Polycyclic aromatic hydrocarbon mutagenesis of human epidermal keratinocytes in culture.

Abstract

We have developed a culture system for detecting and isolating rare hypoxanthine phosphoribosyltransferase-deficient mutants of human epidermal keratinocytes. A thioguanine-resistant variant, 3T3M1, of the Swiss mouse fibroblast line 3T3 was used as a feeder layer to support clonal growth of mutant keratinocytes. A near-diploid, epidermal squamous cell carcinoma line, SCC-13Y, was used as a prototype to determine mutagen treatment conditions, plating density, and phenotypic expression time for maximum mutant recovery. To extend this system to normal keratinocytes, we improved the culture conditions by adding insulin, adenine, and Ham's nutrient mixture F-12, which increased colony-forming efficiencies to 30% in early passage and made feasible the detection of rare mutants in normal epidermal keratinocyte populations. We have quantitated mutation in SCC-13Y and three strains of normal human epidermal keratinocytes after exposure to polycyclic aromatic hydrocarbons, which are activated to their mutagenic forms by cellular mixed-function oxidases.7,12-Dimethylbenz[a]anthracene and benzo[a]pyrene caused almost no cytotoxicity, but induced thioguanine-resistant mutants at frequencies as much as 50-fold higher than the spontaneous frequency of 10-6. The mutants were aminopterin-sensitive and possessed no measurable hypoxanthine phosphoribosyltransferase activity; their behavior was indistinguishable from that of keratinocytes cultured from individuals with Lesch-Nyhan syndrome. This mutagenesis assay system should also be applicable to other feeder layer-dependent human epithelial cell types, such as urothelial, mammary, and tracheal epithelial cells.The epidermis and many other epithelial tissues possess a family of mixed-function oxidases (flavoprotein-linked monoxygenase, EC 1.14.14.1) and related enzymes, which metabolize complex organic chemicals to excretable and generally less toxic intermediates (1-4). Although the mixed-function oxidase system probably evolved as a protective mechanism, some classes of chemicals, such as the polycyclic aromatic hydrocarbons (PAHs), are converted by these enzymes to intermediates that are more cytotoxic, mutagenic, and carcinogenic than the parent compounds (5-7). More than half of all human cancers arise in surface epithelia that are in direct contact with the environment (i.e., the epithelia of the epidermis, esophagus, trachea, bronchus, cervix, and intestine) (8). Exposure of these tissues to PAHs and other environmental agents is thought to contribute significantly to the initiation and/or progression of human carcinomas (for review, see ref. 9).A criterion such as frequency of induced mutation at a defined genetic locus or acquisition of a phenotype associated with malignant transformation in cultures of chemically treated human epithelial cells could be useful in identifying both potentially dangerous chemicals and individuals particularly susceptible to chemical carcinogenesis. The proliferative capacity and clonal growth ability of normal human epithelial cell...

Expression of beta 1, beta 3, beta 4, and beta 5 integrins by human epidermal keratinocytes and non-differentiating keratinocytes.

“…), 0.5 ltg/ml hydrocortisone (Calbiochem, Cambridge Bioscience, Cambridge, England), 10-1°M cholera toxin (ICN Biomedicals, High Wycombe, Berks, UK), and 10 mg/ml EGF (a gift from Dr. GeorgeNascimento, Chiron Corporation, Emeryville, CA) (FAD + FCS + HICE ; Allen-Hoffmann and Rheinwald, 1984) . ndk (passages 5-12), a strain of keratinocytes which are unable to undergo terminal differentiation, were cultured in the same medium as the normal keratinocytes, but without a feeder cell layer (Adams and Watt, 1988) .…”

Application of Chemically Accelerated Biotreatment to Reduce Riskin Oil-Impacted Soils

“…The wastes sites are broken down into waste or reserve pits at the wellhead, dehydrator sites, and gas processing plants (responsible for separating gas liquids from the gas stream and/or remove of nitrogen/sulfur from the gas). 1 A potential site of soil contamination for the petroleum industry follows a similar pattern.…”

“…The 16 PAH-mixture were quantified using fully-deuterated internal standards for each PAH. Five concentrations (1,5,20,50, and 100 ng/µl) were used to generate each calibration curve. All calibration curves had r 2 values of at least 0.999, with the exception of pyrene (r 2 = 0.994).…”

“…Therefore, many PAHs are genotoxic and/or carcinogenic (1,2,8,14,23), and promote similar effects of other compounds (6). Thus, a total of 16 PAHs have been included on the United States EPA's priority pollutant list (13).…”

Identification of a Novel Cis-acting Negative Regulatory Element Affecting Expression of the CYP1A1 Gene in Rat Epidermal Cells

“…In experiments using low passage cells, the feeder layer was removed either by rinsing with 0.5 mM EDTA in PBS or by the advancing keratinocytes colonies as they reached confluence and therefore did not complicate interpretation of the results. Cells were grown in a 3:1 mixture of Dulbecco-Vogt Eagle's and Ham's F-12 media supplemented with 5% fetal bovine serum, hydrocortisone (0.4 g/ml), epidermal growth factor (10 ng/ml), cholera toxin (10 ng/ml), transferrin (5 g/ml), insulin (5 g/ml), adenine (0.18 mM), triiodothyronine (20 pM), and antibiotics (36). Mouse hepatoma (Hepa1c1c7) cells were grown as described previously (18,19).…”