Cell damage by UVA radiation of a mercury microscopy lamp probed by autofluorescence modifications, cloning assay, and comet assay

Koenig, Karsten; Krasieva, Tatiana B.; Bauer, Eckhard; Fiedler, Ursula; Berns, Michael W.; Tromberg, Bruce J.; Greulich, Karl-Otto

doi:10.1117/12.233373

Cited by 19 publications

(11 citation statements)

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“…The accumulation of these molecular species highlights a condition of metabolic impairment, due to the dysfunction of mitochondrial electron transport [24]. The increase of NAD(P)Hb by comparing the wavelength ratios (450/480, 450/525, and 480/525 nm), is consistent with the efflux of the coenzyme from mitochondria, its diffusion and aspecific binding to cytoplasmic and nuclear proteins [23].…”

Section: Discussionmentioning

confidence: 73%

“…5C), might be attributed to membrane damage, thus explaining the diffuse fluorescence haloes observed in hypogravity cells. It is known that membrane damage causes the leakage of coenzyme molecules, as NAD(P)H and flavins from mitochondria, and their diffusion in all the parts of the cell, nucleus included [11,23]. Under 365 nm excitation, the visible autofluorescence is mainly due to NAD(P)H, blue component, and oxidized flavins, yellow-green component [15].…”

Section: Discussionmentioning

confidence: 99%

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Simulated hypogravity impairs the angiogenic response of endothelium by up-regulating apoptotic signals

Morbidelli

Monici²,

Marziliano

et al. 2005

Biochemical and Biophysical Research Communications

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Health hazards in astronauts are represented by cardiovascular problems and impaired bone healing. These disturbances are characterized by a common event, the loss of function by vascular endothelium, leading to impaired angiogenesis. We investigated whether the exposure of cultured endothelial cells to hypogravity condition could affect their behaviour in terms of functional activity, biochemical responses, morphology, and gene expression. Simulated hypogravity conditions for 72 h produced a reduction of cell number. Genomic analysis of endothelial cells exposed to hypogravity revealed that proapoptotic signals increased, while antiapoptotic and proliferation/survival genes were down-regulated by modelled low gravity. Activation of apoptosis was accompanied by morphological changes with mitochondrial disassembly and organelles/cytoplasmic NAD(P)H redistribution, as evidenced by autofluorescence analysis. In this condition cells were not able to respond to angiogenic stimuli in terms of migration and proliferation. Our study documents functional, morphological, and transcription alterations in vascular endothelium exposed to simulated low gravity conditions, thus providing insights on the occurrence of vascular tissue dysregulation in crewmen during prolonged space flights. Moreover, the alteration of vascular endothelium can intervene as a concause in other systemic effects, like bone remodelling, observed in weightlessness. Ó 2005 Elsevier Inc. All rights reserved.Keywords: Endothelial cell; Hypogravity; Gene expression; Autofluorescence; Apoptosis; Angiogenesis; Migration; Proliferation Angiogenesis, a process which leads to formation of new vessels, has a relevant physiological role during development and in the adult life [1,2]. Endothelial cells are the true player of the angiogenesis process. Several cardiovascular pathologies are caused by endothelial dysfunction (myocardial ischemia, atherosclerosis), thus growth factors and strategies able to restore the integrity of the endothelium and to promote angiogenesis can be viewed as innovative therapeutic approaches [3,4].Indications that mechanical stimulation of cells, and endothelium in particular, is able to change some of their functions at biochemical and molecular level are reported in the literature [5]. In spite of the evidence that cells are sensitive to mechanical stimuli, the molecular mechanisms by which individual cells recognize and respond to external forces are not well understood. Our interest on hypogravity originates from the recognition that several disturbances associated to endothelium-0006-291X/$ -see front matter Ó

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Section: Discussionmentioning

confidence: 73%

Section: Discussionmentioning

confidence: 99%

Simulated hypogravity impairs the angiogenic response of endothelium by up-regulating apoptotic signals

Morbidelli

Monici²,

Marziliano

et al. 2005

Biochemical and Biophysical Research Communications

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“…Our results also show that the arrest is not due to alterations in the culture medium, as unimaged embryos in the same drop remain viable. Cellular damage caused by light has been shown in other cell types for various wavelengths, ranging from 295 to 500 nm (one photon) 22,23 or 730 nm to 800 nm (two photons) 17 . Therefore, we sought to determine a possible endogenous source of this phototoxicity.…”

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confidence: 99%

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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“…For a given excitation intensity, UV light is more toxic than visible or near-infrared (NIR) light because there are a variety of endogenous fluorescent species present in cells that can absorb UV light and sensitize ROS production, including flavins, NAD(P)H and porphyrins. 108 For example, Gorgidze et al observed wavelength-dependent, mitotic inhibition in pig kidney embryo cells with near-UV (360 nm) light being 5 times more inhibitory than blue light (423–488 nm) and >50 times more inhibitory than green light (>500 nm). 109 …”

Section: Photoxicity Of Uv Excitation Light Used In Lanthanide Imagingmentioning

confidence: 99%

Lanthanide-Based Imaging of Protein–Protein Interactions in Live Cells

2013

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In order to deduce the molecular mechanisms of biological function, it is necessary to monitor changes in the sub-cellular location, activation and interaction of proteins within living cells in real time. Förster resonance energy transfer (FRET)-based biosensors that incorporate genetically encoded, fluorescent proteins permit high spatial resolution imaging of protein–protein interactions or protein conformational dynamics. However, non-specific fluorescence background often obscures small FRET signal changes, and intensity-based biosensor measurements require careful interpretation and several control experiments. These problems can be overcome by using lanthanide (Tb(III) or Eu(III)) complexes as donors and green fluorescent protein (GFP) or other conventional fluorophores as acceptors. Essential features of this approach are the long-lifetime (~ms) luminescence of Tb(III) complexes and time-gated luminescence microscopy. This allows pulsed excitation followed by a brief delay that eliminates nonspecific fluorescence before detection of Tb(III)-to-GFP emission. The challenges of intracellular delivery, selective protein labeling, and time-gated imaging of lanthanide luminescence are presented, and recent efforts to investigate the cellular uptake of lanthanide probes are reviewed. Data is presented showing that conjugation to arginine-rich, cell penetrating peptides (CPPs) can be used as a general strategy for cellular delivery of membrane impermeable lanthanide complexes. A heterodimer of a luminescent Tb(III) complex, Lumi4, linked to trimethoprim (TMP) and conjugated to nonaarginine via a reducible disulfide linker rapidly (~10 min) translocates into the cytoplasm of Maden Darby canine kidney cells from culture medium. With this reagent, the intracellular interaction between GFP fused to FK506 binding protein 12 (GFP-FKBP12) and the rapamycin binding domain of mTOR fused to Escherichia coli dihydrofolate reductase (FRB-eDHFR) was imaged at high signal-to-noise ratio with fast (1–3 s) image acquisition using a time-gated luminescence microscope. The data reviewed and presented here show that lanthanide biosensors enable fast, sensitive and technically simple imaging of protein-protein interactions in live cells.

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Cell damage by UVA radiation of a mercury microscopy lamp probed by autofluorescence modifications, cloning assay, and comet assay

Cited by 19 publications

References 20 publications

Simulated hypogravity impairs the angiogenic response of endothelium by up-regulating apoptotic signals

Simulated hypogravity impairs the angiogenic response of endothelium by up-regulating apoptotic signals

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

Lanthanide-Based Imaging of Protein–Protein Interactions in Live Cells

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