[CANCER RESEARCH 39, 1980-1984, June 1979]

ABSTRACT

Primary cultures from tracheal explants and a nontumorigenic tracheal epithelial cell line (2Cl), derived from 12-0-tetradecanoylphorbol-1 3-acetate-exposed tracheal explants,metabolized benzo(a)pyrene to qualitatively similar organic solvent-soluble and water-soluble metabolites. Similar metaboliteswere formed by short-term organ cultures of tracheas. In contrast, a tumonigenic tracheal epithelial cell line (1000 W) didnot metabolize benzo(a)pyrene to any significant extent.

The major metabolites formed by these different trachealsystems were 9, 10-dihydno-9, 1O-dihydroxybenzo(a)pyrenewith smaller amounts, mainly as their glucuronide conjugates,of 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene,3-hydroxybenzo(a)pyrene, and 9-hydroxybenzo(a)pyrene. The higherformation of dihydnodiols, precursors of highly reactive diolepoxides, relative to monohydroxybenzo(a)pyrenes, togetherwith low rates of detoxification of the dihydrodiols by conjugation, may in part explain the high susceptibility of the tracheato carcinogenesis.

This study indicates that nontumonigenic respiratory epithehum at different levels of organization possesses similar activating and detoxifying enzymes. Since it is the balance of theseoxidative and conjugating enzymes which determines howmuch of a metabolite is available for reaction with criticalcellular macromolecules, these systems appear to be suitablefor metabolic studies of factors affecting initiation and transformation of respiratory tract epithelium.

INTRODUCTION

In humans, the respiratory tract is the most common targetfor carcinogenic agents known to be of environmental origin ofwhich tobacco smoke is, by far, the most prevalent (31).Considerable efforts have been made to study the mechanismsof tumor induction in this target tissue, using various experimental models (21). A key issue concerns the metabolic activation and detoxification of carcinogens by respiratory tracttissue, more specifically, the epithelium of the conductingairways. Previous studies of our own and several other laboratonies have been concerned with the metabolism of BP@as a

Received November 20, 1978; accepted February 26, 1979.1 Research sponsored jointly by the National Cancer Institute under Intera

gency Agreement 40-5-63, the National Institute of Environmental Health Sciences, and the Office of Health and Environmental Research, United StatesDepartment of Energy, under Contract W-7405-eng-26 with the Union CarbideCorporation, the Medical Research Council, and the Cancer Research Campaignof Great Britain.

2 Permanent address: Department of Biochemistry, University of Surrey, Guild

ford GU25XH, Surrey, England.3To whom requests for reprints should be addressed.4 The abbreviations used are: BP, benzo(a)pyrene: 7,8-dihydrodiol, 7,8-dihy

drodihydroxybenzo(a)pyrene: 9, 10-dihydrodiol, 9, 10-dihydrodihydroxybenzo(a)pyrene: 4,5-dihydrodiol, 4,5-dihydrodihydroxybenzo(a)pyrene: DMBA,

prototype polycyclic carcinogen suspected to be of environmental significance. Such studies have been conducted withhuman bronchus (9, 11, 32) as well as with tnacheas from ratsand hamsters (3, 20, 24), which have been shown to be goodexperimental models for morphological investigations of respiratony tract tumor development (8, 15, 25, 33).

BP is metabolized to a wide variety of both organic solventsoluble metabolites, which include expoxides, monohydroxybenzo(a)pyrenes and their sulfate ester conjugates, dihydrodiols, quinones, diol-epoxides, tniols, and tetrahydrotetnols, aswell as to water-soluble glucuronide and glutathione conjugates (6, 12, 27). Recent evidence strongly implicates theimportance of further metabolism of dihydrodiols (28) andmonohydroxybenzo(a)pyrenes (2) to reactive mutagenic or carcinogenic intermediates (16, 22). The most important of theseappears to be 7,8-dihydnodiol, which is further metabolized toa highly reactive 7,8-dihydroxy-9,1 O-epoxy-7,8,9, 10-tetrahydrobenzo(a)pyrene and which appears to be the major ultimatecarcinogen derived from BP (14). It has been shown that shortterm organ cultures of rodent trachea or human bronchusmetabolize BP to ethyl acetate-soluble metabolites, includingprimarily 9, 10-dihydrodiol with smaller amounts of 7,8-dihydrodiol and little, if any, 4,5-dihydrodiol or monohydroxybenzo(a)pyrenes (3, 4, 32).@More recently, we have shown thatrodent trachea will also metabolize BP to water-soluble glucuronide conjugates mainly of monohydroxybenzo(a)pyrenes(20). When DNA or RNA is isolated from short-term culture ofbronchus (9, 11) or other intact cell systems (9, 27) treatedwith [3HIBP, the major hydrocarbon-deoxyribonucleoside product is the 7,8-dihydroxy-9, 10-epoxy-7,8,9, 10-tetrahydrobenzo(a)pyrene bound predominantly to the exocyclic aminogroup of guanine (23, 30).

In any intact tissue, oncell, xenobiotic metabolism follows anumber of detoxifying and acitivation steps, and it is the balance of oxidative and conjugating enzymes present after treatment with the carcinogen which will determine how much of areactive metabolite will be formed and available for reactionwith critical cellular macromolecules. To pursue this problem,cellular systems in which the relationship between metabolismand quantitative transformation can be correlated are needed.

The purpose of the present investigation was to study themetabolism of BP to both oxidative (primarily ethyl acetatesoluble) and conjugated (primarily water-soluble) metabolitesin cells derived from rat tracheas at different levels of tissueand cell organization. We chose to study: short-term organcultures of tracheas in which the normal histotypic organization

7,12-dimethylbenz(a)anthracene: HPLC, high-pressure liquid chromatography;TPA, 12-O-tetradecanoylphorbol-1 3-acetate; 3-OH-BP, 3-hydroxybenzo(a)-pyrene; 9-OH-BP, 9-hydroxybenzo(a)pyrene; PAH, polyaromatic hydrocarbons.

5M. C. MacLeod, G. M. Cohen, and J. K. Selkirk. The metabolism andmacromolecular binding of the carcinogen benzo(a)pyrene and its relatively inertisomer benzo(e)pyrene by hamster embryo cells, submitted for publication.

1980 CANCER RESEARCH VOL. 39

Comparative Metabolism of Benzo(a)pyrene in Organ and Cell CulturesDerived from Rat Tracheas1

Gerald M. Cohen,2 Ann C. Marchok, Paul Nettesheim, Vernon E. Steele, Fred Nelson, Shilling Huang,and James K. Selkirk3

Biology Division. Oak Ridge National Laboratory, Oak Ridge, Tennessee37830

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BP Metabolism in Tracheal Cultures

of epithelium, connective tissue, and cartilage was maintained;primary cultures from tracheal explants which consist mostlyof epithelial cells; a nontumorigenic pure epithelial cell line(2C1) derived from tracheal explants (29); and a tumonigenicepithelial cell line (1000 W) derived from DMBA-exposed tracheas (19).

The results indicate that these systems, with the exceptionof the neoplastic cell line, possess both oxidative and conjugating enzymes and metabolize BP in a way similar to that ofthe intact trachea. The in vitro cell systems therefore appear tobe well suited for investigations combining the studies of metabolic activation and transformation of respiratory epithelium.

MATERIALS AND METHODS

Chemicals

[3HJBP(25 Ci/mmol) was obtained from Amersham/SearleCorp., Arlington Heights, Ill., and diluted with unlabeled BP(Aldrich Chemical Co., Milwaukee, Wis.) to the appropriateconcentration. Purity was monitored by HPLC and yielded asingle BP peak with purity greaten than 98%.

Media

The primary tracheal cell cultures and cell line cultures weremaintained and labeled in an enriched Waymouth's MB 752/1medium plus 10% fetal bovine serum as described elsewhere(18). This medium includes 10.0 @sginsulin and 0.1 @tghydrocortisone per ml with additional amino acids, sodium pyruvate,fatty acids, and putrescine. All cultures were maintained andlabeled at 37°in water-saturated 5% CO2 and air.

Animals

Specific-pathogen-free Fischer 344 rats born and raised ina barrier facility were used in all experiments. Animals werefed Purina 501 OCautoclavable diet and allowed food and waterad ilbitum.

Short-Term Organ Culture of Tracheal Grafts

To determine the metabolism of BP in the intact trachea, weused tracheal grafts and compared the results with thosepreviously obtained from intact tracheas used immediately afterremoval from rats (4, 20). Tracheas from male Fischer ratswere grafted s.c. into recipient hosts as previously described(8, 15). After 4 weeks, the grafted tnacheas were removed andcultured for 24 hr with 2 @tM[3H]BP in Leibovitz L-15 mediumwith 2 mM L-glutamime. After 24 hr, the medium was removedanalyzed for ethyl acetate-soluble and water-soluble metabolites.

Primary Cultures of Tracheal Epithelial Cells

To determine whether the tissue organization of the intacttrachea was necessary for metabolism, we studied the metabolism of BP by primary cultures of tracheal epithelial cells.Tracheas were removed under sterile conditions, and trachealexplants were prepared as previously described (17). Five 2-x 3-mm tracheal pieces were planted per 60-mm dish andreplanted weekly 3 times. The multiple primary cultures established from the outgrowths of these explants were used at 1

month. Fibroblasts were removed selectively by trypsinizationand/or scraping (18). When the primary tracheal epithelialcells had almost reached confluency, the medium was replacedby a similar medium containing 1.4 @sM[3H]BP and incubatedfor a further 24 hr. The medium was then extracted with ethylacetate and analyzed for organic solvent-soluble and watersoluble metabolites.

Tracheal Epithelial Cell Lines

Finally, we wished to compare the metabolism of BP in botha tumonigenic (1000 W) and a nontumonigenic (2CI) trachealepithelial cell line to determine if such cell lines formed similarmetabolites to primary cultures. The 2Cl cell line was nontumonigenic and was derived from a tracheal explant exposedfor 1 hr on Days 9, 15, 21 , and 27 of organ culture TPA (1.0@.tg/ml).An epithelial outgrowth from this explant was subcultuned 92 days following exposure (see Ref. 29 for details). Theresulting cell line was at passage 20 when used for this metabolism study. Primary cultures from tracheal explants not exposed to TPA oncarcinogen could not be subcultured. The 2CIline did not produce tumors when inoculated at the 27thpassage into immunosuppressed syngeneic hosts.

The I 000 W cells were derived from a tracheal transplantexposed for 2 weeks to 640 @sgDMBA (18). These cells aretumonigenic when inoculated at the 25th passage (19) intoimmunosuppressed animals and were used at the 50th passagein the present study. Both these cell lines were grown almost

to confluency, and then the medium was replaced with freshmedium containing 4@ [3H]BP and incubated at 37°for 24hr. Little toxicity was observed at this concentration of BP with

Chart 1. Metabolism of BP [B(a)pJ to ethyl acetate-soluble metabolites (A) andwater-soluble metabol'des (B) by short-term organ culture of trachea. Trachealgrafts removed after 4 weeks In a host animal were cultured with 2kM [3H] BP Inthe medium for 24 hr. The medium was then extracted with ethyl acetate. Theextracts were dried with sodium sulfate and concentrated, and the ethyl acetatesoluble metabolites (A) were separated by HPLC using a linear 30 to 70%methanol: water gradient. The medium, remaining after the Initial ethyl acetateextractions, was hydrolyzed overnight at 37°wIth $-glucuronldase and thenreextracted with ethyl acetate. The water-soluble glucuronide conjugates, nowreleased as their free unconjugated metabolites (B), were separated by HPLC asdescribed above. 9, 1O-diol, 9,10-dihydrodiol; 7,8-diol, 7,8-dihydrodlol; 3-OH, 3-OH-BP; 9-OH, 9-OH-BP.

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either cell line. The medium was again extracted with ethylacetate and analyzed for metabolites.

Metabolism of BP

Ethyl Acetate-soluble Metabolltes. The ethyl acetate-soluble metabolites from the media were dried with anhydrousNa2SO4,concentrated by rotary evaporation, and separatedusing a Chromatronix 3500 high-pressure liquid chromatograph fitted with a Dupont 1-m ODS Permaphase column.Elution was by reverse phase using a methanol:water gradient(initial, 30% methanol; final, 70% methanol) as previouslydescribed (26), using a sweep time of 30 mm. Metabolitestandards (‘4C)were mixed with extracted metabolites immediately before HPLC and injected simultaneously into the column. Fractions (0.2 ml) were collected and then measured forradioactivity in a Seanle Mark Ill scintillation counter usingAquasol (New England Nuclear, Boston, Mass.) as the countingsolution.

Water-soluble Metabolites. The medium remaining afterethyl acetate extraction contained both water-soluble metabolites and radioactivity covalently bound to macromolecules inthe medium. This medium was reextracted with ethyl acetateto remove traces of [3H]BPthat may have remained in the smallvolume of ethyl acetate-soluble metabolite that was dissolvedin the water. This would have interfered with subsequent anal

ysis of water-soluble metabolites. The medium was divided into2 equal volumes and incubated overnight at 37°in a shakingwater bath, with an equal volume of either f1-glucuronidase or0.1 M acetate buffer (pH 5.0) (3). The incubation mixture wasextracted twice with 1 volume ethyl acetate and analyzed byHPLC.

RESULTS

Short-Term Organ Culture of Tracheal Grafts

Ethyl Acetate-soluble Metabolites. After short-termorganculture (6 to 24 hn)of whole trachea with 2 @tMBP, both watersoluble metabolites and ethyl acetate-soluble metabolites wereobtained at a percentage ratio of 31 :69, respectively. Analysisof the ethyl acetate-soluble metabolites by HPLC showed thatthe major metabolite was 9,10-dihydrodiol (Chart 1A). Smalleramounts of 7,8-dihydrodiol and much smaller amounts of 4,5-dihydrodiol, quinones, and 3-OH-BP were also obtained (Chart1A). These results are in good agreement with our earlierstudies using short-term organ culture of rodent trachea immediately after removal from the animal and thin-layer chromatognaphy to separate the metabolites (4). A major band ofradioactivity also chromatognaphed prior to 9, 10-dihydrodiol(Chart 1A). This material has been shown to contain organsolvent-soluble sulfate ester conjugates of monohydroxybenzo(a)pyrenes, mainly benzo(a)pynen-3-yI hydrogen sulfate,as well as other uncharactenized metabolites, in particular,tniols and tetrahydrotetrols.5 However, we have not yet observed sulfate metabolites in these tracheal systems.

Water-soluble Metabolites. The materials associated withthe water-soluble radioactivity were hydrolyzed with $-glucuronidase, extracted with ethyl acetate (50.8% of the watersoluble radioactivity), and separated by HPLC. The resultingmetabolite profile (Chart 1B) was strikingly different from that

seen following analyses of organic solvent-soluble metabolites(Chart 1A). Mainly 3-OH-BP and quinones, with smalleramounts of 9-OH-BP and 7,8-dihydrodiol and very smallamounts of 4,5-dihydnodiol, but very little or no detectable9,10-dihydrodiol, were found (Chart 1B). The quinones mostprobably have arisen from oxidation of monohydroxybenzo(a)pynenes or BP during either storage or hydrolytic procedunes, since HPLC analysis of the medium following hydrolysiswith acetate buffer detected small amounts of quinones andmuch smaller amounts of monohydroxybenzo(a)pyrene, but nodetectable 7,8-dihydrodiol. Thus, the major water-soluble metabolites obtained from short-term incubation of tracheal graftswith BP are the glucunonide conjugates of 3-OH.-BP,9-OH-BP,and 7,8-dihydrodiol.

Primary Cultures of Tracheal Epithelial Cells

Ethyl Acetate-soluble Metaboiltes. Primaryculturesof tracheal epithelial cells metabolized [3H]BP to ethyl acetate-soluble and water-soluble metabolites at a percentage ratio of 62:37.9, respectively. The ethyl acetate-soluble radioactivity wasconcentrated and separated by HPLC. The major ethyl acetatesoluble metabolite was 9, 10-dihydrodiol with only very smallamounts of 7,8-dihydrodiol, 3-OH-BP, and 9-OH-BP detected(Chart 2A).

Water-soluble Metabolites. The materials associated withthe water-soluble radioactivity were hydrolyzed with /.@-glucuronidase, extracted with ethyl acetate into which was extracted45% of the water-soluble radioactivity, and separated by HPLC.The resulting metabolic profile (Chart 28) was again markedlydifferent from that obtained following analysis of the ethylacetate-soluble metabolites (Chart 2A). Monohydroxybenzo(a)pyrenes, i.e. , 3-OH-BP and 9-OH-BP, were the major metabolites, with significant amounts of both 7,8-dihydrodiol and

0E

Chart 2. Metabolism of 4 @LMBP [B(a)P] by primary cultures of trachealepithelial cells to ethyl acetate-soluble metabolites (A) and water-soluble metabolites (B). The primary cultures of tracheal epithelial cells were prepared fromtracheal explants as described previously (18). The metabolites, after 24 hrincubation, were determined as described in legend to Chart 1. 9. 10-diol, 9,10-dihydrodiol; 7,8-diol, 7,8-dihydrodiol; 3-OH, 3-OH-BP; 9-OH, 9-OH-BP.

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quinones detected, but with no detectable 9,10-dihydrodiol(Chart 2B). The origin of the quinones may again be due tooxidation of monohydnoxybenzo(a)pyrenes or BP. Thus, themajor water-soluble metabolites obtained from primary culturesof tracheal epithelial cells are the glucuronide conjugates of 3-OH-BP, 9-OH-BP, and 7,8-dihydnodiol.

2CICells

Ethyl Acetate-Soluble Metabolites. The 2Cl cells (the nontumorigenic tracheal epithelial cell line derived from exposureof tracheal explants to TPA) also metabolized BP to ethylacetate-soluble and water-soluble metabolites at a percentageratio of 82.8:1 7.2, respectively. The major organic solventsoluble metabolite after 24 hr incubation was again 9,10-dihydnodiol (Chart 3A), with almost no other metabolites foundexcept for very small amounts of 7,8-dihydrodiol.

Water-soluble Metabolites. Hydrolysisof the water-solublemetabolites with $-glucuronidase released 42% of the watersoluble radioactivity, and HPLC analysis again showed that thewater-soluble metabolites (Chart 3B) were different from ethylacetate-soluble metabolites (Chart 3A). Significant amounts of7,8-dihydrodiol, quinones, 9-OH-BP, and 3-OH-BP were found.9,10-Dihydrodiol was not present (Chart 3B). Thus, 2C1cellsare also able to form water-soluble glucuronide conjugates of7,8-dihydrodiol, 9-OH-BP, and 3-OH-BP.

1000 W Cells

This tumonigenic cell line (derived from exposure of trachealtransplants to DMBA), in contrast to the tracheas, trachealprimary cultures, or the TPA-exposed trachea cells, formedinsignificant amounts (<1 % metabolism) of BP metabolites,either ethyl acetate soluble or water soluble (results not shown).

DISCUSSION

Chart 3. MetabolIsm of 4 @&MBP [B(a)P] by 2C1cells (an epithellal cell linederived from TPA-exposed tracheal explants) to ethyl acetate-soluble metabolites(A) and water-soluble metabolites (B). 9, 10-diol, 9,10-dihydrodlol; 7,8-diol, 7,8-dihydrodlol; 4,5-diol, 4,5-dihydrodial; 3-OH, 3-OH-BP; 9-OH. 9-OH-BP.

metabolism of polycyclic hydrocarbons in several biologicalsystems in use in respiratory cancinogenesis studies, since it iscritical to understand the biochemical potential of assay systems that may be used to extrapolate carcinogenic risk tohumans. The major criterion used in this assessment was theability of tracheal epithelial cells to activate and detoxify metabolically, by both oxidative and conjugating reactions, a prototype PAH carcinogen, i.e. , BP. Short-term organ culture oftracheas, primary cultures of tracheal epithelial cells, and anontumonigenic tracheal epithelial cell line (2Cl) all metabolized[3H]BP in a way qualitatively similar to those of ethyl acetatesoluble and water-soluble metabolites. The metabolites obtamed in these studies are also qualitatively similar to thosefound in earlier work (4, 20). Only the tumonigenic cell line,1000 W, was unable to metabolize BP. It has been shown thattransformed liver cell lines are variable in their ability to metabolize PAH (10). Perhaps the ability to metabolize xenobiotics isa highly differentiated function which is repressed in sometransformed cells.

The major ethyl acetate-soluble metabolite formed by all thetracheal systems in the present study was 9,10-dihydrodiol,with much smaller amounts of 7,8-dihydrodiol and very little orno monohydnoxybenzo(a)pynenes (Charts 1A, 2A, and 3A). Instriking contrast, enzymic hydrolysis of the water-soluble fraction with fl-glucuronidase released 3-OH-BP, quinones, and7,8-dihydrodiol as the major metabolites, with smaller amountsof 9-OH-BP and little or no detectable 9,10- on4,5-dihydrodiol.Thus, the major water-soluble metabolites formed by thesedifferent systems are the glucuronide conjugates of 3-OH-BP,9-OH-BP, and 7,8-dihydnodiol. 9,10-Dihydrodiol, formed metabolically by the cells, is a very poor substrate for UDP-glucunonosyltransferase and forms no detectable glucuronide conjugate. This is in agreement with earlier observations usingshort-term organ cutt@preon rodent lung and trachea (3, 20),isolated rat hepatocytes (13), hamster embryo cells (1),5 andmicrosomes fortified with UDP-glucuronic acid (7). These resuIts also suggest that the relative rates of conjugation withUDP-glucuronic acid of the metabolites of BP are monohydroxybenzo(a)pyrenes > 7,8-dihydrodiol@ 9,10-dihydrodiol.

In the systems used in the present study, at the end of theexperiment, most of the material associated with radioactivityin the medium was ethyl acetate-soluble. Thus, dihydrodiols, inparticular 9,10-dihydrodiol with smaller amounts of 7,8-dihydrodiol, are quantitatively the major metabolites formed bytrachea. However, recent studies from our laboratory haveshown important intracellular and extracellular differences inmetabolite profiles (5, 13). In particular, 9,10-dihydrodiol appears to egress very easily into the medium, whereas monohydnoxybenzo(a)pyrenes are retained intracellularly. Thus, funthen studies of intracellular metabolites are required to determine if similar differences in intracellular and extracellularmetabolites are also found with the tracheal cells.

It is of interest that the water-soluble products showed similaramounts of conjugated 7,8-dihydrodiol and monohydroxybenzo(a)pynenes (Charts 2B and 38). This is in marked contrast to our earlier studies which have shown significantly moreconjugated monohydroxybenzo(a)pyrenes than 7,8-dihydrodiol (3, 5, 13, 20). When one considers both ethyl acetatesoluble and water-soluble products, monohydroxybenzo(a)pyrenes are the major metabolites in short-term organ cultuneof hamster lung (3), isolated hepatocytes (13), and hamster

The major aim of the present study was to assess the

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embryo cells,5 whereas dihydrodiols are quantitatively mostimportant in short-term organ culture of rodent trachea (20),primary cultures of tracheal epithelial cells (Chart 2), and 2C1cells, i.e. , the tracheal cell line induced by TPA (Chart 3). Thus,dihydrodiols, in particular 9,10- and 7,8-dihydrodiols, precursonsof highly reactive diol-epoxides, are the major metabolitesformed by the area of the respiratory tract which is highlysusceptible to carcinogenesis. This may, as one of us hassuggested before (4), in part explain the higher susceptibility,particularly if the dihydrodiols are more slowly conjugated,allowing relatively more diol-epoxide to be formed. The pneponderance of dihydrodiols relative to monohydroxybenzo(a)pyrenes may in part be due to a particular species ofcytochrome P.450 (7),

The metabolite profiles obtained by the different respiratorysystems used in this study are also similar to those obtainedfollowing short-term organ culture of human bronchus with[3H]BP (24, 32),@ although the water-soluble metabolitesformed by human bronchus have not yet been investigated.Thus, the above system appears to activate and detoxify PAHin a manner similar to human bronchus. Since our studies showthat primary cultures of tracheal epithelial cells and the nontumonigenic 2CI cells possess the necessary enzymes for metabolic activation of PAH, they would appear to be suitablesystems for transformation studies. Experiments presently inprogress indicate that 2CI cells are indeed transformable byBP and support the use of studies of metabolic activation anddetoxification of carcinogens as a possible means of predictingboth potential biological activity and the suitability of modelsystems for experiments on in vitro transformation.

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1979;39:1980-1984. Cancer Res   Gerald M. Cohen, Ann C. Marchok, Paul Nettesheim, et al.   Cultures Derived from Rat Tracheas

)pyrene in Organ and CellaComparative Metabolism of Benzo(

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