Metabolism of benzo(a) pyrene by human lung microsomal fractions

Life Sciences, Vol . 21, pp . 1629-1636 Printed is the U.S .A .

Pergamon Press

MfiTABOLISM OF BSNZO(a)PYRENE BY HUMAN LUNG MICROSOMAL FRACTIONS R . A. Prough, Z . ~tpa1 1 ' f , and S. ü. Jakobeaone Department of Bioohemiatry, The University of Tex~a Health 3oienoe Center, 5323 Harry Hinea Blvd ., Dallas, Texan 75235 and Department of Forensic Medicine, [Carolinaka Inatitutet, 5-104 01, Stookholm, Stireden (Racelved is final form October 20, 1977) Summary The metabolism of benzo(s)pyrene was studied using miorosomal fraotiona obtained from human lung derived at either reaeotion or autopsy . The rates of metabolism and metabolite distribution were monitored using high pressure liquid chromatography and the metabolic rates were noted to be similar to those obtained using rat lung miorosomea . In oontraet to the rat, human lung mioroaomea appear to form a higher percentage of the 7,8-dihydro-7,8-diol or 9,10-dihydro-9,10-diol of benzo(a)pyrene as a fraction of the total metabolites . However, there was a significant variation among the human lung microaomal preparations which might reflect the olinioal diagnosis and/or individual variation . In recent years it has become apparent that the metabolism of polyoyolio hydrocarbons, such as benzo(e)pyrene, H(a)P, is an obligatory step in oonverting the hydrocarbons into ultimate carcinogens (1) . The dihydrodiol metabolites of B(a)P have been shown to be further metabolized to diol-epoxides which are oapable of reacting with nuoleio acids and proteins to form covaleatly-linked B(a)P-adducts (2-5) . A number of the various metabolites (epoxides, diol-epoxides, and some phenols) have also been shown to be uniquely active as mutagena in bacterial or cultured hamster cell teat systems (6,7) " The chemical reactivity of a number of these metabolites still precludes the identification of the "ultimate" carcinogenic form of H(a)P. However, the possibility that a variation in auaoeptibility to benzo(a)pyrene oarçinogenesis between human lung and rat lung might be due to differences in the metabolic pattern, prompted us to compare the metabolism by lung microsomes from the two species . This report will document that the ratio of dihydrodiol produota to total B(a)P metabolites is mush higher when B(a)P is metabolized by human lung mierosomea oompared to rat lung miorosomea . Materials ~ Methods L7,10- 14C]-Benzo(a)pyrene was purchased from the Ameraham/Searle Corp . and was diluted with unlabeled B(a)P from the Aldrieh Chemioal Co . to a specific radioaotivity of 3-8 mCi/mmole . Authentic B(a)P metabolite standards were obtained from the National Canoer Institute Carcinogeneais Reaearoh Program, Hetheada, Md . All other reagents were purchased commercially in the purest grade available . 1. Present address : Department of Hiochemiatry, Charles University, Prague, 2, Czeohoalovakia .

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12

840

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Han Lung B(a)P Metabolism

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Male Sprague-Dawley rata (150-250 g) were obtained from the Charles River Breeding Laboratory and maintained on lab ohow and water gd libitum. Mioroaomea were prepared from rat lung as previously reported (8) . Hunan male lung (from non-diseased areas) obtained from lung resection or autopsy after sudden death ryas perfuaed with 0 .9~ saline and homogenized in 0 .05 M potassium phosphate buffer, pH T .7 containing 1 mM SDTA and 10x glycerol (v/v), using first a waring blender (20 a) followed by a Potter-Blvehjem homogenizer . After dilution to a 20x (w/v) homogenate, the mixture was filtered through gauze and wea centrifuged using standard differential centrifugation techniques (8), except the centrifugal force used was 78,000xg in Dallas and 105,000xg in Stockholm . The miorosomal pellet was washed by resuapenaion 1n 0.15 M 1< loss of aotivity . Protein concentration was determined by the method of Lowry g.~C ~,,, (9) .

The NADPH-cytochrome g reduntaae aotivity was measured by the method of Mnatera g~ ~ (10) . The apecifio oontent of mioroeomal oytochromea and P-450 xere determined as described by Omura and Sato (11) and Mataubara (12) uaiag an ~minQo DN~2 apeotrophoto~ete~ ; t~e extinction coefficients used were 1 .85 x 10 M- om and 1 .04 x 10 M om , respectively . The procedure used to analyze the metabolism of H(a)P has been described in detail (13) ; the concentration of human or rat lung mioroaomea used in this study was 1 .0-5 .0 mg/ml . Authentic H(a)P derivatives were added to the extracts of the samples in order to identify the B(a)P metabolites ; cochromatography of radioactivity with the added reference compounds is the only criterion for metabolite identification used in this report due to the low amount of metabolites formed by lung microsomes . The radioactivity obtained in the metabolite region of the zero time points was leas than 5-7i and was subtracted from all subsequent time points . The radioactivity recovered in the ethyl acetate phase was >97x and the total recovery on the HPLC analysis was >85x in all cases . The apecifio activity was determined by using the linear portion of several time points between 0 and 30 minutes as described previously (13) . Raaulta Lung microsomea from human tissue derived at autopsy were characterized by comparing several miorosomal components with those reported for rat lung microsomea . Aa noted in Table I, the recovery of lung mioroaomal protein was low compared to that obtained from rat liver (20-30 ng/g wet tissue weight) (14) . The but was similar to the yield noted with rodent lung miorosomea NADPH-oytochrome 4 reduotaae activity was loner than that obtained with rat lung mioroaomea (TO nmolea/min/mg protein) while the apeoific oontent of oytoohrome was similar in human lung and rodent lung microaomea (8,14) . The cytochrom~ P-450 was either not detectable or its content was somewhat lower than that reported in rat lung mioroaomea . Figure 1 shows a reduced-minus-oxidized difference spectra of carbon monoxide-gassed human lung miorosomea . A CO-binding pigment which resembles microsomal oytonhrome P-450 can be detected only by using eztremelq high recorder sensitivity ; a fact that cakes the accurate determination of the apeoific content of eytoehrome P-450 somewhat tenuous . In addition, the presence of another material with an abaorbanoe spectrum in this region can not be excluded . The assay eonditiona for H(a)P metabolism were initially determined using microaomea from rat lung . The optimal pH was fairly broad, pH 7 .4-8 .0, and The assay the effect of protein concentration was linear up to 5 .0 m8/ml . with human lung microsomea was linear with time up to at least 15 min in

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Svmaa Lung B(a)P Metabolism

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TABLE I Activities or Contenta of Human Lung Mierosomal Fractions Activity or Contenta

Dallas

Microsomal Protein Yields NADPH-Cytochrome ç Reductaaeb 0 Cytochrome ~5 Cytochrome P-450° a. b. c. d.

Stockholm

1 .05 t 0 .31 (5) 25 .8 t 7 .3 (5) 0 .11 t 0.030 (5) N .D . -0 .016 (5)

2.27 t 0 .38 (4) 43 " 6 f 15 .9 (4) 0 .082 t 0 .008 (4) 0 .007-0 .012 (4)

mg/g tissue weight after organ perfusion . The Samples in Dallas, Tezas, xere obtained at autopsy and the samples in Stockholm, Sweden were obtained at lung resection . nmolea cytochrome Q rednoed/min/mg mioroeomal protein . nmolea oytoohrome/mg mioroeomal protein . N .D .-not detected .

nearly all oases . The effect of variation of the assay parameters (time, protein concentration, and order of substrate addition) was tested using human lung microsomea and found to be similar to that noted for rat lung . îable II shows the profile and rates of formation of the various olaaaea of B(a)P metabolites isolated Pram incubation mizturea containing lung mioroeomaa obtained from rata pretreated with corn oil (0 .2 ml/animal for three days) . The metabolite profile of the quinones and total dihydrodiola acoounted for 30x and 15-20% of the metabolites, reapeetively, using either rat liver or lung mioroeomaa . However, the ratios of the dihydrodiola are different between the lung and the liver (13) " ühile the percentage of total metabolites represented by the 9,10-dihydrodiol is 1 .8 times larger in liver miorosomes, the percentage of total metabolites represented by the 4,5- and 7,8-dihydrodiola are 3" 5- and 1 .5-fold higher in lung mierosomea relative to liver microsomea . TABLE II The Rates of Formation of Henzo(a)pyrene Metabolites by ~iver and Lung Mieroaomea from Rata Pretreated with Corn 011 Organ Aateb Percent Distribution of Metabolites Dihydrodiola

Quinones

Phenols

Lung

23 .2 °

4 .7

7.8

6 .5

30 .6

16 .0

34 .5

Liver

4720 4

8 .5

2.3

4 .5

31 .6

7.0

46 .2

a.

b. c. d.

The H(a)P metabolites are grouped into three classes of dihydrodiola and two classes of phenols since the radioactive peaks aochromatographed with the authentic standards of the dihydrodiola : 9,10-dihydrodiol (1), 4,5-dihydrodiol and 7,8-dihydrodiol (3) and phenols: (2), 9-phenol (1) and 3-phenol (2) metabolites of benzo(a)pyrene . The values are expressed as picomolea of product formed/min/mg mioroeomal protein . The results represent the average of four preparations with a relative standard deviation of less than 14x. The values are taken from reference 13 and represent the mean of three preparations with a relative standard deviation of leas than 10Z.

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Humas Luag B(a)P Metabolism

Fig .

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1

A The Differenoe Spectrum of Human Lung Microsomal Cytochrome P-450 . suspension of human lung miorosomea (2 .0 mg protein per ml) containing 18 u moles of sodium sucoinate and 6 Vmolea of NADH xas gassed xith carbon After establishing a monoxide and 3 ml xas planed in each of txo cuvettes . baseline of equal light absorption (dashed line), a small amount of solid sodium dithionite xas added to the sample ouvette and a difference spectrum The insert ahoxs the same spectrum recorded at (solid line) xas recorded . higher sensitivity .

Human Luag B(a)P Metabolism

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Table III shows the rates of metabolism of B(a)P oatalyzed by human lung mioroaomea . The rate of B(a)P monoozygenation varies among individuals and is slightly loner with human than with rat lung miorosomea, ezoept in cases 12 and 15 . The rates of formation of the dihydrodiola obtained using human lung the oiorosomea are similar to those obtained from oontrol rata . However, iaoubation mizturea with human lung mioroaomea total dihydrodiola from represent a larger percentage of the total metabolites (approximately 30x) than those obtained from rat lung microsomal fractions (19'x) . In contrasting 7,8- and 9,10-dihydrodiol formation between the two species, the percentage of total metabolites due to benzo-ring dihydrodiola (namely, 7,8- and 9,10-diola) is significantly higher in the samples from reaction mutates with human lung In addition, the rate of formation and x mioroaomea than rat lung (P<0 .01) . of metabolites for the phenols are 20-30x lower using human lung mioroaomea ca®pared to rat lung microsomea which are reflected in the increase in the dihydrodiola ; quinone formation was similar with the two microaomal fractions (with the ezception of cease 12 and 15) " TABLE III The Rates of Formation of Henzo(a)pyrene Metabolites by M~oroaomea From i~man Luag Derived at Autopsy or Lung Resection Sample

Diagnosiab

Rate n

Percent Distribution of Matabolitea Quinoaea

Dihydrodiola

1d 2d e 3 4e 5e 6e 7e 8e e 9 10e 11 e 12f 13f 14f 15f

Adeno as Adeno as CHD Intox ETON (~nahot head Auto CHD Gunshot head Drug CHD CHD Squam ca Undiff ca Undiff ca Squam oa

9.1 15 .4 9 .3

5 .6 6 .9 16 .3 9"2 11 .0 13 .5 15 .5 6 .9 36 .0 13 .1 6 .1 39 .3

1

2

15 .4 4 .5 14 .0

5.5 1 .0 5" 4 1 .8 7 .2 4 .9 14 .1 7 .3 5 .2 4 .5 7 .3 1" 7 3 .1 3"3 1 .8

17 .9 7 .2 7.4 18 .5 8.1 3 .0 11 .0 7 .3 5"3 16 .0 18 .1 12 .0

3 29 .7 6 .5 14 .0 21 .'4 8.7 16 .6 22 .8 10 .0 5 .9 13 " 6 8" 7 5 .3 13 .0 18 .0 17 .3

Phenols 1

14 .3 31 .2 37 .0 35 .7 39 .1 12 .3 25 " 0 41 .8 51 .1 34 " 8 39 " 1 14 .6 23 " 7 26 .2 12 .2

4.4 10 .4 9.7 1 .8 15 " 9 30 .6 2.2 10 .9 16 .3 8 .4 15 .9 46 .7 21 .4 13 " 1 3"3

2 30 .8 45 .5 19 .4 21 .4 21 .7 28 .3 17 .4 21 .8 18 .5 27 .7 21 .7 26 .9 22 :9 21 .3 53 .4

a. See Table II, footnote a . b. Adeno ca -- Adenocaroinoma, CHD -- Coronary heart disease, Intoz ETON -_ Intozication by alcohol, Cunahot head = Death caused by gunshot to head, Auto -_ Death resulting from automobile accident, Drug -_ Death due to drug overdone, Squam ca -Squamoua cell carcinoma, and Undiff oa = Undifferentiated carcinoma. o. The values are expressed ea pioomolea of metabolite formed per min per mg of lung microeomal protein . d. Obtained at lung resection 1n Dallas, Tezaa . e. Obtained at autopsy in Dallas, Texas . f. Obtained at lung resection 1n Stockholm, S~teden .

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Human Lung B(a)P Metabolism

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Diaouasion The preparation of microsomea from human lung tissue can be performed in a manner similar to the procedure for the preparation of rodent lung mioroaomal fractions . The three common microsomal proteins, NADPH-cytochrome ç reductaae, cytochrome , and oytochrome P-450, exist in human lung miarosomea and have differe~ specific activity or content than the same proteins in rodent lung miorosomes . However, the yield of microaomal protein from either human or rat lung is low compared to rodent liver . This probably reflects the large amount of connective and fibrous tissue and the heterogeneity of the cell population in the lung (8,14) . Recently, Seilried g.~ ~ (15) have studied the metabolism of benzo(a)pyrene by miorosomal fractions from lungs of three mouse strains using HPLC teahniquea . The ratio of dihydrodiols to total metabolites formed was greater with microsomes from mouse lung than that noted with mouse liver . In addition, there was a difference noted in the metabolite profile obtained Prom the three strains studied . The increase in dihydrodiol metabolites was postulated to be due to the difference in the relative concentrations of the epoxide hydratase and the cytochrome P-450 ; the relative proportions of the two enzymes, epoxide hydratase to cytochrome P-450-dependent monooxygenaae activity, in liver and lung were 1 .4 and 70, respectively . This report has documented that human lung microsomes form a higher percentage of H(a)P dihydrodiols as metabolites when compared to lung miorosomes from rats . In light of the current studies with the very mutagenio and reactive diol-epoxide metabolites of benzo(a)pyrene, species which have a higher rate of dihydrodiol formation or a larger ratio of dihydrodiol to total metabolites might be more susceptible to polycyolio hydrocarbon-Induced careinogenesia than those species which have lower rates of dihydrodiol formation . The results of this communication would be consistent with the above hypothesis, since human lung is thought to be more susceptible to H(a)P-induced careinogenesia than rat lung . Since the initial steps involved in chemical careinogenesia are currently unknown, any assignment of an "ultimate" carcinogen or description of a difference in metabolic rate which may affect differential susceptibility to hydrocarbon-induced earcinogeneais would be speculative . The variation in the profiles of the various classes of B(a)P metabolites formed by human lung microsomal fractions might also be explained by the clinical diagnosis and/or individual variations among humans . Askno~rle daementa This research was supported in part by NCI Contracts N01-CP-33362 (RAP) and N01-CP-33363 (SÜJ) and by Grants from the NIH (HL 19654, RAP) and from The Robert A . üeleh Foundation (I-616) . RAP is a Research Career Development Awardee, HLCA 00255 . The Authors wish to thank Professors R . W . Satabrook, B. S . S. Masters, Sten Orrenius, and Luis Rodrigues for their encouragement and cooperation . Refereneea 1. 2. 3. 4. 5.

C. HSIDELBSRGBR, Amn . Aev . Bioohem. ~,, 79-121 (1975) " P. SIMS, P. L. GROVSR, A. SüAISLAND, R . PAL, and A . HBWSR, ~ture ~, 326-327 (1974) . R . FI . JENNETTS, A . M . JEFFREY, S. H. BLOBSTEIN, F. A. BSLAND, R. G. HARVEX, and I . H . WSINSTSIN, Biochemiatrv ]~, 932-938 (1977) " S. K. YANG, D. W . MoCOURT, P. P . ROLLSR, and H. V. GSLBOIN, Proo . Nat . Acad . Sai. ysg y3, 2594-2598 (1976) . D. R . THAKICSR, H. YAGI, A. Y. H. LU, il . LEVIN, A. H. CONNSY, and D. M.

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6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 .

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