Alterations In The Biomolecular Signatures Of Cell Injury Or Developing Tissue As Determined By Biospectroscopy

Biological tissue or cell is comprised of proteins, nucleic acids, lipids and other biological molecules. Biomolecule that contain different chemical bonds with an electric dipole moment has its own characteristic vibration bands of molecules in biospectroscopy. Changes in components and/or conformation can give rise to a detailed biomolecular fingerprint of the cells or tissue under investigation in the form of an biospectroscopy, such as attenuated total reflection Fourier-transform infrared (ATR-FTIR) or Raman spectrum relating to chemical structures of biological molecules. Different biospectroscopy, though fundamental different in experimental design and physical background, the predominant advantages of the biospectroscopy techniques are rapid, objective, non-invasive, reproducible and produce datas on multiple biomarkers.Biospectroscopy experiments generate complex biochemical datasets containing hundreds or thousands of spectra, and analysis is best achieved by multivariate approaches such as principal component analysis (PCA) and/or linear discriminant analysis (LDA). This allows for data reduction to facilitate the identification of wavenumber-related biomarkers such as glycogen content, lipid content, conformational protein changes and phosphorylation characteristics, and structural alterations in DNA/RNA. At present, the incidence of cancer induced by environmental chemicals is increasing year by year. It is very important for the prevention and treatment of cancer to detect the concentrations of environmental carcinogenic chemicals consistent with background levels of human exposure and better understood relevant mechanisms of action. The environmental polycyclic aromatic hydrocarbon (PAH), Benzo[a]pyrene (B[a]P) is a environmental pro-mutagenic and pro-carcinogenic contaminant. Traditionally, short-term assessments of genotoxicity have tested high-dose (≥μmol/L) treatments, but how these reflect low-dose contaminant-induced effects consistent with background levels of human exposure remains obscure. Methods capable of delineating and shedding light into relevant mechanisms of action of real-world exposures in target cells are urgently required. In this study, we identify alterations induced by low concentration (the lowest concentration is nmol/L) of benzo[a]pyrene in MCF-7breast cancer cells using ATR-FTIR spectroscopy coupled with computational analysis and shed light into relevant mechanisms of action.The growth and development of tissue is investigated not only to better understand structure-function relationships in different tissue but also to have a deeper understanding of the pathological change of tissue, the prevention and treatment of disease. In this study, we also used ATR-FTIR spectroscopy and Raman microspectroscopy together to obtain biomolecular signatures at2-day intervals from embryonic day10to day18of incubation when the tissue is undergoing embryonic growth and transition into a mature transparent tissue. This study was investigated not only to better understand structure-function relationships in corneal growth and development but also to provide the new insight into studying normal histology and pathology using biospectroscopy. In summary, this study demonstrates that biospectroscopy, ATR-FTIR spectroscopy and Raman microspectroscopy can be used to reveal significant spectral discrimination and changing signatures in cell injury or the developing tissue and that, by the use of multivariate analysis, the biomarkers responsible for the segregation of between-category can be discriminated. Biospectroscopy is a novel approach to determine biomolecular signatures of cell or tissue. Objective Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy can be employed as a novel approach towards interrogating the biomolecular signature of cells in a non-destructive fashion. In the current study, we employed ATR-FTIR spectroscopy with subsequent multivariate analysis to investigate alterations in cycle-associated MCF-7cells induced by various doses of B[a]P. Dose-related effects, the mode of action of different concentrations of B[a]P on MCF-7cells, cycle-associated susceptibility to contaminant and discriminating biomarkers were investigated to determine the magnitude of induced alterations. The aim of the study is to explore a novel approach to investigate the mechanistic effects of low-dose levels of environmental contaminants as well.Methods1. MCF-7cells were treated for24h with the vehicle control (DMSO) and range of B[a]P concentrations (10-9M,10-8M,10-7M,10-6M, or10-5M) in S-phase or G0/G1-phase of the cell cycle. After then, cells were washed, trypsinized, re-suspended, fixed and interrogated with ATR-FTIR spectroscopy.2. Cells were exposed to B[a]P at concentrations of10-9M or10-6 M compared to vehicle control (DMSO) for24h. Cells were then washed, trypsinized, re-suspended, fixed and interrogated with ATR-FTIR spectroscopy.3. Resultant spectra were analyzed for variance by using principal component analysis and linear discriminant analysis (PCA-LDA).4. MCF-7cells were seeded (≈1.0×105) in T25flasks and were grown for24h. Cells were then exposed to B[a]P at concentrations of10-9M or10-6M compared to vehicle control (DMSO) in5ml of complete medium, in three independent experiments. Cells were washed, trypsinized, re-suspended and cell number determined at indicated timepoints (0,12,24,48h) employing a haemocytometer.Results1. Spectral points in1D or2D scores plots representing cells treated with higher concentrations (10-6M or10-5M) are more segregated from the corresponding vehicle control compared to those treated with lower levels, suggesting that dose-related effects were observed. ANOVA tests indicate that all treatment categories are significantly different (P≤0.001) from the corresponding vehicle control.2. Both1-D and2-D scores plots demonstrated that the10-6M group presented a greater degree of separation than the10-9M group whether in S-phase or in G0/G1-phase. ATR-FTIR spectroscopy revealed biomarkers induced by10-6M B[a]P compared to those induced by corresponding control in S-phase, these were changes in DNA/RNA, lipid, secondary structures of proteins (Amide Ⅰ and Amide Ⅲ) and COO-symmetric stretching vibrations of fatty acids and amino acid. While low-dose (10-9M) B[a]P resulted in marked alterations associated with secondary structures of proteins (Amide Ⅰ, Amide Ⅱ and Amide Ⅲ) and lipid. However, B[a]P-induced spectral alterations at both test exposure concentrations in cell populations in G0/G1-phase were associated with secondary structures of proteins, DNA/RNA and COO-symmetric stretching vibrations of fatty acids and amino acid.3. The different effects of B[a]P on the growth kinetics of MCF-7associated with time-and concentration-dependent (10-6M B[a]P vs.10-9M B[a]P vs. DMSO). Following a12-h treatment, there are no marked differences between different treatment categories. After a further12h, high-dose B[a]P gave rise to an elevated cell number, compared to the other two treatment categories. However, following48-h treatment, high-dose B[a]P resulted in marked decreases in cell numbers; in contrast, low-dose B[a]P exposure resulted in a similar if not slightly higher level of cell numbers compared to the corresponding vehicle control.Conclusion The evident alterations of MCF-7cells in different cycle phases induced by benzo[a]pyrene and dose-related effects were observed. The major changes in the spectral regions associated with DNA/RNA, secondary protein structure and lipid were identified. The application of IR spectroscopy with computational analysis offers the possibility to investigate the mechanistic effects of low-dose levels of environmental contaminants. The novel approach could produce an integrated response profile to environmental effects. Objective Biospectroscopy tools are increasingly being recognized as novel approaches toward interrogating complex biological structures in a non-destructive fashion. This study was conducted to apply these tools to interrogate alterations in the molecular signatures of developing chick corneas during the onset and development of transparency. The aim of the study is not only to better understand structure-function relationships in corneal growth and development but also to provide the new insight into studying normal histology and pathology using biospectroscopy.Methods Embryonic chick corneas (n=46) were obtained at two-day intervals from embryonic day10to day18of incubation and interrogated with attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy and Raman microspectroscopy. Resultant spectra were analyzed for variance by using principal component analysis and linear discriminant analysis (PCA-LDA).Results1. Mean spectra following ATR-FTIR spectroscopy or Raman microspectroscopy derived from corneas at each developmental stage showed some overlap, however, notable by both techniques was the increasing intensity of DNA signal (1080cm-1) from day10onwards;2. Using ATR-FTIR spectroscopy or Raman microspectroscopy, a clear cluster segregation of the chick corneas at different developmental days was observed in1-D,2-D and3-D PCA-LDA scores plots. In particular, the PCA-LDA data indicated that there was less overlap of spectral points between developmental stages with greater intervals. ANOVA tests indicate that other developmental stages are significantly different (P≤0.001) from the embryonic day10;3. Two-category discriminant analysis results of ATR-FTIR or Raman spectra from chick corneas at different stages of embryonic development clearly identified the alterations in the biomolecular signatures;4. Major segregating biomarkers identified by ATR-FTIR spectroscopy between day10and day18in the DNA/RNA (1126cm-1), glycogen (1045cm-1), protein (1470cm-1), and Amide II (1512cm-1and1524cm-1) spectral regions;5. Raman spectroscopy also identified major distinguishing vibrational modes in the developmental period which included proteins, amino acids (tyrosine, proline phenylalanine, valine), and secondary structures of proteins (Amide I and Amide II).Conclusion Developing chick cornea undergoes significant changes in its biomolecular composition in the day10-day18developmental period, with the major changes in the spectral regions associated with DNA/RNA, proteins, glycogen, and secondary protein structures.