In vitro toxicology evaluation of pharmaceuticals using Raman micro‐spectroscopy

Abstract

Raman micro-spectroscopy combined with multivariate analysis was employed to monitor real-time biochemical changes induced in living cells in vitro following exposure to a pharmaceutical. The cancer drug etoposide (topoisomerase II inhibitor) was used to induce double-strand DNA breaks in human type II pneumocyte-like cells (A549 cell-line). Raman spectra of A549 cells exposed to 100 microM etoposide were collected and classical least squares (CLS) analysis used to determine the relative concentrations of the main cellular components. It was found that the concentrations of DNA and RNA significantly (P < 0.05) decreased, whilst the concentration of lipids significantly (P < 0.05) increased with increasing etoposide exposure time as compared to control untreated A549 cells. The concentration of DNA decreased by 27.5 and 87.0% after 24 and 48 h exposure to etoposide respectively. Principal components analysis (PCA) successfully discriminated between treated and untreated cells, with the main variance between treatment groups attributed to changes in DNA and lipid. DNA fragmentation was confirmed by Western blot analysis of apoptosis regulator protein p53 and cell metabolic activity determined by MTT assay. The over-expression of p53 protein in the etoposide treated cells indicated a significant level of DNA fragmentation and apoptosis. MTT tests confirmed that cellular metabolic activity decreased following exposure to etoposide by 29.4 and 61.2% after 24 and 48 h, respectively. Raman micro-spectroscopy may find applications in the toxicology screening of other drugs, chemicals and new biomaterials, with a range of cell types.

Evaluation of antibiotic effects on Pseudomonas aeruginosa biofilm using Raman spectroscopy and multivariate analysis

“…This result might indicate denaturation and conformational changes in proteins related to cell death. It was previously proposed that decrease of these Raman intensity were related to the denaturation and conformational changes of proteins, which correlated with the cell death [43][44][45] The reduction of Raman intensity corresponding to DNA, such as the bands at 780 and 1100 cm −1 , arose from the destruction of the ring structure, indicating degradation of the DNA. It indicates another evidence for cell death.…”

A simple and rapid detection of tissue adhesive-induced biochemical changes in cells and DNA using Raman spectroscopy

“…The main changes related to the proteins can be observed at 1002, 1257, and 1656 cm corresponds to the ring stretching of phenylalanine, which is very important component of proteins. It has been shown that this peak is very sensitive to the death of cells [15,31,32]. The peaks at 1257 and 1656 cm −1 represent amide III (β sheet) and amid I (α-helix), respectively, which are basic components of protein structure and are also extremely sensitive to changes in the structure of the protein.…”

High throughput toxicity screening and intracellular detection of nanomaterials

“…CRM imaging was used to follow the different stages of the cell cycle,36 DNA condensation in late stages of apoptosis and also for the assessment of cell viability 37, 38. DNA damage, lipid changes, and protein denaturation were analyzed as a response to drugs and chemicals 39, 40. However, the spatial resolution and sensitivity of CRM are limited compared with TEM which typically has a threshold of ~60 nm 41…”