Polycyclic Aromatic Hydrocarbon Toxicity and Induction of Metabolism in Cultivated Esophageal and Epidermal Keratinocytes1

[CANCER RESEARCH 43, 4856-4862, October 1983]

Ruth Heimann2 and Robert H. Rice3

Charles A. Dana Laboratory of Toxicology, Harvard School of Public Health, Boston, Massachusetts 02115

ABSTRACT cer, and serves as a model subject for study of carcinogen action. Recent experiments have shown that cultured keratino Serially cultivated keratinocytes of human and rat epidermis cytes of human (16, 25, 32) and rodent skin (6, 15) readily and esophagus were compared with respect to their sensitivity metabolize PAH." In these studies, considerable information has to toxic effects of 3-methylcholanthrene and ability to metabolize accumulated on the types of metabolites that are formed from benzo(a)pyrene. 3-Methylcholanthrene was highly toxic to the the carcinogenic agents BP and dimethylbenzanthracene, but human keratinocytes and to early-passage rat epidermal keratin correlating specific products (e.g., DNA adducts) with biological ocytes, as evidenced by markedly reduced growth upon contin effects has not been straightforward. In addition, differences in uous exposure or reduced colony-forming ability after 1-day toxic responses among keratinocytes derived from different ep exposure to concentrations of 0.4 to 40 UMin the culture medium. ithelia have not as yet received much attention. Rat esophageal and late-passage rat epidermal cells appeared Variation among species with respect to carcinogen target site insensitive to 3-methylcholanthrene by these criteria. All the cell and sensitivity is well documented but often enigmatic, due to types except late-passage rat epidermal cells metabolized our limited understanding of the underlying toxic mechanisms. benzo(a)pyrene at comparable rates, and expressed aryl hydro As an initial step in exploring such phenomena, we chose to carbon hydroxylase maximally inducible by similar concentrations study the response of human and rat epidermal and esophageal of 3-methylcholanthrene. Biotransformation in the human cells cells in culture to treatment with PAH. Recently, we have found was greatly inhibited by a-naphthoflavone, a specific inhibitor of that the 2 rat keratinocyte types are serially cultivable in the 3T3 aryl hydrocarbon hydroxylase. The lack of toxicity of 3-methyl feeder layer system and express intrinsic differences in their cholanthrene toward late-passage rat epidermal cells can be properties (14). Thus, since the rat is a common surrogate for attributed to the low constitutive rate of biotransformation these the human in testing chemical carcinogens, these cells are ap cells exhibit. The insensitivity of rat esophageal cells despite propriate for comparative mechanistic studies in culture. substantial metabolic activity reflects the importance of intrinsic differences among keratinocytes derived from different epithelia MATERIALS AND METHODS and species in determining toxic response. Human cervical and monkey esophageal keratinocyte cultures also actively metabo Materials. [3H]BP (25 Ci/mmol, general label, New England Nuclear, lized benzo(a)pyrene, illustrating further the utility of the culture Boston, Mass.) was diluted with unlabeled BP to 3 to 10 mCi/mmol and system for exploring differences among species and epithelial purified (99% pure according to high-pressure liquid chromatography) by cell types. passage through a silica column in benzene (35). Repurification of aliquots was performed periodically by extraction with hexane from 0.1 N KOH:57% dimethyl sulfoxide (34). BP, 3-MC, a-naphthoflavone, and INTRODUCTION indomethacin were purchased from the Sigma Chemical Co. (St. Louis, Mo.), and allylisopropylacetamide was generously provided by Dr. P. Recent improvements in technique now permit serial culture Ortiz de Montellano (University of California, San Francisco). of human keratinocytes (26) which exhibit their normal program Cell Culture. Keratinocytes were serially cultivated (26) with support of terminal differentiation under suitable conditions (10). With of a feeder layer of lethally irradiated mouse 3T3 cells (4x105/60-mm support from a feeder layer of lethally irradiated mouse 3T3, dish) in Dulbecco-Vogt Eagle's medium supplemented with hydrocorti- single cells initiate colonies of stratified squamous epithelium sone (0.4 Mg/ml), cholera toxin (9 ng/ml), and epidermal growth factor (27), from which much has been learned about the biology of (15 ng/ml) (11, 27, 28). Under these conditions, human keratinocytes this cell type (12). Keratinocytes of various squamous epithelia can be grown through numerous subcultures with retention of normal can be cultivated from rabbit (31) and human (1) and retain differentiated character (12, 26). For optimal colony morphology during intrinsic differences in culture (7), expression of which can be routine maintenance, fetal bovine serum concentrations were adjusted to 20% for esophageal and 5% for epidermal and cervical cultures. modulated by agents such as vitamin A (9). Human keratinocytes were grown from trypsin-disaggregated autopsy As the primary constituent of epidermis and of epithelia lining (esophagus) or surgical (epidermis and cervix) tissue samples. The cells the oropharynx, esophagus, and lower female genital tract, the were used between the second and fifth passages without noticeable keratinocyte provides a major extrapulmonary barrier function of alteration in ability to metabolize PAH, consistent with results of others the body against the environment. For this reason, it is a major (25). Monkey esophageal tissue was obtained at autopsy and used in target site of environmentally mediated disease, especially can- secondary culture. Rat (Sprague-Dawley) epidermal and esophageal keratinocytes were cultivated from adult tissues (14). Experiments with ' Supported by USPHS grants CA 27287 from the National Cancer Institute and these cells are segregated according to number of subcultivations, AM 27130 from the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases. 2 Present address: Department of Pharmacology, New York University Medical 4 The abbreviations used are: PAH, polycyclic aromatic hydrocarbons; BP, Center, New York, N. Y. 10016. benzo(a)pyrene; 3-MC, 3-methylcholanthrene; SFM, serum-free medium; REP, rat 3To whom requests for reprints should be addressed. epidermal culture; RES, rat esophageal epithelial culture; HEP, human epidermal Received August 24, 1982; accepted July 6, 1983. culture; HES, human esophageal epithelial culture.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1983 American Association for Cancer Research. Keratinocyte PAH Toxicity and Metabolism Induction indicated by subscript. "Early" and "late" passage designate 2 to 4 and increase in colony-forming efficiency upon serial subcultivation, 17 to 24 subcultivations, respectively, each passage representing ap from typically several % to >30%. This transition, yielding con proximately 10 generations (27). tinuous cell lines, usually occurs within 5 to 15 passages (less Cell Toxicity. One day after seeding the cultures, 20 n\ of dimethyl than 200 generations), and in this work was correlated with the sulfoxide alone (control) or 3-MC or BP dissolved in dimethyl sulfoxide loss by REP of marked sensitivity to inhibition of growth by 3- were added to the medium (4 ml/dish). Stock solutions of PAH yielding MC. Colony-forming efficiencies in the REP3 and REP20pictured 0.04,0.4,4, and 40 MMwere 0.002, 0.02, 0.2, and 2 mg/ml, respectively. The medium was changed at 4-day intervals, and fresh solvent or test in Fig. 2 were approximately 1 and 40%, respectively, as seen solution was added each time. At appropriate times, cultures were fixed in Table 1. In several trials, REP of passages 9 to 12 exhibited and stained with Rhodanile blue (27). Epidermal cultures were treated in both intermediate 3-MC sensitivity and colony-forming efficiency. medium containing 5% fetal bovine serum; esophageal cultures were The selective toxicity of 3-MC toward cultured rat epidermal usually treated in the presence of 20% fetal bovine serum but gave the cells of early passage was also seen in measurements of colony- same response in control experiments using 5% serum. forming ability. Near confluent cultures were treated for 24 hr QuantitÃ¤ten of BP Metabolites. Except as noted, all cultures were with medium containing this agent at 0.4, 4, or 40 UMconcentra treated for 1 day in fresh medium containing 20% fetal bovine serum tions, trypsinized, replated with feeder support, and colony- prior to use in metabolism experiments. Newly confluent cultures were forming efficiencies were then scored. As shown in Table 1, the rinsed carefully with isotonic EDTA solution, removing the few (if any) germinative population of fourth-passage epidermal cells was remaining 3T3 cells and contaminating tissue fibroblasts (30). They were reduced 6-fold, while that of the esophageal cells was not then incubated for 10 min in SFM, given an additional rinse in SFM, and significantly affected, even after 48-hr treatment. These findings finally incubated for indicated times in 2 ml of SFM, to which were added 10 M!of [3H]BP in dimethyl sulfoxide. The extensive washing effectively are entirely consistent with the observations on colony expansion removed serum protein, less than 20 ^g of which was detectable in the upon continuous exposure, including at most a slight effect upon last SFM rinse, compared to approximately 2 mg of cellular protein per REP20. Thus, 3-MC is directly toxic to early-passage REP, with culture (60-mm dish). out a need for mediation by the 3T3 feeder layer. The cultures were solubilized by addition of 0.6 ml of 1 N KOH to the Continuous treatment with 3-MC at 4 ^M was quite effective 2 ml of medium and then transferred to glass test tubes. Dimethyl in inhibiting the growth of HEP and HES, as seen in Fig. 3. A sulfoxide (3.4 ml) was added to each sample with subsequent removal similar result was obtained in one experiment, when HEP were of two 50-Ml aliquots for measurement of protein concentration (2), using Ccomassie Brilliant Blue G-250 reagent (Bio-Rad Laboratories, Rich treated continuously with 4 ^M BP, which is known to be toxic to these cells (32). Microscopically, diagnostic changes in culture mond, Calif.). Aliquots of the remaining 5.9 ml were extracted twice by appearance upon 3-MC exposure included elongation of the cells shaking with 10 ml of hexane, followed by brief centrifugation. In control experiments using [3H]BP in medium without cells, or extracting cultures visible by 3 days of treatment, and partial detachment of colonies immediately after treatment, 99% of the added radioactivity was thus at the edges at later times. In some colonies, a general conver removed. A 1-ml aliquot from the aqueous (lower) phases, containing sion to squamous shape followed by cell detachment took place. free and conjugated metabolites (34), was withdrawn and acidified with Such morphological changes were also evident in early-passage 1 ml of 1 N MCI and submitted to scintillation counting in 13 ml of Aquasol REP, but not RES or late-passage REP. The ability of the human (New England Nuclear). Each experiment was performed at least 3 times, cells to grow upon removal of 3-MC just as changes in cell shape with similar results. Representative experiments are presented, in which were becoming visible was tested. Exposure to 3-MC for 3 days the values reported (in molar equivalents of the BP substrate) are (with subsequent omission from the medium) prevented any averages of duplicate or triplicate cultures, differing from each other by significant further growth of HES (Fig. 3) or HEP, indicating less than 20%. essentially complete loss of germinative capacity by that time. BP Metabolism. To elucidate further the selective toxicity that RESULTS 3-MC exhibited toward the keratinocytes, cellular capacity to metabolize PAH was investigated. For this purpose, the rat cell PAH Toxicity. In initial experiments, early-passage REP were cultures were treated with radioactive BP in SFM, and the treated with 3-MC or BP, starting 1 day after seeding the cells. accumulation of total metabolites was measured as a function As shown in Fig. 1, these agents at 0.4 or 4 /Â¿Mmarkedlyreduced of time. As seen in Chart 1, REP of early (fourth) passage were cell growth over the course of 1 week, compared to control considerably more active in producing metabolites than those of cultures not exposed to PAH. 3-MC and BP were similar in later (21st) passage. The RES of early and late passage had activity and gave small but noticeable effects, even at 0.04 UM. approximately the same activity and were nearly as active as Since 3-MC appeared slightly more active in several trials at the lowest concentrations tested, succeeding work has focused on Table 1 this agent. Colony-forming efficiencies of rat keratinocytes To find whether keratinocytes of different epithelial origin After removal of remaining 3T3 and any tissue fibroblasts with isotonic EDTA uniformly exhibit toxic responses to PAH, RES were also treated (30), near-confluent cultures of REP and RES were treated with or without 3-MC with 3-MC. In contrast to REP, these cells were affected little if in medium containing 5% fetal bovine serum and 0.5% dimethyl sulfoxide for 24 hr (REP,, REPuo, and RES,) or 48 hr (RESM). Upon subsequent trypsinization and at all by the agent at 4 Â¿Â¿M(Fig.2) or 40 /UM(not shown) in culture plating with feeder support, colony-forming efficiency was measured in dilutions of medium supplemented with 5 or 20% serum. The insensitivity of the cells giving 50 to 100 colonies/dish. Each value is the average of 2 or 3 dishes, RES was noted for cultures previously passaged up to 23 times each containing 50 to 100 colonies. Replicate dishes agreed in numbers of colonies within 20%. (RES19 is illustrated in Fig. 2), and thus is a stable property, at 3-MCUM)0 least to that extent. However, the epidermal cells of such ad vanced passage did not retain their original sensitivity and, as 0.4 0.2 17 88 shown in Fig. 2 for REP20, were only slightly inhibited in growth 440REP41.3 0.2 24 8 75 by 3-MC. As described elsewhere (14), rat keratinocytes routinely 0.3REP*,38 22RES466 6RES*,74 82

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4.0 12.0 -

8 12 16 20 24 Time (hr) Chart 1. Accumulation of BP metabolites in rat keratinocyte cultures. The cells were incubated in SFM containing 18 Â¿IM[3H]BP for the times indicated. The metabolite content (medium plus cells) was quantitated by solvent extraction, as described in "Materials and Methods." early-passage REP cells. Close inspection of the curves indicated that the late-passage REP metabolized BP at a constant slow rate, whereas the other cultures exhibited a more rapid accu mulation of metabolites, starting about 2 hr after treatment began. In 2 experiments, early-passage REP and RES differed little in production of relative amounts of water-soluble metabo 24 48 lites (conjugates and adducts). After treatment for 24 hr with [3H]BP, the cultures were disrupted by sonication and extracted Time (hr) Chart 2. Accumulation of BP metabolites in human and monkey keratinocyte with ethyl acetate:acetone (2:1), leaving similar amounts of total cultures, a, cells incubated in SFM containing 18 ^M [3H]BP for the times indicated metabolites (approximately 85%) in the aqueous phase. prior to BP metabolite quantitation by solvent extraction. hEs + CY, cycloheximide Chart 2a shows that keratinocytes from human epidermis and (10 *ig/ml) added concomitant with BP at 0 hr. hCv, human cervical epithelial cultures; mÂ£s,esophageal epithelial cultures from M. mulatta (rhesus monkey). esophagus produced metabolites of BP at rates comparable to Inset: comparison of HES cultures without (O) and with (O) 24-hr 3-MC pretreat those of early passage from the rat (Chart 1). Also illustrated is ment. In the latter, 3-MC (4 *IM) was added to fresh medium containing 20% serum the substantial accumulation of metabolites in human cervical in which the cells were incubated for 1 day prior to [3H]BP exposure in SFM. o, cultures were treated as in a, except that at the indicated times the medium (A, and monkey esophageal keratinocyte cultures, emphasizing that HEP; O, HES) and rinsed cells (A, HEP; Â», HES) were analyzed separately for this cell type in general exhibits considerable activity in biotrans metabolite content, c, HEP and HES were treated with 3-MC (4 /IM) in medium containing 20% serum for the times indicated. The cultures were then rinsed, formation. In each case, the rate of metabolism was low for the incubated for 2 hr with 9 >IM[3H]BP in SFM, and analyzed for metabolite content first 3 hours, and then increased markedly. This increase was by solvent extraction. In such experiments, protein content of the dishes usually also apparent in experiments where the medium and cells were differed by <10%. analyzed separately for metabolite content. As seen in Chart 2o for HES and HEP, the majority of metabolites were associated that attained after ~5 hr in the absence of pretreatment (Chart with the cells at the earliest times measured (5 hr), but the 2a, inset). The rate at which the enzyme activity increased was amounts in the 2 compartments rose in parallel to reach similar measured for HEP and HES. As shown by representative exper final totals. Examination of RES gave the same result (not iments in Chart 2c, 2 to 5 hr were required to reach half-maximal shown). activities, and the maximal activities observed remained constant Induction of Metabolism. PAH such as benz(a)anthracene for at least 2 days. The observed rise in activity occurred rapidly gain entrance to cultured cells and reach equilibrium levels somewhat earlier than that seen in Chart 2a, since at a given about 30 min after addition to the medium (21). Hence, the 5- to time point the cultures in Chart 2c have actually been exposed 10-hr time for maximal rates of biotransformation of BP to occur to inducing levels of PAH for 2 hr longer. In such experiments, a appears to reflect BP induction of aryl hydrocarbon hydroxylase. rise in activity to a comparable degree was evident in cultures Several observations support this finding. First, the low basal treated in serum-containing medium or SFM. rate of BP metabolism in HES (shown in Chart 2a) and RES (not As further evidence that the induced activity corresponds to shown) did not increase in the presence of cycloheximide, con aryl hydrocarbon hydroxylase, HES and HEP cultures exposed sistent with a requirement for protein synthesis. Second, when to a-naphthoflavone, a specific inhibitor of this form of cyto- the cells were pretreated with 3-MC, a potent inducer of aryl chrome P-450 (5), were inhibited in biotransformation of BP. hydrocarbon hydroxylase, the initial rate of BP metabolism was Virtually no metabolites were detectable at concentrations of this greatly enhanced and reached approximately the same level as agent greater than 50 MM,as seen in Table 2 for HES. The 50%

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Table2 Inhibition of aryl hydrocarbon hydroxylase Accumulation of [3H]BP metabolites by HES during 24 hr in the presence of a- 300 naphthoflavone or 5 hr in the presence of allylisopropylacetamide. a-Naphthofla- vone was added with, and allylisopropylacetamide 1 hr prior to, the substrate. The protein content of treated cultures differed from that of controls by <15%. metabolites Inhibitoro-NaphtnoflavoneAllylisopropylacetamideConcentration(/IM)00.11.01010007707007000BP(nmol/mgprotein)7.95.73.40.2<0.10.280.360.300.290.19

effective concentration was estimated at 0.7 /Â¿M,inthe range found effective with HEP, where this agent is not cytotoxic up to 10 MM(25), and with microsomal preparations (5). Allylisopro pylacetamide, a suicide substrate specific for drug-inducible cytochrome P-450 (23), was ineffective over the range of 7 /Â¿Mto 7 HIM, as shown in Table 2. In some cell systems, BP is metabolized by prostaglandin cyclooxygenase (29). The specific inhibitor of this enzyme, indomethacin, did not reduce the ob served metabolism in HES over the concentration range of 1 /XM to 1 mM (data not shown). None of these agents produced any detectable effect on cell appearance, cell detachment, or colony morphology during treatment. In preliminary experiments with esophageal cultures, 7-ethoxycoumarin-O-deethylase activity 100 (13) was inducible by 3-MC but not by phÃ©nobarbital (which 3-MC (/Â¿M) appeared highly toxic). Charts. Induction of aryl hydrocarbon hydroxylase as a function of 3-MC concentration. The rat (a) and primate (b) cultures were treated for 24 hr with 3- The inducibility of aryl hydrocarbon hydroxylase was measured MC at the indicated concentrations in 2 ml of SFM. [3H]BP was added directly to as a function of 3-MC concentration. Following 1-day treatment the medium (9 ^M), and after 2 hr the content of radioactive metabolites was of newly confluent cultures with 3-MC at various concentrations, measured. mEs, esophageal epithelial cultures from M. mulatta (rhesus monkey). [3H]BP was added for 2 hr, and the metabolites produced were quantitated. As shown in Chart 3a, maximal activity in BP me shown that differences in cytotoxicity of BP or 7,12-dimethyl- tabolism was obtained at an inducer concentration of approxi benz(a)anthracene in culture generally are correlated with degree mately 4 /IM. The RES of early and late passage and REP of of metabolism of the agent (4, 17), and not with differences in early passage were inducible to a similar extent. Variation among its uptake (4, 24). The finding that REP of late passage are experiments, with little alteration in the shapes of the curves, insensitive to the toxic effect of 3-MC is thus attributable to appeared due mainly to differences in basal activities without cellular loss of inducible aryl hydrocarbon hydroxylase activity. added inducer. Thus, the ratio of maximally induced to basal activities varied from 3- to 10-fold. The esophageal and epidermal This loss has been noted in the 2 REP lines we have passaged extensively (both at >20 subcultivations), and appears analogous curves were distinguishable with respect to the activity exhibited to loss of metabolic capacity by fetal hamster cells (22) and a at the highest concentration of inducer used, where the reduced number of transformed lines (3). Our finding is consistent with activity of epidermal cultures may be a manifestation of toxic effects. In contrast, late-passage REP did not exhibit significant the suggestion that preneoplastic epidermal cells in experimental animals may lack cytochrome P-450 activity (8), at least poten inducibility, consistent with the low basal rate of metabolite tially allowing for selective growth during promotion by certain accumulation seen in Chart 1. Cultures of HES and HEP, as well toxic chemicals in vivo, as is well documented in rat liver (33). as those from human cervix (not shown) and monkey esophagus, The present results with esophageal and epidermal cells reveal exhibited similar responses with respect to induction of aryl hydrocarbon hydroxylase upon treatment with 3-MC (Chart 30). 2 important differences between them that are relevant to their interaction with PAH. First, the 2 keratinocyte types from the rat The ratios of maximal activities at about 4 Â¿IMinducerto control uninduced activities were approximately 10-fold in most experi in early passage exhibit markedly contrasting growth responses to 3-MC, which are not attributable to significant differences in ments. aryl hydrocarbon hydroxylase activity. Insensitive to the toxic DISCUSSION effects seen in the other cultures, RES did not appear much different in proportion of total metabolites either in water- versus Many carcinogenic agents, including PAH, require metabolic organic-soluble or extra- versus intracellular fractions. However, activation for expression of their acute or chronic toxic effects this contrast in response could reflect different formation or (20). Consistent with this principle, surveys of cell lines have deactivation of specific metabolites, interaction of the metabolites

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with different macromolecular targets, or differential repair of formation of 7,12-dimethylbenz(a)anthracene by mouse epidermal cells in culture. Carcinogenesis (Lond.), 1: 41-49,1980. such damage. Thus, examination of qualitative and quantitative 7. Doran, T. I., Vidrich, A., and Sun, T-T. Intrinsic and extrinsic regulation of the differences in profiles of the various free and conjugated metab differentiation of skin, corneal and esophageal epithelial cells. Cell, 22:17-25, olites, of specific adducts, and of the macromolecules damaged 1980. 8. FÃ¤rber,E., and Cameron, R. The sequential analysis of cancer development. may assist in elucidating the reactions responsible for observed Adv. Cancer Res., 31: 125-226, 1980. toxicity. In addition to toxic responses to 3-MC in early passage, 9. Fuchs, E., and Green, H. Regulation of terminal differentiation of cultured human keratinocytes by vitamin A. Cell, 25:617-625,1981. RES and REP also differed in inducibility of aryl hydrocarbon 10. Green, H. Terminal differentiation of cultured human epidermal cells. Cell, 77: hydroxylase at late passage. Three continuous lines of RES, one 405-415, 1977. derived from the same rat as the 2 REP continuous lines, retained 11. Green, H. Cyclic AMP in relation to proliferation of the epidermal cell: a new view. Cell, 75: 801-811, 1978. expression of this metabolic activity. 12. Green, H. The keratinocyte as differentiated cell type. Harvey Lect., 74:101- The present results emphasize the importance of intrinsic 138,1980. cellular differences in target specificity of carcinogenic agents, 13. Greenlee, W. F., and Poland, A. An improved assay of 7-ethyoxycoumarin O- deethylase activity: induction of hepatic enzyme activity Â¡nC57BL/65 and even among keratinocytes from different epithelia of the same DBA/25 mice by phÃ©nobarbital, 3-methylcholanthrene and 2,3,7,8-tetrachlo- animal. The evident contrast in sensitivity between rat esophagus rodibenzo-p-dioxin. J. Pharmacol. Exp. Tner., 205: 596-605,1978. and epidermis in vivo to nitrosamine-induced carcinogenesis (18), 14. Heimann, R., and Rice, R. H. Rat esophageal and epidermal keratinocytes: intrinsic differences in culture and derivation of continuous lines. J. Cell. for example, plausibly reflects such differences. As illustrated by Physiol., in press, 1983. the contrast between RES and HES in sensitivity to 3-MC in the 15. Irmscher, G., and Fusenig, N. E. Metabolism of 7,12-dimethylbenzanthracene (DMBA) by mouse skin keratinocytes, fibroblasts, and carcinoma cells in present work, the uncertainties in extrapolating observations culture. Arch. Toxicol., 44: 181-195,1980. with a given epithelium among species reflect differences of 16. Kuroki, T., Nemoto, N., and Kitano, Y. Metabolism of benzo(a)pyrene in human similar magnitude. Study of metabolic activity and toxic mecha epidermal keratinocytes in culture. Carcinogenesis (Lond.), 7: 559-565,1980. 17. Landolph, J. R., Becker, J. F., Camper, H., Bartholomew, J. C., and Calvin, M. nisms these cells exhibit in culture promises to help elucidate Biochemical basis for the acquisition of resistance to benzo(a)pyrene in clones the biochemical bases of such conspicuous diversity, perhaps of mouse liver cells in culture. Chem.-Biol. Interact., 23: 331-344,1978. 18. Magee, P. N., Montesano, R., and Preussman, R. N-Nitroso compounds and reducing uncertainties in interspecies extrapolation, and facilitat related carcinogens. In: C. E. Searie (ed.), Chemical Carcinogens, pp. 491- ing choice of appropriate animal models for chemical testing. 625. Washington D. C.: American Chemical Society, 1976. Expression of substantial metabolic activity by human cervical 19. McDowell, E. M., Becci, P. J., Barrett, L. A., and Trump, B. F. Morphogenesis and classification of lung cancer. In: C. C. Harris (ed.), Pathogenesis and and monkey esophageal epithelial cells supports further the utility Therapy of Lung Cancer, pp. 445-519. New York: Marcel Dekker, 1978. of the culture system for such an approach. 20. Miller, E. C. Some current perspectives on chemical carcinogenesis in humans Keratinocytes of the stratified squamous epithelia are recog and experimental animals: presidential address. Cancer Res., 38:1479-1496, 1978. nized as major targets for carcinogenic agents, and for this 21. Nebert, D. W., and Bausserman, L. L. Fate of inducer during induction of aryl reason study of their properties in model systems is important. hydrocarbon hydroxylase activity in mammalian cell culture. I. Intracellular entry, binding, distribution and metabolism. Mol. Pharmacol., 6: 293-303, However, the findings in culture experiments may have more 1970. general applicability to neoplastic processes in epithelia in which 22. Nebert, D. W., and Gelboin, H. V. The in vivo and in vitro induction of aryl the keratinocyte is not the major cell type. A specific example hydrocarbon hydroxylase in mammalian cells of different species, tissues, strains, and developmental and hormonal states. Arch. Biochem. Biophys., concerns the keratinocytes arising in regions of squamous me 734: 76-89, 1969. taplasia and having biochemically distinctive character, com 23. Ortiz de Montellano, P. R., Mico, B. A., and Yost, G. S. Suicidal inactivation of pared to nonstratifying surrounding cells. The possibility merits cytochrome P-450. Formation of heme substance covalent adduct. Biochem. Biophys. Res. Commun., 83:132-137,1978. consideration that these cells may serve as actual targets in the 24. Paraskeva, C., and Parkinson, E. K. Sensitivity of normal and transformed rat generation of squamous cell or epidermoid carcinomas. Such a liver epithelial cells to benzo(a)pyrene toxicity. Carcinogenesis (Lond.), 2: 483- hypothesis is consistent, for instance, with the usual association 487,1981. 25. Parkinson, E. K., and Newbold, R. F. Benzo(a)pyrene metabolism and DNA of epidermoid carcinoma with squamous metaplasia in the lungs adduct formation in serially cultivated strains of human epidermal keratino of humans and experimental animals exposed to cigarette smoke cytes. Int. J. Cancer, 26: 289-299, 1980. 26. Rheinwald, J. G. Serial cultivation of normal human epidermal keratinocytes. or similar agents (19). Methods Cell Biol., 27: 229-254,1980. 27. Rheinwald, J. G., and Green, H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell, 6: ACKNOWLEDGMENTS 331-344,1975. 28. Rheinwald, J. G., and Green, H. Epidermal growth factor and the multiplication We thank Drs. J. J. Godleski, H. C. Grillo, L. Reid, and F. G. Vawterfor providing of cultured human epidemial keratinocytes. Nature (Lond.), 265: 421-424, human esophageal tissue samples, Drs. R. T. Branson and K. C. Hayes for 1977. esophageal samples from Macaca mulatta (rhesus monkey), and Dr. E. Eisenstadt 29. Sivarajah, K., Mukhtar, H., and Eling, T. Arachidonic acid-dependent metabo for valuable suggestions. lism of (Â±) frans-7,8-dihydroxy-7,8-dihydro-benzo(a)pyrene (BP-7,8-diol) to 7,10/8,9 tetrols. FEBS Lett., 706: 17-20,1979. 30. Sun, T-T., and Green, H. Differentiation of the epidermal keratinocyte in cell REFERENCES culture: formation of the cornified envelope. Cell, 9: 511-521,1976. 31. Sun, T-T., and Green, H. Cultured epithelial cells of cornea, conjunctiva and 1. Banks-Schlegel, S., and Green, H. Involucrin synthesis and tissue assembly skin: absence of marked intrinsic divergence of their differentiated states. by keratinocytes in natural and cultured human epithelia. J. Cell Biol., 90:732- Nature (Lond.), 269: 489-493,1977. 737,1981. 32. Theall, G., Eisinger, M., and Grunberger, D. Metabolism of benzo(a)-pyrene 2. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram and DNA adduct formation in cultured human epidermal keratinocytes. Carcin quantities of protein utilizing the principle of protein-dye binding. Anal. ogenesis (Lond.), 2: 581-587,1981. Biochem., 72: 248-254, 1976. 33. Tsuda. H., Lee, G., and Farber, E. Induction of resistant hepatocytes as a new 3. Diamond, L. The interaction of chemical carcinogens and cells in vitro. Prog. principle for a possible short-term in vivo test for carcinogens. Cancer Res., Exp. Tumor Res., 11: 364-383,1969. 40:1157-1164,1980. 4. Diamond, L. Metabolism of polycyclic hydrocarbons in mammalian cell cultures. 34. Van Cantfort, J., DeGraeve, J., and Gielen, J. E. Radioactive assay for aryl Int. J. Cancer, 8: 451 -462, 1971. hydrocarbon hydroxylase. Improved method and biological importance. 5. Diamond, L., and Gelboin, H. V. Alpha-naphthoflavone: an inhibitor of hydro Biochem. Biophys. Res. Commun., 79: 505-512, 1977. carbon cytotoxicity and microsomal hydroxylase. Science (Wash. D. C.), 766: 35. Yang, S. K., Gelboin, H. V., Trump, B. F., Autrup, H., and Harris, C. C. 1023-1025,1969. Metabolic activation of benzo(a)pyrene and binding to DNA in cultured human 6. DiGiovanni, J., Viaje, A., Fischer, S., Slaga, T. J., and Boutwell, R. K. Biotrans bronchus. Cancer Res., 37: 1210-1215,1977.

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3-MC (pM) 0.04 0.4 4

Control 0.04 0.4 BP(pM) Fig. 1. Toxic effect of PAH on growth of REP3. The epithelial cells were seeded at 105/60-mm dish, treated with PAH at the concentrations indicated, fixed, and stained after 7 days of treatment.

3-MC rEPi rEs< rEs 19

Fig. 2. Comparative effect of 3-MC (4 ^M) on growth of REP and RES. The cultures were seeded with 10s (REP3),100 (REPM), 7 x 104(RES3), or 600 (RES,9) cells and treated with 20 Â¡Aofdimethyl sulfoxide containing 0 (-) or 4 ^g (+) of 3-MC. The cultures, grown with 5%(REP and RES,9) or 20%(RES3) serum supplementation, were fixed and stained after 7 days of treatment.

OCTOBER 1983 4861

3-MC hEp hEsÂ£ hEs1

Fig. 3. Toxic effect of 3-MC (4 ^M) on growth of HEP and HES. Cultures were seeded with 10* cells and maintained in medium supplemented with 5% serum. HEP and one set of HES (r)Â£s*)were exposed continuously to 3-MC or solvent alone for 7 and 12 days, respectively, before fixing and staining. Another set of HES (Mrs") was treated with 3-MC or solvent alone for 3 days, rinsed, given medium without 3-MC for 8 days, and then fixed and stained.

4862 CANCER RESEARCH VOL. 43

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1983 American Association for Cancer Research. Polycyclic Aromatic Hydrocarbon Toxicity and Induction of Metabolism in Cultivated Esophageal and Epidermal Keratinocytes

Ruth Heimann and Robert H. Rice

Cancer Res 1983;43:4856-4862.

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