Aidan D. Meade

Impaired Clearance and Enhanced Pulmonary Inflammatory/Fibrotic Response to Carbon Nanotubes in Myeloperoxidase-Deficient Mice

Advancement of biomedical applications of carbonaceous nanomaterials is hampered by their biopersistence and pro-inflammatory action in vivo. Here, we used myeloperoxidase knockout B6.129X1-MPO (MPO k/o) mice and showed that oxidation and clearance of single walled carbon nanotubes (SWCNT) from the lungs of these animals after pharyngeal aspiration was markedly less effective whereas the inflammatory response was more robust than in wild-type C57Bl/6 mice. Our results provide direct evidence for the participation of MPO – one of the key-orchestrators of inflammatory response – in the in vivo pulmonary oxidative biodegradation of SWCNT and suggest new ways to control the biopersistence of nanomaterials through genetic or pharmacological manipulations.

Studies of chemical fixation effects in human cell lines using Raman microspectroscopy

AbstractThe in vitro study of cellular species using Raman spectroscopy has proven a powerful non-invasive modality for the analysis of cell constituents and processes. This work uses micro-Raman spectroscopy to study the chemical fixation mechanism in three human cell lines (normal skin, normal bronchial epithelium, and lung adenocarcinoma) employing fixatives that preferentially preserve proteins (formalin), and nucleic acids (Carnoy’s fixative and methanol–acetic acid). Spectral differences between the mean live cell spectra and fixed cell spectra together with principal components analysis (PCA), and clustering techniques were used to analyse and interpret the spectral changes. The results indicate that fixation in formalin produces spectral content that is closest to that in the live cell and by extension, best preserves the cellular integrity. Nucleic acid degradation, protein denaturation, and lipid leaching were observed with all fixatives and for all cell lines, but to varying degrees. The results presented here suggest that the mechanism of fixation for short fixation times is complex and dependent on both the cell line and fixative employed. Moreover, important spectral changes occur with all fixatives that have consequences for the interpretation of biochemical processes within fixed cells. The study further demonstrates the potential of vibrational spectroscopy in the characterization of complex biochemical processes in cells at a molecular level. FigureChemical preservation of cells for Raman microspectroscopy is shown to be strongly dependent on the cell type and the fixative used, in a variety of cell lines, with formalin fixation show to result in spectral content most comparable to that in the live cell

Raman spectroscopy – a potential platform for the rapid measurement of carbon nanotube-induced cytotoxicity

In this study the suitability of Raman spectroscopy for the determination of carbon nanotube mediated toxicity on human alveolar carcinoma epithelial cells (A549) is explored. The exposure of this cell line represents the primary pathway of exposure in humans, that of inhalation. Peak ratio analysis demonstrates a dose-dependent response which correlates to previous toxicological studies. Principal component analysis is employed to further classify cellular response as a function of dose and to examine differences between spectra as a function of exposed concentration. To further illustrate the potential of Raman spectroscopy in this field, Partial Least Squares (PLS) regression and genetic algorithm feature selection have been utilised to demonstrate that clonogenic endpoints, and therefore toxic response, can be potentially predicted from spectra of cells exposed to un-determined doses, removing the need for costly and time consuming biochemical assays. This preliminary study demonstrates the potential of Raman spectroscopy as a probe of cytotoxicity to nanoparticle exposure.

Comparison of Subcellular Responses for the Evaluation and Prediction of the Chemotherapeutic Response to Cisplatin in Lung Adenocarcinoma using Raman Spectroscopy

Confocal Raman Micro-spectroscopy (CRM) is employed to examine the chemical and physiological effects of anticancer agents, using cisplatin and A549 adenocarcinoma cells as a model compound and test system respectively. Spectral responses of the membrane and cytoplasm of the cell are analysed independently and the results are compared to previously reported spectroscopic studies of the nucleus. Moreover, Raman spectra from the proteins extracted from the control and exposed samples are acquired and analysed to confirm the origin of the molecular changes of the cell membrane and cytoplasm of the A549 cells. Multivariate data analysis techniques including Principal Component Analysis (PCA) and Partial Least Squares Regression (PLSR) along with PLS-Jackknifing are used to analyse the data measured from the cell membrane and cytoplasm of the A549 cells and results are correlated with parallel measurements from the cytotoxicity assay MTT. A PLSR model is used to differentiate between the chemical effect of the chemotherapeutic agent and the physiological response of the A549 cells and to identify regions of the spectrum that are associated with these processes respectively. The PLSR model is also employed to predict, on the basis of the Raman spectra, the effective dose as well as the level of physiological response, using spectra data from the cytoplasmic and cell membrane regions. The effectiveness of the models based on spectral datasets from the cell membrane and cytoplasm is compared to similar models constructed using spectral data from the nuclear region as well as one combining spectral data from all regions. In all cases, higher prediction accuracy is found for regression against the cisplatin dose, and for both regression against the dose and the physiological response, nuclear data yield higher precision.

Fourier Transform Infrared Microspectroscopy and Multivariate Methods for Radiobiological Dosimetry

Abstract The scientific literature contains an ever-growing number of reports of applications of vibrational spectroscopy as a multivariate non-invasive tool for analysis of biological effects at the molecular level. Recently, Fourier transform infrared microspectroscopy (FTIRM) has been demonstrated to be sensitive to molecular events occurring in cells and tissue after exposure to ionizing radiation. In this work the application of FTIRM in the examination of dose-dependent molecular effects occurring in skin cells after exposure to ionizing radiation with the use of partial least-squares regression (PLSR) and generalized regression neural networks (GRNN) was studied. The methodology is shown to be sensitive to molecular events occurring with radiation dose and time after exposure. The variation in molecular species with dose and time after irradiation is shown to be non-linear by virtue of the higher modeling efficiency yielded from the non-linear algorithms. Dose prediction efficiencies of approximately ±10 mGy were achieved at 96 h after irradiation, highlighting the potential applications of the methodology in radiobiological dosimetry.

Three dimensional collagen gels as a cell culture matrix for the study of live cells by Raman spectroscopy

Three dimensional collagen gels are evaluated as matrices for the study of live cells by Raman spectroscopy. The study is conducted on a human lung adenocarcinoma (A549) and a spontaneously immortalized human epithelial keratinocyte (HaCaT) cell line. It is demonstrated, using the Alamar Blue assay, that both cell models exhibit enhanced viability in collagen matrices compared to quartz substrates, commonly used for Raman spectroscopy. Using principal component analysis, it is shown that the Raman spectral analysis of cells in collagen matrices is minimally contaminated by substrate contributions and the cell to cell spectral variations are greatly reduced compared to those measured on quartz substrates. Furthermore, the spectral measurements are seen to have little contribution from the cell culture medium, implying that cultures can be kept viable over prolonged measurement or mapping procedures.

Spectroscopic and Chemometric Approaches to Radiobiological Analyses

Vibrational spectroscopy is an attractive modality for the analysis of biological samples, providing a complete non-invasive acquisition of the biochemical fingerprint of the sample. It has been demonstrated that this data provides the means to assay multiple functional responses of a biological system at a spatial resolution as low as a micron within the sample. As the interaction of ionizing radiation with biological systems involves chemical reactions between the products of radiation-induced damage and various structural and functional units within the cell, the vibrational spectroscopic modalities have received attention as potential measurement platforms for the in situ examination of the chemistry of biological species in radiobiology. This presents challenges in relation to sample preparation and the construction of suitable analytical methodologies. In this work protocols for sample preparation and approaches to multivariate analysis of vibrational spectra in radiobiological analysis are detailed and the utility of the methodology in analyzing the evolution of biochemical responses to radiobiological damage are highlighted.

Analyses of Ionizing Radiation Effects In Vitro in Peripheral Blood Lymphocytes with Raman Spectroscopy

The use of Raman spectroscopy to measure the biochemical profile of healthy and diseased cells and tissues may be a potential solution to many diagnostic problems in the clinic. Although extensively used to identify changes in the biochemical profiles of cancerous cells and tissue, Raman spectroscopy has been used less often for analyzing changes to the cellular environment by external factors such as ionizing radiation. In tandem with this, the biological impact of low doses of ionizing radiation remains poorly understood. Extensive studies have been performed on the radiobiological effects associated with radiation doses above 0.1 Gy, and are well characterized, but recent studies on low-dose radiation exposure have revealed complex and highly variable responses. We report here the novel finding that demonstrate the capability of Raman spectroscopy to detect radiation-induced damage responses in isolated lymphocytes irradiated with doses of 0.05 and 0.5 Gy. Lymphocytes were isolated from peripheral blood in a cohort of volunteers, cultured ex vivo and then irradiated. Within 1 h after irradiation spectral effects were observed with Raman microspectroscopy and principal component analysis and linear discriminant analysis at both doses relative to the sham-irradiated control (0 Gy). Cellular DNA damage was confirmed using parallel γ-H2AX fluorescence measurements on the extracted lymphocytes per donor and per dose. DNA damage measurements exhibited interindividual variability among both donors and dose, which matched that seen in the spectral variability in the lymphocyte cohort. Further evidence of links between spectral features and DNA damage was also observed, which may potentially allow noninvasive insight into the DNA remodeling that occurs after exposure to ionizing radiation.

Integration of new biological and physical retrospective dosimetry methods into EU emergency response plans – joint RENEB and EURADOS inter-laboratory comparisons

Abstract Purpose: RENEB, ‘Realising the European Network of Biodosimetry and Physical Retrospective Dosimetry,’ is a network for research and emergency response mutual assistance in biodosimetry within the EU. Within this extremely active network, a number of new dosimetry methods have recently been proposed or developed. There is a requirement to test and/or validate these candidate techniques and inter-comparison exercises are a well-established method for such validation. Materials and methods: The authors present details of inter-comparisons of four such new methods: dicentric chromosome analysis including telomere and centromere staining; the gene expression assay carried out in whole blood; Raman spectroscopy on blood lymphocytes, and detection of radiation-induced thermoluminescent signals in glass screens taken from mobile phones. Results: In general the results show good agreement between the laboratories and methods within the expected levels of uncertainty, and thus demonstrate that there is a lot of potential for each of the candidate techniques. Conclusions: Further work is required before the new methods can be included within the suite of reliable dosimetry methods for use by RENEB partners and others in routine and emergency response scenarios.

Improved protocols for pre-processing Raman spectra of formalin fixed paraffin preserved tissue sections

Although formalin fixed paraffin preserved (FFPP) tissues are a major resource for retrospective studies of disease progression, their use in vibrational spectroscopy studies has been undermined by issues of contributions of substrate and paraffin wax which persist in the spectra and can compromise spectral analysis. Recognising the microcrystalline nature of the wax in the tissue, which are inhomogeneously oriented with respect to the polarisation of the Raman source laser, in this study, we have developed a novel method for removing the paraffin wax contributions to the spectra using matrices of multiple wax spectra. FFPP tissue sections from the oral mucosa were obtained and, with no further chemical processing, the Raman spectral analysis of two regions, epithelium and connective tissue were compared. Matrices of multiple wax spectra were collected from different regions and subtracted from the epithelial and connective tissue spectra using a least squares analysis with non-negative constraints. Spectra of multiple cell components such as DNA and RNA were used in fitting the least squares model to reduce the residual error. The use of a data matrix of multiple wax spectra, as opposed to a single spectrum, results in a more accurate removal of the wax, hence reducing its contribution to spectral analysis. In unprocessed FFPP tissue sections, the contribution of the glass substrate is seen to be minimised in comparison to chemically dewaxed FFPP tissue sections. Contributions of the glass substrate were also successfully removed digitally using the same methodology. The combined results indicate that direct analysis of FFPP tissue sections is feasible using Raman spectroscopy, avoiding the need for chemical dewaxing. Additionally, the ability to use glass slides is very important in translation to the clinic.