 10 h of incubation, 9.3% of the initial activity had been released into the medium as ethylacetate-soluble metabolites, with small b u t measurable amounts being present at the 3- and 6-h time periods (Fig. la). However, during the incubation period, the precentage of unchanged benzo[a]pyrene decreased and the metabolites released had undergone further metabolism and/or conjugation to water-soluble products. Thus, at the end of 24 h, 38.0% of the initial radioactivity in the medium was present as water-soluble metabolites, only 13.6% of the added b e n z o [ a ] p y r e n e remaining unchanged (Fig. la}. Compared to the extraceUular medium, the radioactivity in the tissue associated with ethylacetate-soluble metabolites and especially, watersoluble products, remained low at all time points examined (Fig. lb). Only 5.3% of the added b e n z o [ a ] p y r e n e was found unchanged in the tissue at the end of 24 h.
Time dependence of nature of ethylacetate-soluble
metabolites formed
The ethylacetate-soluble metabolites of b e n z o [ a ] p y r e n e present in the extracellular medium and lung tissue at the various time points studied were analysed by high pressure liquid chromatography (Fig. 2). Benzo[a]pyrene
348 (a) _Medium 100 -
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Fig. 1. Time course of the percentage of initial radioactivity in (a) extracellular medium and in (b) lung tissue associated with either ethylacetate-soluble metabolites (e o), water-soluble metabolites (o o ) or unchanged b e n z o ( a ~ y r e n e (A ~). Short-term organ culture of peripheral lung (100 _+ 10 rag) from patient I were incubated with benzo[a]pyrene (2 /zM) at 37 °. The tissue, after homogenisation in 1.15% KC1, and the medium were extracted with ethylacetate (3 x 1 vol.). The radioactivity not extracted by ethylacetate is termed water-soluble. The ethylacetate-soluble products were separated by high pressure liquid chromatography, and the values for unchanged [3H]benzo[a]pyrene obtained were subtracted from the total ethylacetate-soluble radioactivity to give the values for ethylaeetate-soluble metabolites.
was metabolised mainly t o ethylacetate-extractable metabolites which coc h r o m a t o g r a p h e d with 9,10-dihydro-9,10-dihydroxybenzo[a] pyrene (9,10-
diol) and 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (7,8-diol). The material eluting in the 9,10-diol region may also consist of 7/8,9-trihydroxy-7,8,9,10pentahydrobenzo[a]pyrene (7/8,9-triol) [17]. The appearance of these
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350 metabolites and other as yet unidentified metabolites eluting in fractions 3--10 and 14--26 (Peaks I and II respectively; Fig. 2) was very much dependent u p o n the time of incubation. The material eluting in fractions 3--10 contains very polar metabolites such as sulphate conjugates of monoh y d r o x y b e n z o [ a ] p y r e n e s [ 19] and other as y e t uncharacterised metabolites, whilst that eluting in fractions 14 -26 may consist of tetrols [18,25]. After 10 h of incubation with benzo[a]pyrene, significant amounts of only 9,10-diol and 7,8-diol with small amounts of polar metabolites (Peak I) were present (Fig. 2a). However, following incubation for 21 h a major proportion of the radioactivity eluted as metabolites more polar than 9,10diol which probably represent the secondary metabolism of benzo[a]pyrene metabolites though significant amounts of 9,10-diol, 7,8-diol and unmetabolised b e n z o [ a ] p y r e n e were also present. After incubation for 24 h, the radioactivity eluting with 9,10~1iol and 7,8<1iol had been markedly reduced and most of the radioactivity eluted with Peaks I and II (Fig. 2b). Distributional differences o f metabolites between tissue and medium Certain striking qualitative and quantitative differences were also apparent in the distribution of individual ethylacetate-soluble metabolites between the extracellular medium and tissue (Fig. 3). Thus after 24 h incubation, detectable amounts of both 9-OH-BP (17.5 pmol/100 mg tissue) and 3-OH-BP (70.2 p m o l / 1 0 0 mg tissue} were found only in the tissue (Fig. 3b) b u t not in the medium. It must also be remembered that many factors will contribute to the amounts of any metabolite detected at a given time. Of particular importance in this regard will be the rates of formation and further metabolism, both oxidation and conjugation, of a metabolite, the enzyme activity of the tissue and its ability to generate essential cofactors. The polar metabolites (Peaks I and II) were present both in the tissue and medium, the latter having a greater proportion of these metabolites. Small amounts of quinones were detectable in the medium after incubation periods exceeding 10 h. Whereas more of the material associated with the 9,10-diol peak compared with 7,8-diol was observed in the medium, the ratio of 7,8-diol : 9,10-diol in tissue remained high at all the time points examined (Fig. 3). Thus, the ratio of 7,8-diol : 9,10
351
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Fig. 4. Effect of benzo[a]pyrene concentration on ethyl acetate-soluble metabolite production and the distribution of these metabolites in patient II between (a) extracellular medium and (b) lung tissue. Short-term organ cultures of lung (100 f 10 mg) from patient II were incubated with various concentrations of benzo[a]pyrene for 24 h. The ethylacetate-soluble metabolites were extracted and analysed by HPLC as described in legends to Figs. 1 and 2. c----- 0, Polar metabolites eluting in fractions 3-10; a----- l, tetrols eluting in Fractions 14..-26; A-----A, 9,10-dihydro-9,10-dihydroxybenzo[o Jpyrene and/or 7/8,9-triol; A-A, 7,8-dihydro-7,8-dihydroxybenzo[alpyrene.
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353 Almost all the radioactivity from ethylacetate-soluble extracts of short~ term organ cultures of lung from patient II at varying b e n z o [ a ] p y r e n e concentrations (1--10 pM) eluted as either polar metabolites (Peak I), tetrols (Peak II), 9,10-diol and 7,8-diol in agreement with the time course study in patient I (Fig. 2). Whilst the amounts of 9,10-diol and 7,8-diol increased in both the tissue and medium when the b e n z o [ a ] p y r e n e concentration was increased from 1 to 10 pM, the amounts of the more polar metabolites and tetrols (Peaks I and II) appeared to plateau at higher substrate concentrations (Fig. 4a, b). No detectable levels of phenols or quinones were found at any of the benzo[a] pyrene concentrations studied in this patient. In contrast to the observations made in cultures from patient II, in cultures from patient VI, which showed a very low level of metabolism of b e n z o [ a ] p y r e n e (2 uM) (Ref. 21, Table I). Only very small amounts of 7,8-diol and 9,10-diol were detected in the extraceUular medium at all the substrate concentrations examined, with very little or no secondary metabolism of b e n z o [ a ] p y r e n e metabolites. Thus, in patient VI, the maximal levels of 9,10-diol and 7,8-diol detected in the medium were 60 and 70 p m o l / 1 0 0 mg tissue/24 h respectively at 10 pM b e n z o [ a ] p y r e n e , compared to 1400 and 600 p m o l / 1 0 0 mg tissue/24 h respectively in the medium of cultures from patient II at the same concentration of b e n z o [ a ] p y r e n e . Examination of ethylacetate-soluble extracts from patient VI indicated the presence of significant amounts of 3-OH-BP, 9-OH-BP and quinones in the tissue b u t n o t in the medium at all substrate concentrations studied. These
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Benzo(a;pyrene Concentration OJM) Fig. 5. C o v a l e n t b i n d i n g of [ 3 H ] b e n z o [ a ] p y r e n e r e l a t e d r a d i o a c t i v i t y t o s h o r t - t e r m o r g a n c u l t u r e s o f h u m a n p e r i p h e r a l lung. C u l t u r e s of l u n g f r o m p a t i e n t s II a n d V I w e r e i n c u b a t e d for 24 h w i t h various c o n c e n t r a t i o n s o f b e n z o [ a ] p y r e n e . T h e c o v a l e n t l y b o u n d radioa c t i v i t y was m e a s u r e d as d e s c r i b e d in Materials a n d M e t h o d s a n d is d e s i g n a t e d as (e • ) in p a t i e n t II a n d (o o ) in p a t i e n t VI.
354 amounts increased almost linearly with a rise in the concentration of benzo[a]pyrene (results n o t shown). Thus significant differences were observed in the metabolic profiles of b e n z o [ a ] p y r e n e at different substrate concentrations and these differences were apparently dependent to a large extent on the rates of metabolism of b e n z o [ a ] p y r e n e in different individuals.
Covalently bound benzo[a]pyrene and/or its metabolites Up to 0.5% of the initial radioactivity was b o u n d to cellular macromolecules. The amount of covalently b o u n d b e n z o [ a ] p y r e n e and/or its metabolites increased with increasing concentrations of b e n z o [ a ] p y r e n e (1--10 ~M} in short-term organ cultures from patient II, but n o t in those from patient VI, where the covalent binding appeared to have become saturated {Fig. 5). The levels of binding were also comparatively lower in cultures from the patient with lower rate of metabolism, i.e. patient VI (Fig. 5). This was in agreement with the results of Stoner et al. [18] w h o showed a good correlation between the ability of human lung to metabolise b e n z o [ a ] p y r e n e and the a m o u n t of covalent binding to either DNA or protein. DISCUSSION Benzo[a]pyrene was metabolised b y short-term organ cultures of human peripheral lung to ethylacetate-soluble metabolites which co-chromatographed with 9,10-diol, 7,8-diol, tetrols and other more polar metabolites. The nature of the metabolites found depended markedly on the time of incubation, substrate concentration and the inter-individual differences in rates of metabolism. Similar results have been reported by Holder et al. [26] using liver microsomes from untreated and 3-methylcholanthrene pretreated rats and mice. As expected further metabolism of primary metabolites, such as dihydrodiols, to secondary metabolites increased with the duration of incubation (Fig. 2) and was more extensive in lung cultures of patients with more rapid metabolism (Figs. 2--4). Much recent evidence implicates the diolepoxide 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene 9,10-oxide, derived from 7,8-diol, as a major ultimate carcinogenic and mutagenic metabolite derived from b e n z o [ a ] p y r e n e [9,14,16]. Other studies have identified this diol epoxide b o u n d to the exocyclic amino group of guanine as a major adduct b e t w e e n metabolically activated b e n z o [ a ] p y r e n e and human bronchial DNA [27]. Indirect evidence has also been provided to show the same major adduct is formed with DNA of human peripheral lung [28]. Thus factors affecting the further metabolism of primary metabolites such as 7,8-diol may be of critical importance in determining h o w much of a reactive diolepoxide is formed. In this study it was readily apparent that 7,8-diol was further metabolised presumably via the diol-epoxide to tetrols (Peak II, Figs. 2 and 3), in agreement with results ~in cultured rodent trachea [29], cultured human bronchus [25], cultured human peripheral lung [18] and
355 rat liver microsomes [8]. It was of interest in the present study that this further metabolism could occur even in the presence of significant amounts of unmetabolised benzo[a]pyrene (Fig. 2b) suggesting that the affinity of the mixed function oxidase(s) for 7,8-diol might be higher than for benzo[a]pyrene or that different species of cytochrome P-450 may be responsible for the metabolism of benzo[a]pyrene and 7,8-diol. The loss of radioactivity associated with 9,10-diol or 7/8,9-triol over longer periods of incubation (Figs. 2 and 3) could be due to further metabolism of 9,10-diol to either 9,10-dihydroxybenzo[a]pyrene, 1(3),9,10trihydroxy-9,10-dihydrobenzo[a]pyrene or 7,8,9,10-tetrahydro-7,8,9,10t e t r a h y d r o x y b e n z o [ a ] p y r e n e , which have been shown to be metabolites of 9,10-diol in cultured rat trachea, rat liver microsomes and cultured hamster lung respectively [24,29,30]. It would appear (Fig. 3) that 9,10-diol may be a better substrate for further metabolism in human peripheral lung than in either short-term organ cultures of rodent trachea or lung [31]. This may be of some significance as recent studies have indicated that the anti-isomer of 9,10-dihydro-9,10-dihydroxybenzo [ a ] pyrene 7,8-oxide, a diol epoxide intermediate in the metabolism of 9,10-diol to a tetrol, is highly mutagenic to Salmonella typhimurium TA 98 and moderately mutagenic in Chinese hamster V79 cells, although it did not induce malignant transformation in M2 mouse fibroblasts [32,33]. In respect to the possible further metabolism of either 7,8-diol or 9,10diol to potentially reactive diol epoxides, it is also necessary to consider the relative intracellular retentions of these dihydrodiols. It is interesting to speculate that the higher intracellular retention of 7,8-diol compared to 9,10-diol (Figs. 3 and 4) is an additional factor contributing to the higher biological activity of 7,8-diol, by favouring its further metabolism to reactive metabolites rather than its excretion from the cell. Our previous studies have indicated that both isolated rat hepatocytes [34] and hamster embryo cells [35] also show similar distributional differences of metabolites. In order to explain these differences we have investigated the relative binding affinities of different metabolites to ligandin, small molecular weight binding protein A and lipid bilayers as model membrane systems. It appears that the binding of 9,10-diol to protein A and to lipid bilayers but not to ligandin, is significantly lower than the binding of other metabolites and may explain the relative ease of efflux of 9,10-diol from the cell (E. Tipping et al., unpublished data). The release of either of these dihydrodiols into the extracellular medium (Figs. 3 and 4) may also be of significance in vivo especially in peripheral lung tissue since such metabolites from alveolar cells could escape into the circulatory and lymphatic systems to be distributed around the body, including the bronchus which is considered to be the main site of human lung carcinoma. In addition, such metabolites released extracellularly could be taken up by pulmonary alveolar macrophages which whilst transporting these carcinogens up the mucocilliary tree could release them at or near the bronchial epithelium.
356 It was also of interest to note that the monohydroxybenzo[a]pyrenes, 3-OH-BP and 9-OH-BP, were only detectable in the ethyl acetate-extractable fraction of the tissue at the 24-h time point in patient I (Fig. 3) and in the tissue but not the medium at all the concentrations of benzo[a]pyrene studied in patient VI, who had a relatively low rate of metabolism. The retention of monohydroxybenzo[a]pyrenes intracellularly is in agreement with our earlier observations with both isolated rat hepatocytes and hamster embryo cells [34,35]. A possible explanation for the absence of phenols at earlier time ponts in patient I was their further metabolism primarily to sulphate conjugates. We have recently shown that sulphation is a major route for conjugation of phenols in human lung with little or no glucuronide conjugation taking place [22]. Thus part of the radioactivity eluting in peak I (fractions 3--10, Figs. 2--4) will be due to sulphate esters of monohydroxybenzo[a]pyrenes, which are partially organic solvent-soluble and are formed by human lung [19], and also elute in fractions 3--10. The material associated with the water-soluble radioactivity has also been shown to contain some sulphate ester conjugates of monohydroxybenzo[a]pyrenes [22]. Thus the presence of phenols intracellularly at later time points (Fig. 3) may possibly be due either to saturation of sulphate conjugation or to leakage of cofactors or to release of aryl sulphatase from lysosomes. In conclusion, large interindividual variations exist in the extent of benzo[a]pyrene metabolism by short-term organ cultures of peripheral lung from lung cancer patients. In patients exhibiting high rates of metabolism, a range of metabolites, including secondary metabolites' of benzo[a]pyrene metabolites, are produced, their biosynthesis being dependent upon the period of incubation and the concentration of substrate. Certain of these metabolites, namely 3-OH-BP, 9-OH-BP and 7,8-diol, are preferentially retained within the tissue. Although the significance of this selective retention of metabolites is as yet unexplored, intracellular accumulation of metabolites capable of being metabolised to ultimate carcinogenic forms implies serious biological consequences. ACKNOWLEDGEMENTS
The work was supported in part by grants from the Cancer Research Campaign and the Medical Research Council. The reference metabolites of benzo[a]pyrene were kindly supplied by N.C.I. Carcinogenesis Research Program, U.S.A. REFERENCES
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358 22 R. Mehta and G.M. Cohen, Major differences in the extent of conjugation with glucuronic acid and sulphate in human peripheral lung, Biochem. Pharmacol., 28 (1979) 2479. 23 P. Siekevitz, Uptake of radioactive alanine in vitro into the protein of rat liver fractions, J. Biol. Chem., 195 (1952) 549. 24 G.M. Cohen and B.P. Moore, The metabolism of benzo(a)pyrene, 7,8-dihydro-7,8dihydroxybenzo (a)pyrene and 9,10-dih ydro-9,10-dihydroxy-benzo(a)pyrene by short-term organ cultures of hamster lung, Biochem. Pharmacol., 26 (1977) 1481. 25 S.K. Yang, H.V. Gelboin, B.F. Trump, H. Autrup and C.C. Harris, Metabolic activation of benzo(a)pyrene and binding to DNA in cultured human bronchus, Cancer Res., 37 (1977) 1210. 26 G.M. Holder, H. Yagi, D.M.Jerina,W. Levin, A.Y.H. Lu and A.H. Conney, Metabolism of b e n z o ( a ) p y r e n e . - Effect of substrate concentration and 3-methylcholanthrene pretreatment on hepatic metabolism by microsomes from rats and mice, Arch. Biochem. Biophys., 170 (1975)557. 27 A.M. Jeffrey, I.B. Weinstein, K.W. Jennette, K. Grzeskowiak, K. Nakanishi, R.G. Harvey, H. Autrup and C. Harris, Structures of benzo(a)pyrene-nucleic acid adducts formed in human and bovine bronchial explants, Nature, 269 (1977) 348. 28 K. Shinohara and P.A. Cerutti, Formation of benzo(a)pyrene-DNA adducts in peripheral human lung tissue, Cancer Lett., 3 (1977) 303. 29 B.P. Moore and G.M. Cohen, Metabolism of benzo(a)pyrene and its major metabolites to ethyl acetate-soluble metabolites by cultured rodent trachea, Cancer Res., 38 (1978) 3066. 30 D.R. Thakker, H. Yagi, R.E. Lehr, W. Levin, M. Buening, A.Y.H. Lu, R.L. Chang, A.W. Wood, A.H. Conney and D.M. Jerina, Metabolism of trans-9,10-dihydroxy9,10-dihydrobenzo(a)pyrene occurs primarily by arylhydroxylation rather than formation of a diol epoxide, Mol. Pharmacol., 14 (1978) 502. 31 G.M. Cohen and B.P. Moore, Metabolism of benzo(a)pyrene and its major metabolites by respiratory tissues, in: P.W. Jones and R.I. Freudenthal (Ed.), C a r c i n o g e n e s i s - A Comprehensive Survey, Vol. 3, Raven Press, New York, 1978, pp. 325--340. 32 C. Malaveille, T. Kuroki, P. Sims, P.L. Grover and H. Bartsch, Mutagenicity of isomeric diol-epoxides of benzo(a)pyrene and benzo(a)anthracene in S. typhimurium TA 98 and TA 100 and in V 79 chinese hamster cells, Murat. Res., 44 (1977) 313. 33 H. Marquardt, S. Baker, P.L. Grover an d P. Sims, Malignant transformation and mutagenesis in mammalian cells induced by vicinal diol~epoxides derived from benzo(a)pyrene, Cancer Lett., 3 (1977) 31. 34 C.A. Jones, B.P. Moore, G.M. Cohen, J.R. Fry and J.W. Bridges, Studies on the metabolism and excretion of benzo(a)pyrene in isolated adult rat hepatoeytes, Bioehem. Pharmaeol., 27 (1978) 693. 35 G.M. Cohen, M.C. MacLeod and J.K. Selkirk, Organic solvent-soluble and watersoluble metabolites of benzo(a)pyrene formed by hamster embryo fibroblasts, The Pharmacologist, 20 (1978)157.
345
METABOLISM AND COVALENT BINDING OF BENZO[a]PYRENE IN HUMAN PERIPHERAL LUNG
REKHA MEHTA, M. MEREDITH-BROWN a and G E R A L D M. COHEN
Department of Biochemistry, University of Surrey, Guildford, Surrey, GU2 5XH and aMilford Chest Hospital, Milford, Surrey (United Kingdom) (Received July 21st, 1979) (Accepted September 15th, 1979)
SUMMARY
Short-term organ cultures of peripheral lung from lung cancer patients metabolise benzo[a]pyrene to ethylacetate-soluble metabolites, which covalently bind to tissue macromolecules. The nature and quantities of metabolites formed and the extent of covalent binding are dependent upon the time of incubation, the substrate concentration and interindividual variability in the metabolic activity of the lung. Individuals whose lungs rapidly metabolise the carcinogen exhibit more extensive further metabolism of primary metabolites and higher levels of covalent binding. Certain striking differences in the relative retention iIl the tissue or release into the extracellular medium of different metabolites have also been found as illustrated by the observation that the ratio of 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene to 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene was always significantly higher in the tissue than in the extracellular medium. INTRODUCTION
As environmental contaminants and constituents of cigarette smoke, polycyclic aromatic hydrocarbons such as benzo[a]pyrene have been implicated as major factors in the aetiology of human lung carcinoma [1,2]. Many chemical carcinogens, including polycyclic aromatic hydrocarbons, require metabolic activation to reactive electrophiles to elicit cell transformation, mutagenicity and cytotoxicity [3--6]. Benzo[a]pyrene and related chemicals are metabolised to various hydroxylated derivatives including epoxides, phenols, dihydrodiols, quinones and diol-epoxides [4,5,7--10]. Certain of these metabolites, namely epoxides, diol-epoxides and some phenols have been shown to be mutagenic, carcinogenic and capable of reacting with nucleic acids and proteins [9,11--16]. Inter-individual variation in the ability to activate and deactivate chemical
346 carcinogens, particularly in target tissues, may influence the susceptibility of an individual to hydrocarbon-induced carcinogenesis. Organ cultures of human bronchus [17], peripheral lung [18,19] and cultured alveolar macrophages [20] can metabolise b e n z o [ a ] p y r e n e to intermediates that are bound to cellular macromolecules including DNA. A 75-fold and 9-fold variation between individuals has been found in the binding of b e n z o [ a ] p y r e n e to bronchial cell DNA and alveolar macrophage DNA, respectively [17,18]. Whilst most primary malignancies of the human lung are believed to originate from the bronchial epithelium, some recent reports suggest that a considerable fraction possibly up to 50% may arise in peripheral subpleural regions [1]. Recently, we reported a large interindividual variation in the metabolism of b e n z o [ a ] p y r e n e by short-term organ culture of human peripheral lung from six lung cancer patients [21]. Besides the nature of metabolites formed, other factors such as uptake of benzo[a]pyrene, inter-relationships between activating and deactivating metabolism and the rates of formation and release of free and conjugated metabolites from the cell into the extracellular medium may be crucial in determinmg the levels of reactive intermediates available to interact with cellular macromolecules, and hence, the susceptibility of a particular tissue to polycyclic aromatic hydrocarboninduced carcinogenesis. We have, therefore, investigated the kinetics of formation and covalent binding of b e n z o [ a ] p y r e n e metabolites in short-term organ cultures of human peripheral lung in the presence of different concentrations of benzo[a]pyrene, with particular reference to the distribution of metabolites between the tissue and extracellular medium. MATERIALS AND METHODS
Organ cultures Peripheral lung specimens from lung cancer patients were obtained at the time of surgery. These sepcimens were transported to the laboratory in phosphate buffered saline (lacking calcium and magnesium salts) at 4°C and cultured within 3 h of removal at surgery. Samples of lung (100 -+ 10 mg) that appeared macroscopically normal were cultured for 18 h at 37°C as described previously [21]. The full clinical details of these patients and the ethylacetate-soluble metabolites formed after short-term organ culture of peripheral lung with b e n z o [ a ] p y r e n e (2 uM, final concentration) have been published recently [ 21].
Benzo[a]pyrene metabolism After culture for 18 h, the lung samples were transferred to media containing [3H]benzo[a]pyrene (10--20 ttCi/ml; specific activity, 24 Ci/mmol; Radiochemical Centre, Amersham, U.K.) and unlabelled benzo[a]pyrene (Koch-Light Laboratories Limited, Bucks. U.K.) at final concentrations in the range 1 - 1 0 pM, and incubated in a shaking water-bath at 37°C for periods of up to 24 h. Control incubations containing tissue and similar
347 concentrations of b e n z o [ a ] p y r e n e as the test incubations, were incubated for the same periods at 4°C.
Analysis of ethylacetate-soluble metabolites After culture, the tissue and medium were separated and the tissue homogenised in 2 ml of 1.15% KC1. The tissue homogenate and medium were extracted with 3 × 1 vol. of ethylacetate. The pooled ethylacetate extracts were dried with anhydrous sodium sulphate and concentrated to dryness on a rotary evaporator. The metabolites were redissolved in ethylacetate and separated by high pressure liquid chromatography using a linear methanol/water gradient (1 : 1 to 4 : 1) as previously described [22].
Covalent binding The tissue homogenates, after extraction with ethyl acetate, were stored at - 2 0 ° C until the amount of covalent binding was determined essentially by the m e t h o d of Siekevitz [23] as described previously [24]. RESULTS
Uptake and distribution of benzo[a]pyrene Organ cultures of peripheral lung from patient I were used to study the time course of b e n z o [ a ] p y r e n e metabolism over a period of 24 h. Within 3 h, 56% of the initial radioactivity had been taken up from the medium into lung tissue {Fig. lb). The total activity in the tissue then slowly declined as metabolism of the substrate continued. Examination of the radioactivity in the medium with respect to its association with either ethylacetatesoluble or water-soluble metabolites, or unchanged benzo[a]pyrene, indicated that after 10 h of incubation, 9.3% of the initial activity had been released into the medium as ethylacetate-soluble metabolites, with small b u t measurable amounts being present at the 3- and 6-h time periods (Fig. la). However, during the incubation period, the precentage of unchanged benzo[a]pyrene decreased and the metabolites released had undergone further metabolism and/or conjugation to water-soluble products. Thus, at the end of 24 h, 38.0% of the initial radioactivity in the medium was present as water-soluble metabolites, only 13.6% of the added b e n z o [ a ] p y r e n e remaining unchanged (Fig. la}. Compared to the extraceUular medium, the radioactivity in the tissue associated with ethylacetate-soluble metabolites and especially, watersoluble products, remained low at all time points examined (Fig. lb). Only 5.3% of the added b e n z o [ a ] p y r e n e was found unchanged in the tissue at the end of 24 h.
Time dependence of nature of ethylacetate-soluble
metabolites formed
The ethylacetate-soluble metabolites of b e n z o [ a ] p y r e n e present in the extracellular medium and lung tissue at the various time points studied were analysed by high pressure liquid chromatography (Fig. 2). Benzo[a]pyrene
348 (a) _Medium 100 -
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40:=_ _= 20 ~
(b) Tissue
60-
.> 40-
20~ c
4
8 Incubation
12
2()
16
Time
24
(hr)
Fig. 1. Time course of the percentage of initial radioactivity in (a) extracellular medium and in (b) lung tissue associated with either ethylacetate-soluble metabolites (e o), water-soluble metabolites (o o ) or unchanged b e n z o ( a ~ y r e n e (A ~). Short-term organ culture of peripheral lung (100 _+ 10 rag) from patient I were incubated with benzo[a]pyrene (2 /zM) at 37 °. The tissue, after homogenisation in 1.15% KC1, and the medium were extracted with ethylacetate (3 x 1 vol.). The radioactivity not extracted by ethylacetate is termed water-soluble. The ethylacetate-soluble products were separated by high pressure liquid chromatography, and the values for unchanged [3H]benzo[a]pyrene obtained were subtracted from the total ethylacetate-soluble radioactivity to give the values for ethylaeetate-soluble metabolites.
was metabolised mainly t o ethylacetate-extractable metabolites which coc h r o m a t o g r a p h e d with 9,10-dihydro-9,10-dihydroxybenzo[a] pyrene (9,10-
diol) and 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (7,8-diol). The material eluting in the 9,10-diol region may also consist of 7/8,9-trihydroxy-7,8,9,10pentahydrobenzo[a]pyrene (7/8,9-triol) [17]. The appearance of these
0
m
L-
---J L
350 metabolites and other as yet unidentified metabolites eluting in fractions 3--10 and 14--26 (Peaks I and II respectively; Fig. 2) was very much dependent u p o n the time of incubation. The material eluting in fractions 3--10 contains very polar metabolites such as sulphate conjugates of monoh y d r o x y b e n z o [ a ] p y r e n e s [ 19] and other as y e t uncharacterised metabolites, whilst that eluting in fractions 14 -26 may consist of tetrols [18,25]. After 10 h of incubation with benzo[a]pyrene, significant amounts of only 9,10-diol and 7,8-diol with small amounts of polar metabolites (Peak I) were present (Fig. 2a). However, following incubation for 21 h a major proportion of the radioactivity eluted as metabolites more polar than 9,10diol which probably represent the secondary metabolism of benzo[a]pyrene metabolites though significant amounts of 9,10-diol, 7,8-diol and unmetabolised b e n z o [ a ] p y r e n e were also present. After incubation for 24 h, the radioactivity eluting with 9,10~1iol and 7,8<1iol had been markedly reduced and most of the radioactivity eluted with Peaks I and II (Fig. 2b). Distributional differences o f metabolites between tissue and medium Certain striking qualitative and quantitative differences were also apparent in the distribution of individual ethylacetate-soluble metabolites between the extracellular medium and tissue (Fig. 3). Thus after 24 h incubation, detectable amounts of both 9-OH-BP (17.5 pmol/100 mg tissue) and 3-OH-BP (70.2 p m o l / 1 0 0 mg tissue} were found only in the tissue (Fig. 3b) b u t not in the medium. It must also be remembered that many factors will contribute to the amounts of any metabolite detected at a given time. Of particular importance in this regard will be the rates of formation and further metabolism, both oxidation and conjugation, of a metabolite, the enzyme activity of the tissue and its ability to generate essential cofactors. The polar metabolites (Peaks I and II) were present both in the tissue and medium, the latter having a greater proportion of these metabolites. Small amounts of quinones were detectable in the medium after incubation periods exceeding 10 h. Whereas more of the material associated with the 9,10-diol peak compared with 7,8-diol was observed in the medium, the ratio of 7,8-diol : 9,10-diol in tissue remained high at all the time points examined (Fig. 3). Thus, the ratio of 7,8-diol : 9,10
351
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~
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,
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Fig. 4. Effect of benzo[a]pyrene concentration on ethyl acetate-soluble metabolite production and the distribution of these metabolites in patient II between (a) extracellular medium and (b) lung tissue. Short-term organ cultures of lung (100 f 10 mg) from patient II were incubated with various concentrations of benzo[a]pyrene for 24 h. The ethylacetate-soluble metabolites were extracted and analysed by HPLC as described in legends to Figs. 1 and 2. c----- 0, Polar metabolites eluting in fractions 3-10; a----- l, tetrols eluting in Fractions 14..-26; A-----A, 9,10-dihydro-9,10-dihydroxybenzo[o Jpyrene and/or 7/8,9-triol; A-A, 7,8-dihydro-7,8-dihydroxybenzo[alpyrene.
20(
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353 Almost all the radioactivity from ethylacetate-soluble extracts of short~ term organ cultures of lung from patient II at varying b e n z o [ a ] p y r e n e concentrations (1--10 pM) eluted as either polar metabolites (Peak I), tetrols (Peak II), 9,10-diol and 7,8-diol in agreement with the time course study in patient I (Fig. 2). Whilst the amounts of 9,10-diol and 7,8-diol increased in both the tissue and medium when the b e n z o [ a ] p y r e n e concentration was increased from 1 to 10 pM, the amounts of the more polar metabolites and tetrols (Peaks I and II) appeared to plateau at higher substrate concentrations (Fig. 4a, b). No detectable levels of phenols or quinones were found at any of the benzo[a] pyrene concentrations studied in this patient. In contrast to the observations made in cultures from patient II, in cultures from patient VI, which showed a very low level of metabolism of b e n z o [ a ] p y r e n e (2 uM) (Ref. 21, Table I). Only very small amounts of 7,8-diol and 9,10-diol were detected in the extraceUular medium at all the substrate concentrations examined, with very little or no secondary metabolism of b e n z o [ a ] p y r e n e metabolites. Thus, in patient VI, the maximal levels of 9,10-diol and 7,8-diol detected in the medium were 60 and 70 p m o l / 1 0 0 mg tissue/24 h respectively at 10 pM b e n z o [ a ] p y r e n e , compared to 1400 and 600 p m o l / 1 0 0 mg tissue/24 h respectively in the medium of cultures from patient II at the same concentration of b e n z o [ a ] p y r e n e . Examination of ethylacetate-soluble extracts from patient VI indicated the presence of significant amounts of 3-OH-BP, 9-OH-BP and quinones in the tissue b u t n o t in the medium at all substrate concentrations studied. These
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Benzo(a;pyrene Concentration OJM) Fig. 5. C o v a l e n t b i n d i n g of [ 3 H ] b e n z o [ a ] p y r e n e r e l a t e d r a d i o a c t i v i t y t o s h o r t - t e r m o r g a n c u l t u r e s o f h u m a n p e r i p h e r a l lung. C u l t u r e s of l u n g f r o m p a t i e n t s II a n d V I w e r e i n c u b a t e d for 24 h w i t h various c o n c e n t r a t i o n s o f b e n z o [ a ] p y r e n e . T h e c o v a l e n t l y b o u n d radioa c t i v i t y was m e a s u r e d as d e s c r i b e d in Materials a n d M e t h o d s a n d is d e s i g n a t e d as (e • ) in p a t i e n t II a n d (o o ) in p a t i e n t VI.
354 amounts increased almost linearly with a rise in the concentration of benzo[a]pyrene (results n o t shown). Thus significant differences were observed in the metabolic profiles of b e n z o [ a ] p y r e n e at different substrate concentrations and these differences were apparently dependent to a large extent on the rates of metabolism of b e n z o [ a ] p y r e n e in different individuals.
Covalently bound benzo[a]pyrene and/or its metabolites Up to 0.5% of the initial radioactivity was b o u n d to cellular macromolecules. The amount of covalently b o u n d b e n z o [ a ] p y r e n e and/or its metabolites increased with increasing concentrations of b e n z o [ a ] p y r e n e (1--10 ~M} in short-term organ cultures from patient II, but n o t in those from patient VI, where the covalent binding appeared to have become saturated {Fig. 5). The levels of binding were also comparatively lower in cultures from the patient with lower rate of metabolism, i.e. patient VI (Fig. 5). This was in agreement with the results of Stoner et al. [18] w h o showed a good correlation between the ability of human lung to metabolise b e n z o [ a ] p y r e n e and the a m o u n t of covalent binding to either DNA or protein. DISCUSSION Benzo[a]pyrene was metabolised b y short-term organ cultures of human peripheral lung to ethylacetate-soluble metabolites which co-chromatographed with 9,10-diol, 7,8-diol, tetrols and other more polar metabolites. The nature of the metabolites found depended markedly on the time of incubation, substrate concentration and the inter-individual differences in rates of metabolism. Similar results have been reported by Holder et al. [26] using liver microsomes from untreated and 3-methylcholanthrene pretreated rats and mice. As expected further metabolism of primary metabolites, such as dihydrodiols, to secondary metabolites increased with the duration of incubation (Fig. 2) and was more extensive in lung cultures of patients with more rapid metabolism (Figs. 2--4). Much recent evidence implicates the diolepoxide 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene 9,10-oxide, derived from 7,8-diol, as a major ultimate carcinogenic and mutagenic metabolite derived from b e n z o [ a ] p y r e n e [9,14,16]. Other studies have identified this diol epoxide b o u n d to the exocyclic amino group of guanine as a major adduct b e t w e e n metabolically activated b e n z o [ a ] p y r e n e and human bronchial DNA [27]. Indirect evidence has also been provided to show the same major adduct is formed with DNA of human peripheral lung [28]. Thus factors affecting the further metabolism of primary metabolites such as 7,8-diol may be of critical importance in determining h o w much of a reactive diolepoxide is formed. In this study it was readily apparent that 7,8-diol was further metabolised presumably via the diol-epoxide to tetrols (Peak II, Figs. 2 and 3), in agreement with results ~in cultured rodent trachea [29], cultured human bronchus [25], cultured human peripheral lung [18] and
355 rat liver microsomes [8]. It was of interest in the present study that this further metabolism could occur even in the presence of significant amounts of unmetabolised benzo[a]pyrene (Fig. 2b) suggesting that the affinity of the mixed function oxidase(s) for 7,8-diol might be higher than for benzo[a]pyrene or that different species of cytochrome P-450 may be responsible for the metabolism of benzo[a]pyrene and 7,8-diol. The loss of radioactivity associated with 9,10-diol or 7/8,9-triol over longer periods of incubation (Figs. 2 and 3) could be due to further metabolism of 9,10-diol to either 9,10-dihydroxybenzo[a]pyrene, 1(3),9,10trihydroxy-9,10-dihydrobenzo[a]pyrene or 7,8,9,10-tetrahydro-7,8,9,10t e t r a h y d r o x y b e n z o [ a ] p y r e n e , which have been shown to be metabolites of 9,10-diol in cultured rat trachea, rat liver microsomes and cultured hamster lung respectively [24,29,30]. It would appear (Fig. 3) that 9,10-diol may be a better substrate for further metabolism in human peripheral lung than in either short-term organ cultures of rodent trachea or lung [31]. This may be of some significance as recent studies have indicated that the anti-isomer of 9,10-dihydro-9,10-dihydroxybenzo [ a ] pyrene 7,8-oxide, a diol epoxide intermediate in the metabolism of 9,10-diol to a tetrol, is highly mutagenic to Salmonella typhimurium TA 98 and moderately mutagenic in Chinese hamster V79 cells, although it did not induce malignant transformation in M2 mouse fibroblasts [32,33]. In respect to the possible further metabolism of either 7,8-diol or 9,10diol to potentially reactive diol epoxides, it is also necessary to consider the relative intracellular retentions of these dihydrodiols. It is interesting to speculate that the higher intracellular retention of 7,8-diol compared to 9,10-diol (Figs. 3 and 4) is an additional factor contributing to the higher biological activity of 7,8-diol, by favouring its further metabolism to reactive metabolites rather than its excretion from the cell. Our previous studies have indicated that both isolated rat hepatocytes [34] and hamster embryo cells [35] also show similar distributional differences of metabolites. In order to explain these differences we have investigated the relative binding affinities of different metabolites to ligandin, small molecular weight binding protein A and lipid bilayers as model membrane systems. It appears that the binding of 9,10-diol to protein A and to lipid bilayers but not to ligandin, is significantly lower than the binding of other metabolites and may explain the relative ease of efflux of 9,10-diol from the cell (E. Tipping et al., unpublished data). The release of either of these dihydrodiols into the extracellular medium (Figs. 3 and 4) may also be of significance in vivo especially in peripheral lung tissue since such metabolites from alveolar cells could escape into the circulatory and lymphatic systems to be distributed around the body, including the bronchus which is considered to be the main site of human lung carcinoma. In addition, such metabolites released extracellularly could be taken up by pulmonary alveolar macrophages which whilst transporting these carcinogens up the mucocilliary tree could release them at or near the bronchial epithelium.
356 It was also of interest to note that the monohydroxybenzo[a]pyrenes, 3-OH-BP and 9-OH-BP, were only detectable in the ethyl acetate-extractable fraction of the tissue at the 24-h time point in patient I (Fig. 3) and in the tissue but not the medium at all the concentrations of benzo[a]pyrene studied in patient VI, who had a relatively low rate of metabolism. The retention of monohydroxybenzo[a]pyrenes intracellularly is in agreement with our earlier observations with both isolated rat hepatocytes and hamster embryo cells [34,35]. A possible explanation for the absence of phenols at earlier time ponts in patient I was their further metabolism primarily to sulphate conjugates. We have recently shown that sulphation is a major route for conjugation of phenols in human lung with little or no glucuronide conjugation taking place [22]. Thus part of the radioactivity eluting in peak I (fractions 3--10, Figs. 2--4) will be due to sulphate esters of monohydroxybenzo[a]pyrenes, which are partially organic solvent-soluble and are formed by human lung [19], and also elute in fractions 3--10. The material associated with the water-soluble radioactivity has also been shown to contain some sulphate ester conjugates of monohydroxybenzo[a]pyrenes [22]. Thus the presence of phenols intracellularly at later time points (Fig. 3) may possibly be due either to saturation of sulphate conjugation or to leakage of cofactors or to release of aryl sulphatase from lysosomes. In conclusion, large interindividual variations exist in the extent of benzo[a]pyrene metabolism by short-term organ cultures of peripheral lung from lung cancer patients. In patients exhibiting high rates of metabolism, a range of metabolites, including secondary metabolites' of benzo[a]pyrene metabolites, are produced, their biosynthesis being dependent upon the period of incubation and the concentration of substrate. Certain of these metabolites, namely 3-OH-BP, 9-OH-BP and 7,8-diol, are preferentially retained within the tissue. Although the significance of this selective retention of metabolites is as yet unexplored, intracellular accumulation of metabolites capable of being metabolised to ultimate carcinogenic forms implies serious biological consequences. ACKNOWLEDGEMENTS
The work was supported in part by grants from the Cancer Research Campaign and the Medical Research Council. The reference metabolites of benzo[a]pyrene were kindly supplied by N.C.I. Carcinogenesis Research Program, U.S.A. REFERENCES
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