Effect of Near-Ultraviolet and Visible Light on Mammalian Cells in Culture II. Formation of Toxic Photoproducts in Tissue Culture Medium by Blacklight

Abstract

Near-ultraviolet radiation was found to be lethal for mammalian cells in Dulbecco's modified Eagle's medium without serum or phenol red. Irradiation of the cells with near-ultraviolet light while the cells were in phosphate-buffered-saline abolished the lethal effect. When only the medium was irradiated followed by the addition of unirradiated cells and serum, the cells were still killed. The photoactive components of the medium for this effect were riboflavin, tryptophan, and tyrosine. When riboflavin was deleted from the medium being irradiated and added later, almost no killing was detected. Irradiation of salt solution of riboflavin and tryptophan or riboflavin and tyrosine, resulted in cell killing. Little Irradiation of Cells. The light sources used in this study were two General Electric F15T8 BLB integral filter blacklight tubes with emission in the 300-to 420-nm range peaking at 365 nm (12). Light exposure was carried out in covered Falcon 3002 60-mm dishes placed approximately 20 cm below the light source. The lids of these dishes exclude light below 300 nm (6). Exposure intensity was 4 sW/mm2 at the cell surface, as measured by a Blak-ray meter J-221 (Ultraviolet Products, Inc.). Temperature was monitored and regulated to avoid any effect due to heating by the light source. The cells were trypsinized and plated in medium containing serum but no phenol red, 18 hr before exposure to blacklight. Twenty minutes before irradiation, the medium was removed and replaced with either 3.5 ml of phosphate-buffered saline containing NaCl, KC1, Na2HPO4, and KH2PO4; or MEM free of both phenol red and serum. The dishes were placed in the exposure chamber under 10% CO2 for 20 min before the light was turned on. At various intervals, dishes were removed from the light and kept in the dark in the same chamber. After the last dishes were removed from the light, serum was added to all dishes.Irradiation of Medium or Medium Components. Exposure conditions were similar to those of the last section except that no cells were present and only the aqueous solutions of medium components were exposed. The basic mixture (BaM) employed, to which other components were selectively added, contained NaCl, KC1, CaC12, MgSO4, NaH2PO4, Fe(NO3)3, glucose, inositol, cysteine, and glutamine. For the determination of the medium components responsible for photoproduct formation, other ingredients were added to BaM: (1) aminoacid solution (AA) containing arginine, isoleucine, leucine, lysine, methionine, threonine, valine, glycine, and serine; (2) vitamin solution (Vit) containing nicotinamide, thiamine, choline, D-pantothenic acid and pyridoxine; (3) one or more of the following compounds: histidine, phenylalanine, tryptophan, tyrosine, and riboflavin. After irradiation, the missing MEM ingredients and serum were added to the dishes fol-

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405 nm light exposure of osteoblasts and inactivation of bacterial isolates from arthroplasty patients: potential for new disinfection applications?

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