[CANCER RESEARCH 43, 4856-4862, October 1983] Polycyclic Aromatic Hydrocarbon Toxicity and Induction of Metabolism in Cultivated Esophageal and Epidermal Keratinocytes1 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. 4856 CANCER RESEARCH VOL. 43 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 Quantitäten 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