Richard J. Wang

IDENTIFICATION OF HYDROGEN PEROXIDE AS A PHOTOPRODUCT TOXIC TO HUMAN CELLS IN TISSUE-CULTURE MEDIUM IRRADIATED WITH "DAYLIGHT" FLUORESCENT LIGHT

SummaryHydrogen peroxide, lethal for human cells, is produced in Dulbeccos modified Eagles tissue culture medium when exposed to “daylight” fluorescent light. Addition of pure H2O2 and use of the enzyme catalase demonstrate that about 40% of the toxicity in irradiated medium is due to generated peroxide. Riboflavin and tryptophan, or riboflavin and tyrosine, are the components necessary for formation of lethal levels of H2O2 during light exposure.

LETHAL EFFECT OF “DAYLIGHT” FLUORESCENT LIGHT ON HUMAN CELLS IN TISSUE‐CULTURE MEDIUM

Fluorescent lamps are utilized in virtually every laboratory working with human and mammalian cells in culture. The lamps are also being used as part of the experimental protocol on mammalian cell cultures. Possible deleterious effects of these lamps on such cells have not been seriously examined. Commercially available low-pressure mercury discharge lamps fall in four categories (General Electric, 1970). The most widely used type includes ‘white’ and ‘daylight’ (DL) tubes, which emit in a broad spectrum of 310 to 750nm and provide light for almost a11 cell culture laboratories. The second type, the ‘colored’ tubes, e.g. ‘blue’, ‘green’, ‘gold’, and ‘red‘, contain radiation in the visible range skewed towards more specific regions of the spectrum. The third class are the ‘black-light’ tubes, which emit primarily in the near-UV region. The ‘germicidal’ tubes fall in the last class, which emit below 300nm in the far-UV region. The dangers associated with far-UV irradiation of biological systems are well known. We reported earlier that when mammalian cells in tissue-culture medium (Dulbecco’s Modified Eagle’s Medium) were irradiated with the near-UV light emitted by black-light tubes, the cells were killed both physiologically and reproductively (Wang et al., 1974). In another report, we showed that the killing effect was due to formation of toxic photoproducts from riboflavin, tryptophan, and tyrosine in the medium (Stoien and Wang, 1974). Fluorescent light from white or daylight lamps contains a significant amount of near-UV radiation with emission peaks near 310 and 360nm (General Electric, 1970). The results that we have reported (Wang ct a/.. 1974; Stoien and Wang, 1974) on mammalian cells raised the question of possible effects of such fluorescent light on mammalian cells, since exposure of the cells to the light illuminating the laboratory is ditticult to avoid. The use of fluorescent lights as experimental tools in investigations involving mammalian cells in culture is becoming increasingly popular. Fogel (1973) found that ‘visible’ light increased polyoma virus induction nine-fold. Todd et al. (1973) discovered that marsupial ptK cells in Eagle’s medium were killed when exposed to the black light used for photoreactivation. ‘Visible’ light was found to contribute to inhibition of fermentation and growth of mouse ascites tumor cells (Warburg et al., 1968). When solutions containing riboflavin and methotrexate were inadvertently exposed to light, photochemical reactions took place resulting in modified methotrexate uptake kinetics by mouse leukemic cells (Lichtenstein and Goldman, 1970). A program was initiated in this laboratory to examine the effects of ‘visible’ fluorescent light on tissue culture media and cells. I report here that such light kills human D98/AHz cells in tissue-culture medium.

Effect of room fluorescent light on the deterioration of tissue culture medium

SummaryA major cause of tissue culture medium deterioration is exposure to room fluorescent light. Riboflavin and tryptophan present in Dulbeccos modified Eagles minimum essential medium, when exposed to light, yield toxic photoproducts responsible for loss of the ability of the medium to support clonal growth of human, mouse and Chinese hamster cell lines. Procedures for minimizing medium deterioration are discussed.

FORMATION OF PHOTOPRODUCTS LETHAL FOR HUMAN CELLS IN CULTURE BY DAYLIGHT FLUORESCENT LIGHT AND BILIRUBIN LIGHT

Abstract. Irradiation of Dulbeccos modified Eagles tissue culture medium with “Daylight,”“Special Blue,” or “Bilirubin” fluorescent light produces photoproducts lethal to human cells. Killing is abolished when (1) riboflavin, (2) tryptophan and tyrosine, or (3) riboflavin, tryptophan and tyrosine are deleted from medium prior to irradiation with any of the above fluorescent lamps. Toxic photoproducts are also formed when buffered salt solutions containing (a) riboflavin and tryptophan, (b) riboflavin and tyrosine, or (c) riboflavin, tryptophan and tyrosine are exposed to any of these light sources.

SISTER CHROMATID EXCHANGES AND CHROMOSOME ABERRATIONS IN HUMAN CELLS INDUCED BY H2O2 AND OTHER PHOTOPRODUCTS GENERATED IN FLUORESCENT LIGHT-EXPOSED MEDIUM

Exposure of Dulbeccos modified Eagles tissue culture medium to visible fluorescent light generated photoproducts toxic to human cells in culture. Toxicity manifested at the chromosome level was increased chromosome aberrations and sister chromatid exchanges in cells exposed to the photoproducts. Hydrogen peroxide (H2O2), a major photoproduct, induced SCE but failed to increase chromosome aberrations. Pure H2O2, or the H2O2 generated in light‐exposed medium, was necessary and sufficient for inducing all the increase in SCE. However, H2O2 was necessary but insufficient to cause most of the chromosome aberrations. Only when acting synergistically with other photoproducts did H2O, induce extensive chromosome aberrations. The relatively high cell densities at near confluence levels used in these experiments were less sensitive to light‐induced effects, nevertheless the entire light exposure dosage range effected photoproduct production adequate for inducing SCE and chromosome aberrations. Thus, mammalian tissue and cell culture media can receive sufficient dosage from fluorescent lights illuminating rooms and culture hoods for generation of photoproducts causing gross and insidious SCE and chromosome alterations.

Antigenic domains on the U1 small nuclear ribonucleoprotein-associated 70K polypeptide: A comparison of regions selectively recognized by human and mouse autoantibodies and by monoclonal antibodies

Antigenic regions on the U1 small nuclear ribonucleoprotein (snRNP)-associated 70K polypeptide recognized by human and mouse autoantibodies or by monoclonal antibodies were identified and compared. Using a set of 70K fusion proteins as antigen in enzyme-linked immunosorbent assay and immunoblotting revealed that serum autoantibodies of human and of MRL/Mp mouse origin recognized a common region of the 70K polypeptide. Monoclonal anti-70K antibodies derived from a patient with mixed connective tissue disease, from an autoimmune MRL/Mp mouse, and from a BALB/c mouse immunized with purified U1 snRNP were all shown to bind to a part of the 70K polypeptide rich in charged residues and different from the region recognized by most human and MRL/Mp mouse serum autoantibodies.

A Differential Staining Technique for Simultaneous Visualization of Mitotic Spindle and Chromosomes in Mammalian Cells

A new technique has been devised for staining the mitotic spindle in mammalian cells while preserving spindle structure and chromosome number. The cells are trypsinized and fixed with a 3:1 methanol:acetic acid solution containing 4 mM MgCl2 and 1.5 mM CaCl2 at room temperature. The cells are then placed on slides and treated with 5% perchloric acid before staining with a 10% acetic acid solution containing safranin O and brilliant blue R. The preserved spindles appear dark blue against a light cytoplasmic background with chromosomes stained bright red. Individual chromosomes and chromatids are clearly visible. Positioning of the chromosomes relative to the spindle apparatus is readily ascertained allowing easy study of mitotic spindle and chromosome behavior.

Studies on cell division in mammalian cells: IV. A temperature-sensitive cell line defective in post-metaphase chromosome movement

Abstract A temperature-sensitive mutant with defective post-metaphase chromosome movement, ts-687, was isolated from a hamster cell line. When incubated at the non-permissive temperature of 39 °C, cell number increase stops within 3 days after which time temperature effects become irreversible. Mutant cells at 39 °C continue to enter mitosis, but anaphase chromosome movement is abnormal with lagging chromosomal material remaining in the cleavage furrow preventing its progression. In some cells regression of the furrow occurs resulting in the formation of enlarged mono-nucleate, bi-nucleate and variable sized multi-nucleate cells, while in other cells cytokinesis proceeds to completion after a prolonged delay period. This mutant complements a previously reported metaphase mutant, ts-546, demonstrating that more than one gene locus is active in determining successful post-metaphase mitotic progression in mammalian cells.

Further studies on a mutant mammalian cell line defective in mitosis.

Abstract Experiments have been performed on a temperature-sensitive hamster cell line, ts-546. After the cells are switched to the non-permissive temperature, interphase cells continue through the cell cycle until the cells enter metaphase. Normal mitotic events then fail to occur. Metaphase chromosomes in the cells condense and coalesce into chromatin aggregates. Nuclear membrane re-forms around the aggregates resulting in the formation of mono-, bi- or multi-nucleate interphase-like cells. The conversion of mitotic cells to interphase-like states is completed within a few hours. The initial characterization of the mutant cell line was based on the observation that rounded-up cells accumulate in culture at the non-permissive temperature. The mitotic roundingup process may be utilized as a useful marker for selective isolation of mutant cell lines defective in mitosis.

Effects of chemical smokes on flora and fauna under field and laboratory exposures

Various types of obscurant smokes are used routinely in training by the U.S. Army. Because continued routine use of the smokes could be detrimental to the native flora and fauna at training sites, a preliminary biological and chemical field study of fogoil, hexachloroethane, and tank diesel smokes was conducted. Smoke plumes were sampled and chemically analyzed at distances of 15-150 m from the smoke source where Tradescantia clones 4430 and 03 and the native plant Ambrosia dumosa and the native rodent Dipodomys merriami were exposed for 30 min. In addition, Tradescantia clone 4430 was exposed to tank diesel in the laboratory at concentration levels equivalent to exposure at 15 and 50 m. Tradescantia clones were examined for mutagenic effects indicated by micronuclei induction in developing pollen and pink somatic mutations in stamen hairs. Photosynthetic perturbations were measured in Tradescantia and A. dumosa using variable fluorescence induction. Animals were examined for sister chromatid exchanges and chromosome aberrations. It was found that all of the smokes tested exerted varying degrees of physiological and mutagenic effects in one or more assay system at one or more exposure distance. The studies reported here indicate that exposed ecological systems, or at least components of these systems, are at a higher risk than are unexposed components (e.g., organisms) for several types of damage attributed to obscurant smoke exposure.