Articles

Quantitative Reagent-Free Detection Of Fibrinogen Levels In Human Blood Plasma Using Raman Spectroscopy

Kelvin W. Poon et al. — Thu, 03 Jan 2013 01:55:33 PST

Fibrinogen assays are commonly used as part of clinical screening tests to investigate haemorrhagic states, for detection of disseminated intravascular coagulation and as a predictor of a variety of cardiovascular events. The Clauss assay, which measures thrombin clotting time, is the most commonly used method for measuring fibrinogen levels. Nevertheless, inconsistencies are present in inter-manufacturer reagent sources, calibration standards and methodologies. Automated coagulation analysers, which measure changes in optical density during the prothrombin time (PT-Fg), have found use in many hospitals. However, the PT-Fg method is found to give falsely elevated values due to varying choices of calibrants, reagents and analysers. As an alternative, Raman spectroscopy has previously been applied to the analysis of blood and its various constituents to determine various analyte concentrations such as glucose, urea, triglycerides and cholesterol. In this study, Raman spectroscopy was investigated for its ability to accurately quantify fibrinogen concentration in blood plasma. Samples collected from 34 patients were analysed by Raman spectroscopy and the resultant spectra were fitted with a Partial Least Squares Regression model using target values obtained through a pre-calibrated Clauss fibrinogen assay. Various spectral pre-processing methods were utilised to prepare data to be entered into a calibration model. A root mean square error of prediction of 0.72 ± 0.05 g/L was achieved with as few as 25 spectra. In this pilot study, Raman spectroscopy has been demonstrated to be a robust technique providing rapid and reagent-free quantification of fibrinogen levels in blood plasma and a potential alternative to the Clauss assay.

Reactive Oxygen Species-induced Release of Signalling Factors in Irradiated Cells Triggers Membrane Signalling and Calcium Influx in Bystander Cells.

Fiona Lyng et al. — Fri, 20 Apr 2012 00:39:14 PDT

Purpose: The aim of this study was to elucidate the sequence of very early bystander signalling events and to determine the role of the different signaling molecules in both the production of the bystander signal and the response to this signal.

Materials and Methods: Human keratinocytes (HaCaT cell line) were irradiated (0.005, 0.05 and 0.5 Gy) using a cobalt 60 teletherapy unit, the medium was harvested one hour post irradiation and transferred to recipient HaCaT cells. Membrane permeability and levels of calcium, reactive oxygen species and nitric oxide were measured in the recipient cells immediately after the addition of irradiated cell conditioned medium (ICCM). Inhibitors of reactive oxygen species (ROS), nitric oxide (NO), calcium and membrane signalling were used in both donor and recipient cells to investigate if bystander effects could be blocked.

Results: It was found that membrane signalling followed by calcium influx was the first response in the recipient cells to addition of ICCM. ROS, NO and calcium were all found to be important signalling molecules involved in bystander responses, while ROS and calcium were found to be involved in the production of the bystander signal.

Conclusions: The data suggest that calcium and/or ROS induce irradiated cells to release long lived signalling factors which can trigger membrane signalling and an influx of calcium further inducing ROS in unirradiated cells.

Identifying and Localizing Intracellular Nanoparticles Using Raman Spectroscopy

Jennifer Dorney et al. — Thu, 26 Jan 2012 01:41:17 PST

Raman microscopy is employed to spectroscopically image biological cells previously exposed to fluorescently labelled polystyrene nanoparticles and, in combination with K-means clustering and Principal Component Analysis (PCA), is demonstrated to be capable of localising the nanoparticles and identifying the subcellular environment based on the molecular spectroscopic signatures. The neutral nanoparticles of 50 nm or 100 nm, as characterised by dynamic light scattering, are shown to be non-toxic to a human lung adenocarcinoma cell-line (A549), according to a range of cytotoxicity assays including Neutral Red, Alamar Blue, Coomassie Blue and (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Confocal fluorescence microscopy identifies intracellular fluorescence due to the nanoparticle exposure, but the fluorescence distribution is spatially diffuse, potentially due to detachment of the dye from the nanoparticles, and the technique fails to unambiguously identify the distribution of the nanoparticles within the cells. Raman spectroscopic mapping of the cells in combination with K-means cluster analysis is used to clearly identify and localise the polystyrene nanoparticles in exposed cells, based on their characteristic spectroscopic signatures. PCA identifies the local environment as rich in lipidic signatures which are associated with localisation of the nanoparticles in the endoplasmic reticulum. The importance of optimised cell growth conditions and fixation processes is highlighted. The preliminary study demonstrates the potential of the technique to unambiguously identify and locate nonfluorescent nanoparticles in cells and to probe not only the local environment but also changes to the cell metabolism which may be associated with cytotoxic responses.

Understanding the Molecular Information Contained in Principal Component Analysis of Vibrational Spectra of Biological Systems

Franck Bonnier et al. — Wed, 14 Dec 2011 02:08:36 PST

K-means clustering followed by Principal Component Analysis (PCA) is employed to analyse Raman spectroscopic maps of single biological cells. K-means clustering successfully identifies regions of cellular cytoplasm, nucleus and nucleoli, but the mean spectra do not differentiate their biochemical composition. The loadings of the principal components identified by PCA shed further light on the spectral basis for differentiation but they are complex and, as the number of spectra per cluster is imbalanced, particularly in the case of the nucleoli, the loadings under-represent the basis for differentiation of some cellular regions. Analysis of pure bio-molecules, both structurally and spectrally distinct, in the case of histone, ceramide and RNA, and similar in the case of the proteins albumin, collagen and histone, show the relative strong representation of spectrally sharp features in the spectral loadings, and the systematic variation of the loadings as one cluster becomes reduced in number. The more complex cellular environment is simulated by weighted sums of spectra, illustrating that although the loading become increasingly complex; their origin in a weighted sum of the constituent molecular components is still evident. Returning to the cellular analysis, the number of spectra per cluster is artificially balanced by increasing the weighting of the spectra of smaller number clusters. While it renders the PCA loading more complex for the three-way analysis, a pair wise analysis illustrates clear differences between the identified subcellular regions, and notably the molecular differences between nuclear and nucleoli regions are elucidated. Overall, the study demonstrates how appropriate consideration of the data available can improve the understanding of the information delivered by PCA.

In Vitro Analysis of Immersed Human Tissues by Raman Microspectroscopy

Franck Bonnier et al. — Wed, 16 Nov 2011 04:53:15 PST

Raman microspectroscopy is a powerful tool for the analysis of tissue sections, providing a molecular map of the investigated samples. Nevertheless, data pre-processing and, particularly, the removal of the broad background to the spectra remain problematic. Indeed, the physical origin of the background has not been satisfactorily determined. Using 785 nm as source in a confocal geometry, it is demonstrated for the example of the protein kappa-elastin that the background and resulting quality of the recorded spectrum are dependent on the morphology of the sample. Whereas a fine powder yields a dominant broad background, compressed pellets and solution-cast thin films produce, respectively, improved quality spectra and significantly reduced spectral background. As the chemical composition of the samples is identical, the background is ascribed to stray light due to diffuse scattering rather than an intrinsic photoluminescence.

The recorded spectra from a tissue sample exhibit a large and spatially variable background, resulting in poorly defined spectral features. A significant reduction of the background signal as well as improvement of the spectral quality is achieved by immersion of the sample in water and measurement with an immersion objective. The significant improvement in signal to background is attributed to a reduction of the diffuse scattering due to a change in the effective morphology as a result of an improved index matching at the water/tissue interface compared to the air/tissue interface.

Compared to sections measured in air, the background is reduced to that of the water, and pre-processing is reduced to the subtraction of the substrate and water signal and correction for the instrument response, both of which are highly reproducible. Data pre-processing is thus greatly simplified and the results significantly more reliable.

Correlation of p16INK4A Expression and HPV Copy Number with Cellular FTIR Spectroscopic Signatures of Cervical Cancer Cells

Kamila Ostrowska et al. — Wed, 16 Nov 2011 03:59:52 PST

Cervical cancer, a potentially preventable disease, has its main aetiology in infection by high risk human papillomavirus (HR-HPV). Approaches to improving cervical cancer screening and diagnostic methodologies include molecular biological analysis, targeting of biomarker proteins, but also exploration and implementation of new techniques such as vibrational spectroscopy. This study correlates the biomarker protein p16^INK4A expression levels dependent on HPV copy number with the infrared absorption spectral signatures of the cervical cancer cell lines, HPV negative C33A, HPV-16 positive SiHa and CaSki and HPV-18 positive HeLa. Confocal fluorescence microscopy demonstrated that p16^INK4A is expressed in all investigated cell lines in both nuclear and cytoplasmic regions, although predominantly in the cytoplasm. Flow cytometry was used to quantify the p16^INK4A expression levels and demonstrated a correlation, albeit nonlinear, between the reported number of integrated HPV copies and p16^INK4A expression levels. CaSki cells were found to have the highest level of expression, HeLa intermediate levels, and SiHa and C33A the lowest levels. FTIR spectra revealed differences in nucleic acid, lipid and protein signatures between the cell lines with varying HPV copy number. Peak intensities exhibited increasing tendency in nucleic acid levels and decreasing tendency in lipid levels with increasing HPV copy number, and although they were found to be nonlinearly correlated with the HPV copy number, their dependence on p16^INK4A levels was found to be close to linear. Principal Component Analysis (PCA) of the Infrared absorption spectra revealed differences between nuclear and cytoplasmic spectroscopic signatures for all cell lines, and furthermore clearly differentiated the groups of spectra representing each cell line. Finally, Partial Least Squares (PLS) analysis was employed to construct a model which can predict the p16^INK4A expression level based on a spectral fingerprint of a cell line, demonstrating the diagnostic potential of spectroscopic techniques.

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

Haq Nawaz et al. — Wed, 16 Nov 2011 01:25:18 PST

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 Square Regression (PLSR) along with PLS-Jack knifing 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 dose and physiological response, nuclear data yields higher precision.

The Involvement of Calcium and MAP Kinase Signaling Pathways in the Production of Radiation-Induced Bystander Effects

Fiona M. Lyng et al. — Thu, 27 Jan 2011 06:41:36 PST

Much evidence now exists regarding radiation-induced bystander effects, but the mechanisms involved in the transduction of the signal are still unclear. The mitogen-activated protein kinase (MAPK) pathways have been linked to growth factor-mediated regulation of cellular events such as proliferation, senescence, differentiation and apoptosis. Activation of multiple MAPK pathways such as the ERK, JNK and p38 pathways have been shown to occur after exposure of cells to radiation and a variety of other toxic stresses. Previous studies have shown oxidative stress and calcium signaling to be important in radiation-induced bystander effects. The aim of the present study was to investigate MAPK signaling pathways in bystander cells exposed to irradiated cell conditioned medium (ICCM) and the role of oxidative metabolism and calcium signaling in the induction of bystander responses. Human keratinocytes (HPV-G cell line) were irradiated (0.005–5 Gy) using a cobalt-60 teletherapy unit. The medium was harvested 1 h postirradiation and transferred to recipient HPV-G cells. Phosphorylated forms of p38, JNK and ERK were studied by immunofluorescence 30 min–24 h after exposure to ICCM. Inhibitors of the ERK pathway (PD98059 and U0126), the JNK pathway (SP600125), and the p38 pathway (SB203580) were used to investigate whether bystander-induced cell death could be blocked. Cells were also incubated with ICCM in the presence of superoxide dismutase, catalase, EGTA, verapamil, nifedipine and thapsigargin to investigate whether bystander effects could be inhibited because of the known effects on calcium homeostasis. Activated forms of JNK and ERK proteins were observed after exposure to ICCM. Inhibition of the ERK pathway appeared to increase bystander-induced apoptosis, while inhibition of the JNK pathway appeared to decrease apoptosis. In addition, reactive oxygen species, such as superoxide and hydrogen peroxide, and calcium signaling were found to be important modulators of bystander responses. Further investigations of these signaling pathways may aid in the identification of novel therapeutic targets.

Initiation of Apoptosis in Cells Exposed to Medium from the Progeny of Irradiated Cells: a Possible Mechanism for "Bystander" induced Genomic Instability.

Fiona M. Lyng et al. — Thu, 27 Jan 2011 06:41:34 PST

Vibrational Spectroscopy for Pathology from Biochemical Analysis to Diagnostic Tool

Fiona M. Lyng et al. — Thu, 27 Jan 2011 06:41:33 PST

Cervical cancer is the second most common cancer in women worldwide with 80% of cases arising in the developing world. The mortality associated with cervical cancer can be reduced if this disease is detected at the early stages of development or at the pre-malignant state (cervical intra-epithelial neoplasia, CIN). The aim of this study was to investigate the potential of Raman spectroscopy as a diagnostic tool to detect biochemical changes accompanying cervical cancer progression. Raman spectra were acquired from proteins, nucleic acids, lipids and carbohydrates in order to gain an insight into the biochemical composition of cells and tissues. Spectra were also obtained from histological samples of normal, CIN and invasive carcinoma tissue from 40 patients. Multivariate analysis of the spectra was carried out to develop a classification model to discriminate normal from abnormal tissue. The results show that Raman spectroscopy displays a high sensitivity to biochemical changes in tissue during disease progression resulting in an exceptional prediction accuracy when discriminating between normal cervical tissue, invasive carcinoma and cervical intra-epithelial neoplasia (CIN). Raman spectroscopy shows enormous clinical potential as a rapid non invasive diagnostic tool for cervical and other cancers.

Apoptosis is Initiated in Human Keratinocytes Exposed to Signalling Factors from Microbeam Irradiated Cells

Fiona M. Lyng et al. — Thu, 27 Jan 2011 06:41:31 PST

Purpose: There is now no doubt that bystander signalling from irradiated cells occurs and causes a variety of responses in cells not targeted by the ionising track. However, the mechanisms underlying these processes are unknown and the relevance to radiotherapy and risk assessment remains controversial. Previous research by our laboratory has shown bystander effects in a human keratinocyte cell line, HPV-G cells, exposed to medium from irradiated HPV-G cells. The aim of this work was to investigate if similar mechanisms to those identified in medium transfer experiments occurred in these HPV-G cells when they are in the vicinity of microbeam irradiated cells. Demonstration of a commonality of mechanisms would support the idea that the process is not artifactual. Materials and Methods: HPV-G cells were plated as two separate populations on mylar dishes. One population was directly irradiated using a charged particle microbeam (1 - 10 protons). The other population was not irradiated. Bystander factor induced apoptosis was investigated in both populations following treatment by monitoring the levels of reactive oxygen species and mitochondrial membrane potential using fluorescent probes. Expression of the anti-apoptotic protein, bcl-2, and cytochrome c were determined, as well as apoptosis levels. Results: Microbeam irradiation induced increases in reactive oxygen species and decreases in mitochondrial membrane potential at 6 hours post exposure, increased expression of bcl-2 and cytochrome c release at 6.5 hours and increased apoptosis at 24 hours. Conclusion: This study shows that similar bystander signalling pathways leading to apoptosis are induced following microbeam irradiation and following medium transfer. This demonstrates that the mechanisms involved are common across different radiation qualities and conditions and indicates that they may be relevant in vivo.

Medium from Irradiated Cells Induces a Dose Dependent Expression of Mitochondrial Changes and BCL2 Responses in Unirradiated Human Keratinocytes

Paula Maguire et al. — Thu, 27 Jan 2011 06:41:30 PST

Exposure of unirradiated human keratinocytes to irradiated cell conditioned medium (ICCM) is known to cause a cascade of events that leads to reproductive death and apoptosis. This study investigates the effect of ICCM on clonogenic survival, mitochondrial mass and BCL2 expression in unirradiated keratinocytes. Exposure to 5 mGy, 0.5 Gy and 5 Gy ICCM resulted in a significant decrease in clonogenic survival. Human keratinocytes incubated with ICCM containing an antioxidant, N-acetylcysteine, showed no significant decrease in clonogenic survival. HPV-G cells incubated with ICCM containing a caspase 9 inhibitor showed no significant decrease in clonogenic survival when the ICCM dose was #0.5 Gy. A significant increase in mitochondrial mass per cell was observed after exposure to 5 mGy and 0.5 Gy ICCM. A change in the distribution of the mitochondria from a diffuse cytoplasmic distribution to a more densely concentrated perinuclear distribution was also observed at these doses. No significant increase in mitochondrial mass or change in distribution of the mitochondria was found for 5 Gy ICCM. Low BCL2 expression was observed in HPV-G cells exposed to 5 mGy or 0.5 Gy ICCM, whereas a large significant increase in BCL2 expression was observed in cells exposed to 5 Gy ICCM. This study has shown that low-dose irradiation can cause cells to produce medium-borne signals that can cause mitochondrial changes and the induction of BCL2 expression in unirradiated HPV-G cells. The dose dependence of the mitochondrial changes and BCL2 expression suggests that the mechanisms may be aimed at control of response to radiation at the population level through signaling pathways

Modulation of Radiation Responses by Pre-Exposure to Irradiated Cell Conditioned Medium

Paula Maguire et al. — Thu, 27 Jan 2011 06:41:28 PST

The aim of this study was to investigate whether exposure of HPV-G cells to irradiated cell conditioned medium (ICCM) could induce an adaptive response if the cells were subsequently challenged with a higher ICCM dose. Clonogenic survival and major steps in the cascade leading to apoptosis, such as calcium influx and loss of mitochondrial membrane potential, were examined to determine whether these events could be modified by giving a priming dose of ICCM before the challenge dose. Clonogenic survival data indicated an ICCMinduced adaptive response in HPV-G cells ‘‘primed’’ with 5 mGy or 0.5 Gy ICCM for 24 h and then exposed to 0.5 Gy or 5 Gy ICCM. Reactive oxygen species (ROS) were found to be involved in the bystander-induced cell death. Calcium fluxes varied in magnitude across the exposed cell population, and a significant number of the primed HPV-G cells did not respond to the challenge ICCM dose. No significant loss of mitochondrial membrane potential was observed when HPV-G cells were exposed to 0.5 Gy ICCM for 24 h followed by exposure to 5 Gy ICCM for 6 h. Exposure of HPV-G cells to 5 mGy ICCM for 24 h followed by exposure to 0.5 Gy ICCM for 18 h caused a significant increase in mitochondrial mass and a change in mitochondrial location, events associated with the perpetuation of genomic instability. This study has shown that a priming dose of ICCM has the ability to induce an adaptive response in HPV-G cells subsequently exposed to a challenge dose of ICCM.

Increased Mitochondrial Mass in Cells with Functionally Compromised Mitochondria after Exposure to both Direct y Radiation and Bystander Factors

Sharon Nugent et al. — Thu, 27 Jan 2011 06:41:27 PST

The bystander effect describes radiation-like damage in unirradiated cells either in the vicinity of irradiated cells or exposed to medium from irradiated cells. This study aimed to further characterize the poorly understood mitochondrial response to both direct irradiation and bystander factor(s) in human keratinocytes (HPV-G) and Chinese hamster ovarian cells (CHO-K1). Oxygen consumption rates were determined during periods of state 4, state 3 and uncoupled respiration. Mitochondrial mass was determined using MitoTracker FM. CHO-K1 cells showed significantly reduced oxygen consumption rates 4 h after exposure to 5 Gy direct radiation and irradiated cell conditioned medium (ICCM) and an apparent recovery 12–24 h later. The apparent recovery was likely due to the substantial increase in mitochondrial mass observed in these cells as soon as 4 h after exposure. HPV-G cells, on the other hand, showed a sustained increase in oxygen consumption rates after ICCM exposure and a transient increase 4 h after exposure to 5 Gy direct radiation. A significant increase in mitochondrial mass per HPV-G cell was observed after exposure to both direct radiation and ICCM. These findings are indicative of a stress response to mitochondrial dysfunction that increases the number of mitochondria per cell.

Solar Simulated Radiation Induced Cell Death Depends on Spectral Distribution and Irradiance But Not Output Delivery

Alanna Maguire et al. — Thu, 27 Jan 2011 06:41:26 PST

Photo biological investigations are dependent on calibration and characterisation to determine the relevance of an artificial irradiator to the study at hand. The importance of this has been voiced in the literature. However, the importance of output delivery is relatively unknown. The biological relevance of a high energy, rapidly pulsing solar simulator was investigated using the clonogenic assay and was found to be reciprocity law compliant despite an exaggerated UV irradiance in excess of 1600 Wm-2 delivered per pulse. In fact, it was found to be the least cytotoxic irradiator compared to a second solar simulator and a UVB fluorescent lamp with continuous UV irradiances of 55 Wm-2 and 6.4 Wm-2 respectively. The reduced survival observed with the continuous irradiators is attributed to differences in spectral irradiance and distribution, particularly in the UVB, which in the absence of thorough calibration and characterisation may have resulted in erroneous conclusions.

Ionizing Radiation Induces a Stress Response in Primary Cultures of Rainbow Trout Skin

Fiona M. Lyng et al. — Thu, 27 Jan 2011 06:41:24 PST

Fish skin is very vulnerable to damage from physical and chemical pollutants because it is in direct contact with the aquatic environment. In this study, the effect of g radiation on primary cultures of rainbow trout skin was investigated. Primary cultures containing two cell types, epidermal cells and goblet mucous cells, were exposed to doses ranging from 0.5–15 Gy 60Co g radiation. Expression of PCNA, c-myc and BCL2 was investigated as well as growth and levels of apoptosis and necrosis. Morphological and functional changes were also studied. The irradiated cultures showed evidence of a dose-dependent increase in necrosis and enhanced proliferation as well as morphological damage. In addition, mucous cell area was found to decrease significantly after irradiation. The study shows the value of these primary cultures as in vitro models for studying radiation effects. They provide an effective alternative to whole-animal exposures for radiation risk assessment.

A Dose Threshold for a Medium Transfer Bystander Effect for a Human Skin Cell Line

Zhengfeng Liou et al. — Thu, 27 Jan 2011 06:41:23 PST

The existence of radiation-induced bystander effects mediated by diffusible factors is now accepted but the mechanisms and precise behavior at low doses remain unclear. We exposed cells to gamma doses in the range 0.04 mGy – 5 Gy, harvested the culture medium and transferred it to unirradiated reporter cells. Calcium fluxes and clonogenic survival were measured in the recipients. We show evidence for a dose threshold around 2 mGy for the human skin cell line used with a suggestion of increased survival below that dose. Similar experiments using direct gamma irradiation showed no reduction in survival until the dose exceeded 7 mGy. Preliminary data for neutrons where the gamma dose was kept below the bystander threshold, does not show significant bystander effect in the dose range 1 – 33 mGy. A lack of a bystander response with neutrons occurred at high dose around 1 Gy where significant cell killing from direct irradiation was observed. The result may have implications for understanding the role of bystander effects at low doses.

In-vitro screening of organotin compounds and sediment extracts for cytotoxicity to fish cells

Michelle Giltrap et al. — Mon, 24 Jan 2011 04:32:09 PST

The present study reports an in vitro screening method for contaminants in sediment samples utilizing an RTG-2 cell line. This technique integrates cytotoxicity testing with analytical chemistry with the aim of achieving a toxicity evaluation of the sediment sample. The toxic effect of individual organotin (OT) compounds and their presence in the sediment sample is the focus of the present study; however, other contaminants are also discussed. The following OT compounds: tributyltin (TBT), dibutyltin (DBT), monobutyltin (MBT), triphenyltin (TPT), diphenyltin (DPT), and a sediment solvent extract are exposed to the RTG-2 fish cell line. Both the alamar blue (AB) and neutral red (NR) assays are used to assess cytotoxicity after 24-h and 96-h exposure. Methodology for preparation of a sediment solvent extract suitable for biological testing and analytical determination is also described. With the RTG-2 cells, the AB and NR assays had comparable sensitivity for each individual OT compound exposure after 24 h, with TPT being the most toxic compound tested. The individual OT compound concentrations required to induce a 50% toxic effect on the cells (369 ng ml1 TBT, 1,905 ng ml1 DBT) did not equate to the concentrations of these contaminants present in the sediment extract that induced a 50% effect on the cells (294 ng ml1 TBT, 109 ng ml1 DBT). The solvent extract therefore exhibited a greater toxicity, and this suggests that the toxic effects observed were not due to OT compounds alone. The presence of other contaminants in the solvent extract is confirmed with chemical analysis, warranting further toxicity testing of contaminant mixtures and exposure to the cell line to further elucidate a complete toxicity evaluation.

Reactive Oxygen Species Induced Cytokine Production and Cytotoxicity of PAMAM Dendrimers in J774a.1 Cells

Pratap Naha et al. — Fri, 07 Jan 2011 06:53:27 PST

The immunotoxicity of three generations of polyamidoamine (PAMAM) dendrimers (G-4, G-5 and G-6) was evaluated in mouse macrophage cells in vitro. Using the Alamar blue and MTT assays, a generation dependent cytotoxicity of the PAMAM dendrimers was found whereby G-6 > G-5 > G-4. The toxic response of the PAMAM dendrimers correlated well with the number of surface primary amino groups, with increasing number resulting in an increase in toxic response. An assessment of intracellular ROS generation by the PAMAM dendrimers was performed by measuring the increased fluorescence as a result of intracellular oxidation of Carboxy H2DCFDA to DCF both quantitatively using plate reader and qualitatively by confocal laser scanning microscopy. The inflammatory mediators macrophage inflammatory protein-2 (MIP-2), tumour necrosis factor-α (TNF-α) and interleukin-6, (IL-6) were measured by the enzyme linked immunosorbant assay (ELISA) following exposure of mouse macrophage cells to PAMAM dendrimers. A generation dependent ROS and cytokine production was found, which correlated well with the cytotoxicological response and therefore number of surface amino groups. A clear time sequence of increased ROS generation (maximum at not, vert, similar 4 h), TNF-α and IL-6 secretion (maximum at not, vert, similar 24 h), MIP-2 levels and cell death (not, vert, similar 72 h) was observed. The intracellular ROS generation and cytokine production induced cytotoxicity point towards the mechanistic pathway of cell death upon exposure to PAMAM dendrimers.

Imaging Live Cells Grown on a Three Dimensional Collagen Matrix Using Raman Microspectroscopy

Franck Bonnier et al. — Tue, 04 Jan 2011 06:38:45 PST

Three dimensional collagen gels have been used as matrices for the imaging 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. The lateral resolution of the system has been estimated to be <1.5 μm making it possible to access the subcellular organization. Using K-means clustering analysis, it is shown that the different subcellular compartments of individual cells can be indentified and differentiated. The biochemical specificity of the information contained in the Raman spectra allows the visualization of differences in the molecular signature of the different sub-cellular structures. Furthermore, to enhance the chemical information obtained from the spectra, principal component analysis has been employed, allowing the identification of spectral windows with a high variability. The comparison between the loadings calculated and spectra from pure biochemical compounds enables the correlation of the variations observed with the molecular content of the different cellular compartments.