Identification of hydrogen peroxide as a photoproduct toxic to human cells in tissue-culture medium irradiated with “daylight” fluorescent light

Wang, Richard J.; Nixon, B. Tracy

doi:10.1007/bf02616168

Cited by 117 publications

(56 citation statements)

References 26 publications

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“…The principal mechanism of phototoxicity is thought to involve the generation of "O # and probable subsequent oxidation of the amino acids tryptophan and tyrosine [35,36]. It has since been demonstrated that fluorescent light may cause a build up of toxic H # O # levels in DMEM or RPMI-1640 medium, both of which contain riboflavin [21,37]. In the present experiments, the SOD-sensitive quench of the NO signal produced by riboflavin alone (in the light) in Tris buffer increased in proportion with riboflavin concentration, as expected from a simple photosensitive generation of O # d − .…”

Section: Discussionmentioning

confidence: 99%

Superoxide-dependent consumption of nitric oxide in biological media may confound in vitro experiments

Keynes

Griffiths

Garthwaite

2003

Biochemical Journal

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NO functions ubiquitously as a biological messenger but has also been implicated in various pathologies, a role supported by many reports that exogenous or endogenous NO can kill cells in tissue culture. In the course of experiments aimed at examining the toxicity of exogenous NO towards cultured cells, we found that most of the NO delivered using a NONOate (diazeniumdiolate) donor was removed by reaction with the tissue-culture medium. Two NO-consuming ingredients were identified : Hepes buffer and, under laboratory lighting, the vitamin riboflavin. In each case, the loss of NO was reversed by the addition of superoxide dismutase. The effect of Hepes was observed over a range of NONOate concentrations (producing up to 1 µM NO). Furthermore, from measurements of soluble guanylate cyclase activity, Hepes-dependent NO consumption remained significant at the low nanomolar NO concentrations relevant to physiological NO

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Section: Discussionmentioning

confidence: 99%

Superoxide-dependent consumption of nitric oxide in biological media may confound in vitro experiments

Keynes

Griffiths

Garthwaite

2003

Biochemical Journal

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“…Occasionally, embryos in the imaged sample were out of the field of view and therefore not directly subjected to the LSCM irradiation. Among this subset of embryos, all but one (n = 12) developed to at least the morula stage, indicating that the detrimental effect of imaging with these wavelengths occurs directly on the embryos rather than by altering the culture medium 21 . Development was inhibited by each of the three wavelengths (514, 532, and 568 nm) used to excite this fluorophore.…”

Section: Lscm Inhibited Development Whereas Tplsm Maintained Embryo mentioning

confidence: 98%

Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability

et al. 1999

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A major challenge for fluorescence imaging of living mammalian cells is maintaining viability following prolonged exposure to excitation illumination. We have monitored the dynamics of mitochondrial distribution in hamster embryos at frequent intervals over 24 h using two-photon microscopy (1,047 nm) while maintaining blastocyst, and even fetal, developmental competence. In contrast, confocal imaging for only 8 h inhibits development, even without fluorophore excitation. Photo-induced production of H 2 O 2 may account, in part, for this inhibition. Thus, twophoton microscopy, but not confocal microscopy, has permitted long-term fluorescence observations of the dynamics of three-dimensional cytoarchitecture in highly photosensitive specimens such as mammalian embryos.Keywords two-photon microscopy; laser scanning confocal microscopy; live cell fluorescence imaging; embryo; mitochondrial dynamics; mammal; hamster The detection of specific cellular components by imaging techniques such as wide-field epifluorescence or laser scanning confocal microscopy (LSCM) requires exposure to high intensity light that can cause cellular damage 1 . Consequently, the quantity or quality of images that can be collected is limited or, even worse, the reliability of the images may be compromised. This is a particular problem when imaging events that occur over periods of time ranging from hours to days, such as embryonic development. For this reason, much of our current understanding of subcellular morphological changes during mammalian embryonic development is based on images of fixed or static specimens at different developmental stages [2][3][4][5][6] . Thus, it can be difficult to interpret dynamic processes accurately, because the continuity of events must be inferred. The establishment of long-term fluorescence imaging methods that maintain the viability of live specimens is critical for advancing our understanding of cell biology and embryonic development in areas such as ion dynamics 7 , cytoplasmic reorganization, compaction and blastocoel formation, embryonic development in exotic species (where specimens are heterogeneous and difficult * Corresponding author (jsquirre@students.wisc.edu). to obtain), use of fluorescent tags for the preselection of embryos for subsequent embryo transfer 8 , and studies of protein expression in living cells using green fluorescent protein 9 . HHS Public AccessEmbryos of some mammals, particular hamsters, are very sensitive to culture conditions 10 . Furthermore, studies suggest that mammalian oocytes and embryos are adversely affected by exposure to visible light [11][12][13] . Because of this sensitivity, mammalian embryos are ideal to test live-cell imaging techniques. In addition, there are obvious morphological changes associated with differentiation, namely compaction and blastocoel formation, which can be used to assess viability. The embryo must undergo cell division during and after imaging as well as maintain a level of developmental competence that allows it to initiate dif...

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“…It is well known that the light‐induced degradation of Rf in interplay with Trp and Tyr can trigger the formation of toxic radicals, including hydrogen peroxide and singlet oxygen species 5, 6, 7, 8, 15, 16. In order to test whether the photosensitized reactions of Rf in CDPM and DMEM induce a higher level of ROS/RNS, the total radical burden in media samples was determined by fluorimetric assay.…”

Section: Resultsmentioning

confidence: 99%

“…Rf is contained in most culture media in typical concentrations up to 1.5 µmol L −1 , and has a key function in oxygen reduction, energy production, vitamin metabolism and other antioxidative protection mechanisms 22. It is one of the few well‐described examples of media components that are prone to photolysis with proven impact on culture performance 4, 8, 23, 24. However, owing to an exceptionally high quantum yield, Rf could eventually mask other constituents in the EEM spectrum, which should be considered for interpretation 12…”

Section: Discussionmentioning

confidence: 99%

“…Riboflavin (Rf), tryptophan (Trp) and tyrosine (Tyr) are among the most prominent examples of substances involved in light‐induced degradation processes and in the photosensitized generation of reactive oxygen species 3, 4, 5, 6, 7, 8. While effects of this kind have first been described more than four decades ago, the mechanistic basis of medium deterioration and especially the resultant impact on bioprocess performance, culture physiology and productivity is still poorly understood.…”

Section: Introductionmentioning

confidence: 99%

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Media photo‐degradation in pharmaceutical biotechnology – impact of ambient light on media quality, cell physiology, and IgG production in CHO cultures

Neutsch

Kröll

Brunner

et al. 2018

J of Chemical Tech & Biotech

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BACKGROUNDMany vital components in bioprocess media are prone to photo‐conversion or photo‐degradation upon exposure to ambient light, with severe negative consequences for biomass yield and overall productivity. However, there is only limited awareness of light irradiation as a potential risk factor when working in transparent glass bioreactors, storage vessels or disposable bag systems. The chemical complexity of most media renders a root‐cause analysis difficult. This study investigated in a novel, holistic approach how light‐induced changes in media composition relate to alterations in radical burden, cell physiology, morphology, and product formation in industrial Chinese hamster ovary (CHO) bioprocesses.RESULTSTwo media formulations from proprietary and commercial sources were tested in a pre‐hoc light exposure scenario prior to cultivation. Using fluorescence excitation/emission (EEM) matrix spectroscopy, a photo‐sensitization of riboflavin was identified as a likely cause for drastically decreased IgG titers (up to −80%) and specific growth rates (−50% to −90%). Up to three‐fold higher radical levels were observed in photo‐degraded medium. On the biological side, this resulted in significant changes in cell morphology and aberrations in the normal IgG biosynthesis/secretion pathway.CONCLUSIONThese findings clearly illustrate the underrated impact of room light after only short periods of exposure, occurring accidentally or knowingly during bioprocess development and scale‐ up. The detrimental effects, which may share a common mechanistic cause at the molecular level, correlate well with changes in spectroscopic properties. This offers new perspectives for online monitoring concepts, and improved detectability of such effects in future. © 2018 The Authors. Journal of Chemical Technology & Biotechnology published by JohnWiley & Sons Ltd on behalf of Society of Chemical Industry.

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Identification of hydrogen peroxide as a photoproduct toxic to human cells in tissue-culture medium irradiated with “daylight” fluorescent light

Cited by 117 publications

References 26 publications

Superoxide-dependent consumption of nitric oxide in biological media may confound in vitro experiments

Superoxide-dependent consumption of nitric oxide in biological media may confound in vitro experiments

Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability

Media photo‐degradation in pharmaceutical biotechnology – impact of ambient light on media quality, cell physiology, and IgG production in CHO cultures

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