Abstract
The cellular response of a normal human keratinocyte cell line exposed to non-cytotoxic doses of a deltamethrin-based pesticide was investigated by means of two different electrical ...impedance data spectroscopy approaches: Nyquist plot and broadband dielectric spectroscopy. The measurements have shown that the membrane capacity increases with pesticide concentration and this facilitates the electric current through cell membranes. Furthermore, the impedance of the extracellular matrix also increases with pesticide concentration, thus reducing the electric current outside the cell. Dielectric permittivity changes in the cellular samples at frequencies larger than 100 Hz. Fluorescence (FL) measurements emphasized an increase in neutral membrane lipids as a consequence of the pesticide exposure. Comparison of FL response of pesticide exposed cells with the control ones showed a time increase in the emission intensity, suggesting the existence of a membrane lipid response aimed at repairing the cell damage due to pesticide exposure. Therefore, both spectroscopic techniques have been demonstrated as potential means to investigate the response to cell stress and damage. This opens up new possibilities in the early diagnosis of cellular modifications related to the pesticide exposure of cells.
•Raman spectroscopy on nuclear and membrane regions of single human neuroblastoma cells.•Measurements are performed after irradiation by clinically relevant X-ray doses.•The acquired spectra are ...analyzed by PCA and interval-PCA methods.•The most relevant effects are evidenced in the spectra from the cell nucleus region.•Potential markers of an apoptotic process could be singled out for some X- ray doses.
Raman micro-spectroscopy was performed in vitro on nuclear and membrane regions of single SH-SY5Y human neuroblastoma cells after irradiation by graded X-ray doses (2, 4, 6, 8 Gy). The acquired spectra were analyzed by principal component analysis (PCA) and interval-PCA (i-PCA) methods. Biochemical changes occurring in the different regions of single cells as a consequence of the radiation exposure were observed in cells fixed immediately after the irradiation. The most relevant effects arose from the analysis of the spectra from the cell nucleus region. The observed changes were discussed in terms of the modifications in the cell cycle, resulting in an increase in the DNA-related signal, a protein rearrangement and changes in lipid and carbohydrates profiles within the nucleus. Potential markers of an apoptotic process in cell population irradiated with 6 and 8-Gy X-ray doses could have been singled out. No significant effects were found in spectra from cells fixed 24 h after the irradiation, thus suggesting the occurrence of repairing processes of the X-ray induced damage.
Blood is a fluid connective tissue of human body, where it plays vital functions for the nutrition, defense and well-being of the organism. When circulating in peripheral districts, it is exposed to ...some physical stresses coming from outside the human body, as electromagnetic fields (EMFs) which can cross the skin. Such fields may interact with biomolecules possibly inducing non thermal-mediated biological effects at the cellular level. In this study, the occurrence of biochemical/biological modifications in human peripheral blood lympho-monocytes exposed in a reverberation chamber for times ranging from 1 to 20 h to EMFs at 1.8 GHz frequency and 200 V/m electric field strength was investigated. Morphological analysis of adherent cells unveiled, in some of these, appearance of an enlarged and deformed shape after EMFs exposure. Raman spectra of the nuclear compartment of cells exposed to EMFs revealed the onset of biochemical modifications, mainly consisting in the reduction of the DNA backbone-linked vibrational modes. Respirometric measurements of mitochondrial activity in intact lympho-monocytes resulted in increase of the resting oxygen consumption rate after 20 h of exposure, which was coupled to a significant increase of the FoF1-ATP synthase-related oxygen consumption. Notably, at lower time-intervals of EMFs exposure (i.e. 5 and 12 h) a large increase of the proton leak-related respiration was observed which, however, recovered at control levels after 20 h exposure. Confocal microscopy analysis of the mitochondrial membrane potential supported the respiratory activities whereas no significant variations in the mitochondrial mass/morphology was observed in EMFs-exposed lympho-monocytes. Finally, altered redox homeostasis was shown in EMFs-exposed lympho-monocytes, which progressed differently in nucleated cellular subsets. This results suggest the occurrence of adaptive mechanisms put in action, likely via redox signaling, to compensate for early impairments of the oxidative phosphorylation system caused by exposure to EMFs. Overall the data presented warn for health safety of people involved in long-term exposure to electromagnetic fields, although further studies are required to pinpoint the leukocyte cellular subset(s) selectively targeted by the EMFs action and the mechanisms by which it is achieved.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Purpose
The response of human cells to applied electrical signals depends on the cellular health status, because it is influenced by the composition and structure of the main cellular components. ...Therefore, electrical impedance‐based techniques can be considered as sensitive tools to investigate healthy or disease state at cellular level. The goal of this study is to show that different types of in vitro cellular lines, related to different health status, can be differentiated using impedance spectra analysis.
Methods
Three different types of human breast cell line, corresponding to healthy, cancerous, and metastatic adenocarcinoma cells, were measured by means of electrical impedance spectroscopy. By modeling the investigated cells with proper resistive and capacitive circuital elements, the magnitude of the cell electrical components and spectra of real and imaginary part of dielectric permittivity were obtained. The latter were subsequently examined with a commonly adopted mathematical model, in order to estimate the values of specific dielectric parameters for the three different cellular lines.
Results
The relative variation of cellular capacitance with respect to that of the culture medium, estimated at 100 Hz, has a larger value for the two types of cancerous cells with respect to the noncancerous type. Furthermore, the ratio between the real and imaginary part of the dielectric permittivity function has larger values for metastatic cells with respect to the normal and nonmetastatic ones. Therefore, the mentioned relative capacitance allows to discriminate between normal and cancerous cells, whereas the results obtained for the dielectric function can discriminate between metastatic and nonmetastatic cells.
Conclusions
This study can be considered as an exploratory investigation of evaluating in vitro the health status of humans cells using selected electrical impedance parameters as potential markers. The obtained results highlight that a standard cultureware system, provided with interdigitated electrodes and appropriate impedance parameters, that is, cellular capacitance and the ratio between the imaginary and real part of cellular dielectric function, can be used to discriminate between healthy and cancerous breast cell lines, as well as different malignancy degrees.
Among different radiotherapy techniques, proton irradiation is an established and effective method for treatment of several types of cancer, because less healthy tissue is exposed with respect to ...conventional radiotherapy by photons/electrons. Recently, proton therapy has been proposed for the treatment of breast cancer. In vitro studies of proton irradiated normal human breast cells can provide information about cellular radioresponse, particularly as far as healthy tissue is concerned. In this paper, a study of the effects at different time points, following proton irradiation at different doses, of human normal MCF10A breast cells is performed by Raman spectroscopy. The aim of this investigation is to detect the unwanted effects of proton treatment and to investigate the possibility of monitoring them and of making an assessment of the cellular sensitivity by means of such a technique. The obtained results seem to indicate a rather significant sensitivity of MCF10A cells to proton irradiation. In fact, even at doses as low as 0.5 Gy, biological effects are clearly detectable in Raman spectra. In particular, ratiometric analysis of the Raman spectra measured from the nucleoplasm compartment showed that DNA/RNA damage increases with time, suggesting that most cells are unable to repair DNA/RNA broken bonds. The results obtained by the Raman spectroscopy analysis exhibit a similar trend with regard to dose to those obtained by commonly used radiobiological assays (i.e. MTT, clonogenic assay, senescence, apoptosis and necrosis). The results of this study strongly suggest the possibility that the Raman technique can be used to identify molecular markers predicting radiation response.
Oral squamous cell carcinoma is a widespread cancer disease whose survival rate is strongly dependent on early diagnosis and on the degree of malignancy. The conventional histopathology methods, ...which are currently the standard ones for diagnosis, are very invasive so that they can be hardly proposed as screening methods for an early and accurate detection of disease. Raman microspectroscopy and atomic force microscopy can be potentially considered as useful tools for cancer diagnosis and detection of the malignancy degree because they provide information about the biochemical cellular content and nanomechanical properties, respectively, which would be modified by the onset and progression of pathology. The present work shows that both techniques can successfully discriminate the two cellular types of cells characterized by different degrees of oral squamous cell carcinoma. The discrimination by Raman microspectroscopy occurs according to a larger content of nucleic acids and a minor content of protein components in cells characterized by a larger degree of disease, whereas the discrimination by atomic force microscopy is achieved because of a decrease of stiffness as the degree of disease increases. Overall, both techniques could provide useful diagnostic information related to the degree of malignancy of the oral squamous cell carcinoma disease.
Two cellular types characterized by different degree of oral squamous cell carcinoma can be discriminated by Raman microspectroscopy and Atomic Force Microscopy, according to the content of nucleic acids and proteins as well as the membrane stiffness.
•l-DOPA eumelanin aggregates as filamentous and granular structures on glass and mica.•Both types of structures have several tents nanometers size.•Point-like deposits having few nanometers size are ...found on mica.
Bright field microscopy and atomic force microscopy techniques are used to investigate morphological properties of synthetic eumelanin, obtained by oxidation of l-DOPA solution, deposited on glass and mica substrates. Deposits of eumelanin are characterized by aggregates with different shape and size. On a micrometric scale, filamentous as well as granular structures are present on glass and mica substrates, with a larger density on the former than on the latter. On a nanometric scale, filamentous aggregates, several microns long and about 100nm wide and high, and granular aggregates, ∼50nm high and 100nm wide, are found on both substrates, whereas point-like deposits less than 10nm high and less than 50nm wide are found on mica substrate. Dynamic light scattering measurements and atomic force microscopy images support the evidence that eumelanin presents only nanometric point-like aggregates in aqueous solution, whereas such nanoaggregates organize themselves according to granular and filamentous structures when deposition occurs, as a consequence of interactions with the substrate surface.
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