ABSTRACT Polycyclic aromatic hydrocarbons (PAHs) induce cytochrome P-450 monoxygenase enzymes that catalyze the formation of DNA adducts. We investigated the effects benzo(α)pyrene (B[α]P) alone or in combination with ethanol on normal human keratinocyte (NHK) growth, induction of cytochrome P-4501A1 (CYP1A1), and modulation of these treatments by retinoic acid (RA) in a serum-free culture medium. Growth-arrested confluent NHK serum-free cultures were treated with B[α]P alone or in combination with ethanol and RA. The effects on CYP1A1 enzyme activity were investigated. B[α]P treatment alone was not toxic to post-confluent cells; sub-toxic ethanol stimulated cell growth regardless B[α]P treatment. No CYP1A1 activity was detected in control or ethanol-treated NHK cell cultures. B[α]P alone induced CYP1A1 activity, and B[α]P plus ethanol treatment further enhanced B[α]P-induced CYP1A1 activity. Pretreatment with all-trans-RA (t-RA) abolished ethanol enhancement of CYP1A1 activity. There is a synergistic action of ethanol in combination with PAH on induction of P-450 cytochrome enzymes. By contrast, RA reverses ethanol enhancement implying a role for retinoid therapy in counteracting the risk posed by combined alcohol and PAH exposure on epidermal cell carcinogenesis.
Cite this paper
J. Wille and J. Park, "Retinoid and Ethanol-Sensitive Benzo(α)Pyrene Induction of Cytochrome P450 in Human Keratinocytes," Journal of Cancer Therapy, Vol. 3 No. 6, 2012, pp. 1080-1085. doi: 10.4236/jct.2012.36141.
 L. Loeb and C. Harris, “Advances in Chemical Carcinogenesis: A Historical Review and Prospective,” Cancer Research, Vol. 68, No. 17, 2008, pp. 6863-6890.
 X. Ding and L. Kaminski, “Human Extrahepatic Cytochromes P450: Function in Xenobiotic Metabolism and Tissue-Selective Chemical Toxicity in the Respiratory and Gastrointestinal Tracts,” Annual Review of Pharmacology and Toxicology, Vol. 43, 1993, pp. 149-173.
 Q. Ma and A. Lu, “CYP1A Induction and human risk assessment: An Evolving Tale of in Vitro and in vivo Studies,” Drug Metabolism and Disposition, Vol. 3, No. 5, 2007, pp.1009-1016. doi:10.1124/dmd.107.015826
 K. Tatematsu, A. Koide, M. Hirose, A. Nishikawa and Y. Mori, “Effect of Cigarette Smoke on Mutagenic Activation of Environmental Carcinogens by Cytochrome P450 2A8 and Inactivation by Glucuronidation in Hamster Liver,” Mutagenesis, 2010.
 T. Shimada, Y. Oda, E. Gillam, F. Guengerich and K. Inoue, “Metabolic Activation of Polycyclic Aromatic Hydrocarbons and Other Procarcinogens by Cytochromes P450 1A1 and P450 1B1 Allelic Variants and Other Human Cytochromes P450 in Salmonella typhimurium NM2009,” Vol. 29, 2001, pp. 1176-1182.
 M. Finnen, C. Lawrence and S. Shuster, “Human Skin Aryl Hydrocarbon Hydroxylase,” British Journal of Dermatology, Vol. 110, No. 3, 1984, pp. 339-342.
 J. Reiners , Jr, A. Cantu and A. Pavone, “Modulation of Constitutive Cytochrome P-450 Expression in Vivo and in Vitro in Murine Keratinocytes as a Function of Differentiation and Extracellular Ca2+ Concentration,” Proceedings of the National Academy of Sciences, Vol. 87, No. 5, 1990, pp. 1825-1829. doi:10.1073/pnas.87.5.1825
 J. Guo, R. Brown, C. Rothwell and I. Bernstein, “Levels of Cytochrome P-450-Mediated Aryl Hydrocarbon Hydroxylase (AHH) Are Higher in Differentiated than in Germinative Cutaneous Keratinocytes,” Journal of Investigative Dermatology, Vol. 94, No. 1, 1990, pp. 86-93.
 T.Kometani, I. Yoshino, N. Miura, H. Okazaki, T. Ohba, T. Takenaka, F. Shoji, T. Yano and Y. Maehara, “Benzo-[a]pyrene Promotes Proliferation of Human Lung Cancer Cells by Accelerating the Epidermal Growth Factor Receptor Signaling Pathway,” Cancer Letters, Vol. 278, No. 1, 2009, pp. 27-33. doi:10.1016/j.canlet.2008.12.017
 W. Jiang, L. Wang, S. Kondraganti, I. Fazili, X. Couroucli, E. Felix and B. Moorthy, “Disruption of the Gene for CYP1A2, Which Is Expressed Primarily in Liver, Leads to Differential Regulation of Hepatic and Pulmonary Mouse CYP1A1 Expression and Augmented Human CYP1A1 Transcriptional Activation in Response to 3-Methylcholanthrene in Vivo,” Journal of Pharmacology and Experimental Therapeutics, Vol. 201, 2010, pp. 369-379. doi:10.1124/jpet.110.171173
 I. Khan, D. Bickers, T. Haqqi, and H. Mukhtar, “Induction of CYP1A1 mRNA in Rat Epidermis and Cultured Human Epidermal Keratinocytes by Benz(a)Anthracene and Beta-Naphthoflavone,” Drug Metabolism and Disposition, Vol. 20, No. 5, 1992, pp. 620-624.
 B. Allen-Hoffmann and J. Rheinwald, “Polycyclic Aromatic Hydrocarbon Mutagenesis of Human Epidermal Keratinocytes in Culture,” Proceedings of the National Academy of Sciences, Vol. 81, No. 24, 1984, pp. 7802-7806. doi:10.1073/pnas.81.24.7802
 M. Stampfer and J. Bartley, “Induction of Transformation and Continuous Cell Lines from Normal Human Mammary Epithelial Cells after Exposure to Benzo[a]pyrene,” Proceedings of the National Academy of Sciences, Vol. 82, No. 8, 1985, pp. 2394-2398.
 J. Park, J. Muscat, Q. Ren, S. Schantz, R. Harwick, J. Stern, V. Pike, J. Richie Jr and P. Lazarus, “CYP1A1 and GSTM1 Polymorphisms and Oral Cancer Risk,” Cancer Epidemiology Biomarkers & Prevention, Vol. 6, No. 10, 1997, pp. 791-797.
 B. Rodu and C. Jansson, “Smokeless Tobacco and Oral Cancer: A Review of the Risks and Determinants,” Critical Reviews in Oral Biology & Medicine, Vol. 15, No. 5, 2004, pp. 252-263. doi:10.1177/154411130401500502
 N. Maserejian, K. Joshipura, B. Rosner, E. Giovannucci and A. Zavras, “Prospective Study of Alcohol Consumption and Risk of Oral Premalignant Lesions in Men,” Cancer Epidemiology, Biomarkers & Prevention, Vol. 15, No. 4, 2006, pp. 774-781.
 M. Sporn, N. Dunlop, D. Newton and J. Smith, “Prevention of Chemical Carcinogenesis by Vitamin A and Its Synthetic Analogs (Retinoids),” The FASEB Journal, Vol. 35, 1976, pp. 1332-1338.
 G, Zhou G, M. Richardson, I. Fazilil, J. Wang, K. Donnelly, F. Wang, B. Amendt and B. Moorthy, “Role of Retinoic Acid in the Modulation of Benzo(a)pyrene-DNA Adducts in Human Hepatoma Cells: Implications for Cancer Prevention,” Toxicology and Applied Pharmacology, Vol. 249, No. 3, 2010, pp. 224-230.
 P. Mrass, M. Rendl, M. Mildner, F. Gruber, B. Lengauer, C. Ballaun, L. Eckhart and E. Tschachler E, “Retinoic Acid Increases the Expression of p53 and Proapoptotic Caspases and Sensitizes Keratinocytes to Apoptosis: A Possible Explanation for Tumor Preventive Action of Retinoids,” Cancer Research, Vol. 64, No. 18, 2004, pp. 6542-6548. doi:10.1158/0008-5472.CAN-04-1129
 K. Bogos, F. Renyi-Vamos, G. Kovacs, J. Tovari and B. Dome, “Role of Retinoic Receptors in Lung Carcinogenesis,” Journal of Experimental & Clinical Cancer Research, Vol. 27, No. 18, 2008.
 J. Wille and D. Chopra, “Reversal by Retinoids of Keratinization Induced by Benzo[alpha]pyrene in Normal Hamster Tracheal Explants: Comparison with the Assay Involving Organ Culture of Tracheas from Vitamin A-Deficient Hamsters,” Cancer Letters, Vol. 40, No. 3, 1988, pp. 35-46. doi:10.1016/0304-3835(88)90082-1
 D. Ramya, M. Siddikuzzaman and V. Berlin Grace, “Chemoprotective Effect of All-Trans Retinoic Acid (ATRA) on Oxidative Stress and Lung Metastasis Induced by Benzo(a)pyrene” Immunopharmacology and Immunotoxicology, Vol. 34, No. 2, 2012, pp. 317-325.
 J. Wille Jr., M. Pittelkow, G. Shipley and R. Scott, “Integrated Control of Growth and Differentiation of Normal Human Prokeratinocytes Cultured in Serum-Free Medium: Clonal Analyses, Growth Kinetics, and Cell Cycle Studies,” Journal of Cellular Physiology, Vol. 121, No. 1, 1984, pp. 31-44. doi:10.1002/jcp.1041210106
 M. Pittelkow, J. Wille Jr. and R. Scott, “Two Functionally Distinct Classes of Growth Arrest States in Human Prokeratinocytes That Regulate Clonogenic Potential,” Journal of Investigative Dermatology, Vol. 86, 1986, pp. 410-417. doi:10.1111/1523-1747.ep12285684
 Q. Ren, S. MurphyA. Dannenberg, J. Park, T. Tephly and P. Lazarus, “Glucuronidation of the Lung Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol by Rat UDT-Glucuronosyltransferase 2B1,” Drug Metabolism and Disposition, Vol. 27, 1986, pp. 1010-1016.
 G. Bowden, T. Slaga, B. Shapas and R. Boutwel, “The Role of Aryl Hydrocarbon Hydroxylase in Skin Tumor Initiation by 7,12-Dimethylbenz(a)anthracene and 1,2,5,6-Dibenzanthracene Using DNA Binding and Thymidine-3H Incorporation into DNA as Criteria,” Cancer Research, Vol. 34, No. 10, 1974, pp. 2634-2642.
 O. Lowry, N. Rosebrough, A. Farr and R. Randall, “Protein Measurement with the Folin Phenol Reagent,” Journal of Biological Chemistry, Vol. 193, No. 1, 1951, pp. 265-275.
 A. Verma , H. Rice, B. Shapas and R. Boutwell, “Inhibition of 12-O-Tetradecanoylphorbol-13-Acetate-Induced Ornithine Decarboxylase Activity in Mouse Epidermis by Vitamin A Analogs (Retinoids),” Cancer Research, Vol. 38, No. 3, 1978, pp. 793-801.
 U. Goswami, and N. Sharma, “Efficiency of a Few Retinoids and Carotenoids in Vivo in Controlling Benzo-[a]pyrene-Induced Forestomach Tumour in Female Swiss Mice,” British Journal of Nutrition, Vol. 94, No. 4, 2005, pp. 540-543. doi:10.1079/BJN20051484
 M. Kuratsune, S. Kohchi and H. Horie, “Carcinogenesis in the Esophagus. I. Penetration of Benzo(a)pyrene and Other Hydrocarbons into the Esophageal Mucosa,” Gann, Vol. 56, 1965, pp. 177-187.