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ATR-FTIR spectroscopy detects alterations induced by organotin(IV) carboxylates in MCF-7 cells at sub-cytotoxic/-genotoxic concentrations.

Ahmad MS, Mirza B, Hussain M, Hanif M, Ali S, Walsh MJ, Martin FL - PMC Biophys (2008)

Bottom Line: Elevated micronucleus-forming activities were also observed.Our results demonstrate that ATR-FTIR spectroscopy can be applied to detect molecular alterations induced by organotin(IV) compounds at sub-cytotoxic and sub-genotoxic concentrations.This biophysical approach points to a novel means of assessing risk associated with environmental contaminants.PACS codes: 87.15.-v, 87.17.-d, 87.18.-h.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lancaster Environment Centre, Lancaster University, Bailrigg, Lancaster LA1 4YQ, UK. f.martin@lancaster.ac.uk.

ABSTRACT
The environmental impact of metal complexes such as organotin(IV) compounds is of increasing concern. Genotoxic effects of organotin(IV) compounds (0.01 mug/ml, 0.1 mug/ml or 1.0 mug/ml) were measured using the alkaline single-cell gel electrophoresis (comet) assay to measure DNA single-strand breaks (SSBs) and the cytokinesis-block micronucleus (CBMN) assay to determine micronucleus formation. Biochemical-cell signatures were also ascertained using attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy. In the comet assay, organotin(IV) carboxylates induced significantly-elevated levels of DNA SSBs. Elevated micronucleus-forming activities were also observed. Following interrogation using ATR-FTIR spectroscopy, infrared spectra in the biomolecular range (900 cm-1 - 1800 cm-1) derived from organotin-treated MCF-7 cells exhibited clear alterations in their biochemical-cell fingerprint compared to control-cell populations following exposures as low as 0.0001 mug/ml. Mono-, di- or tri-organotin(IV) carboxylates (0.1 mug/ml, 1.0 mug/ml or 10.0 mug/ml) were markedly cytotoxic as determined by the clonogenic assay following treatment of MCF-7 cells with >/= 1.0 mug/ml. Our results demonstrate that ATR-FTIR spectroscopy can be applied to detect molecular alterations induced by organotin(IV) compounds at sub-cytotoxic and sub-genotoxic concentrations. This biophysical approach points to a novel means of assessing risk associated with environmental contaminants.PACS codes: 87.15.-v, 87.17.-d, 87.18.-h.

No MeSH data available.


Related in: MedlinePlus

Average IR spectra of MCF-7 cells with corresponding scores plots plotted on PCs 1,2 and 3 and loadings plots following 24-h exposure to 0.0001 μg Me3Sn(L3)/ml. Cells were seeded into 60-mm Petri dishes and allowed to attach for 24 h prior to exposure. Following exposure, cells were disaggregated with trypsin/EDTA solution, fixed in 70% EtOH, and applied to Low-E Microscope slides whereupon the suspensions were allowed to air-dry. A) 10 IR spectra were derived (vehicle control in blue, treated in red). B) From subsequent multivariate analysis, a 3-D scores plot on PCs selected to demonstrate best segregation of vehicle control (blue) vs. exposure (red) was constructed. C) Following cluster analysis, loadings plots were constructed on each relevant PC to identify the wavenumbers most responsible for segregation of clusters.
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Figure 6: Average IR spectra of MCF-7 cells with corresponding scores plots plotted on PCs 1,2 and 3 and loadings plots following 24-h exposure to 0.0001 μg Me3Sn(L3)/ml. Cells were seeded into 60-mm Petri dishes and allowed to attach for 24 h prior to exposure. Following exposure, cells were disaggregated with trypsin/EDTA solution, fixed in 70% EtOH, and applied to Low-E Microscope slides whereupon the suspensions were allowed to air-dry. A) 10 IR spectra were derived (vehicle control in blue, treated in red). B) From subsequent multivariate analysis, a 3-D scores plot on PCs selected to demonstrate best segregation of vehicle control (blue) vs. exposure (red) was constructed. C) Following cluster analysis, loadings plots were constructed on each relevant PC to identify the wavenumbers most responsible for segregation of clusters.

Mentions: The effects of 24-h exposure of MCF-7 cells with 0.0001 μg/ml Bu3Sn(L1) (a mono-organotin carboxylate) (Figure 4), Ph3Sn(L2) (a di-organotin carboxylate) (Figure 5) or Me3Sn(L3) (a tri-organotin carboxylate) (Figure 6) on the consequent IR spectrum of 70% EtOH-fixed cellular material, compared to vehicle control are shown. To see effects induced by all the organotin(IV) carboxylates tested at all exposures (0.0001 μg/ml, 0.01 μg/ml or 1.0 μg/ml), see Additional file 1. Following fixation, MCF-7 cells were applied to Low-E reflective glass slides and interrogated using the ≈ 250 μm × 250 μm octagon-shaped sampling area employed in this study for ATR-FTIR spectroscopy [18]. An IR spectrum of cellular biochemistry was obtainable from such 70% EtOH-fixed cells [19]. In the biomolecular region (900 cm-1 – 1800 cm-1), clear induced alterations in the "biochemical-cell fingerprint" were associated with organotin-treated cells (Figures 4, 5, 6; see Additional file 1). In this study, variability was noted in control spectra between 1400 cm-1 and 1500 cm-1; however, in order to distinguish intra-class differences (i.e., vehicle control vs. treatment) one needs to look at the direction of the PCs. The most marked differences were noted in the spectral region ≈ 1300 cm-1 – 1500 cm-1; these would be associated with amide III absorptions (predominantly C-N stretching) with significant contributions from CH2 stretching vibrations of carbohydrate residues (≈ 1280 cm-1 – 1360 cm-1), but some of these wavenumbers would be associated with intra-class as opposed to inter-class variance (Figures 4, 5, 6). However, in Figure 5 PC1 mostly excludes this aforementioned region as a contributor to variance but highlights 1225 cm-1 (asymmetric phosphate), which consistently contributed to variance. Additionally, in some cases [Bu2Sn(L1)2, Et2Sn(L2)2, Me2Sn(L2)2, BuSn(L3)3 and Me3Sn(L3)], glycogen (≈ 1030 cm-1) was observed as an important contributing factor. Of note was the fact that except for BuSnCl(L1)2, separation of IR spectra derived from 0.0001 μg/ml exposed and control MCF-7 cells was readily achievable along PCs 1, 2 and 3 (see Additional file 1). This suggested that organotin(IV) carboxylates (tri-, di- or mono-derivatives) are capable of inducing profound biomolecular alterations even at low concentrations.


ATR-FTIR spectroscopy detects alterations induced by organotin(IV) carboxylates in MCF-7 cells at sub-cytotoxic/-genotoxic concentrations.

Ahmad MS, Mirza B, Hussain M, Hanif M, Ali S, Walsh MJ, Martin FL - PMC Biophys (2008)

Average IR spectra of MCF-7 cells with corresponding scores plots plotted on PCs 1,2 and 3 and loadings plots following 24-h exposure to 0.0001 μg Me3Sn(L3)/ml. Cells were seeded into 60-mm Petri dishes and allowed to attach for 24 h prior to exposure. Following exposure, cells were disaggregated with trypsin/EDTA solution, fixed in 70% EtOH, and applied to Low-E Microscope slides whereupon the suspensions were allowed to air-dry. A) 10 IR spectra were derived (vehicle control in blue, treated in red). B) From subsequent multivariate analysis, a 3-D scores plot on PCs selected to demonstrate best segregation of vehicle control (blue) vs. exposure (red) was constructed. C) Following cluster analysis, loadings plots were constructed on each relevant PC to identify the wavenumbers most responsible for segregation of clusters.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2666631&req=5

Figure 6: Average IR spectra of MCF-7 cells with corresponding scores plots plotted on PCs 1,2 and 3 and loadings plots following 24-h exposure to 0.0001 μg Me3Sn(L3)/ml. Cells were seeded into 60-mm Petri dishes and allowed to attach for 24 h prior to exposure. Following exposure, cells were disaggregated with trypsin/EDTA solution, fixed in 70% EtOH, and applied to Low-E Microscope slides whereupon the suspensions were allowed to air-dry. A) 10 IR spectra were derived (vehicle control in blue, treated in red). B) From subsequent multivariate analysis, a 3-D scores plot on PCs selected to demonstrate best segregation of vehicle control (blue) vs. exposure (red) was constructed. C) Following cluster analysis, loadings plots were constructed on each relevant PC to identify the wavenumbers most responsible for segregation of clusters.
Mentions: The effects of 24-h exposure of MCF-7 cells with 0.0001 μg/ml Bu3Sn(L1) (a mono-organotin carboxylate) (Figure 4), Ph3Sn(L2) (a di-organotin carboxylate) (Figure 5) or Me3Sn(L3) (a tri-organotin carboxylate) (Figure 6) on the consequent IR spectrum of 70% EtOH-fixed cellular material, compared to vehicle control are shown. To see effects induced by all the organotin(IV) carboxylates tested at all exposures (0.0001 μg/ml, 0.01 μg/ml or 1.0 μg/ml), see Additional file 1. Following fixation, MCF-7 cells were applied to Low-E reflective glass slides and interrogated using the ≈ 250 μm × 250 μm octagon-shaped sampling area employed in this study for ATR-FTIR spectroscopy [18]. An IR spectrum of cellular biochemistry was obtainable from such 70% EtOH-fixed cells [19]. In the biomolecular region (900 cm-1 – 1800 cm-1), clear induced alterations in the "biochemical-cell fingerprint" were associated with organotin-treated cells (Figures 4, 5, 6; see Additional file 1). In this study, variability was noted in control spectra between 1400 cm-1 and 1500 cm-1; however, in order to distinguish intra-class differences (i.e., vehicle control vs. treatment) one needs to look at the direction of the PCs. The most marked differences were noted in the spectral region ≈ 1300 cm-1 – 1500 cm-1; these would be associated with amide III absorptions (predominantly C-N stretching) with significant contributions from CH2 stretching vibrations of carbohydrate residues (≈ 1280 cm-1 – 1360 cm-1), but some of these wavenumbers would be associated with intra-class as opposed to inter-class variance (Figures 4, 5, 6). However, in Figure 5 PC1 mostly excludes this aforementioned region as a contributor to variance but highlights 1225 cm-1 (asymmetric phosphate), which consistently contributed to variance. Additionally, in some cases [Bu2Sn(L1)2, Et2Sn(L2)2, Me2Sn(L2)2, BuSn(L3)3 and Me3Sn(L3)], glycogen (≈ 1030 cm-1) was observed as an important contributing factor. Of note was the fact that except for BuSnCl(L1)2, separation of IR spectra derived from 0.0001 μg/ml exposed and control MCF-7 cells was readily achievable along PCs 1, 2 and 3 (see Additional file 1). This suggested that organotin(IV) carboxylates (tri-, di- or mono-derivatives) are capable of inducing profound biomolecular alterations even at low concentrations.

Bottom Line: Elevated micronucleus-forming activities were also observed.Our results demonstrate that ATR-FTIR spectroscopy can be applied to detect molecular alterations induced by organotin(IV) compounds at sub-cytotoxic and sub-genotoxic concentrations.This biophysical approach points to a novel means of assessing risk associated with environmental contaminants.PACS codes: 87.15.-v, 87.17.-d, 87.18.-h.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lancaster Environment Centre, Lancaster University, Bailrigg, Lancaster LA1 4YQ, UK. f.martin@lancaster.ac.uk.

ABSTRACT
The environmental impact of metal complexes such as organotin(IV) compounds is of increasing concern. Genotoxic effects of organotin(IV) compounds (0.01 mug/ml, 0.1 mug/ml or 1.0 mug/ml) were measured using the alkaline single-cell gel electrophoresis (comet) assay to measure DNA single-strand breaks (SSBs) and the cytokinesis-block micronucleus (CBMN) assay to determine micronucleus formation. Biochemical-cell signatures were also ascertained using attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy. In the comet assay, organotin(IV) carboxylates induced significantly-elevated levels of DNA SSBs. Elevated micronucleus-forming activities were also observed. Following interrogation using ATR-FTIR spectroscopy, infrared spectra in the biomolecular range (900 cm-1 - 1800 cm-1) derived from organotin-treated MCF-7 cells exhibited clear alterations in their biochemical-cell fingerprint compared to control-cell populations following exposures as low as 0.0001 mug/ml. Mono-, di- or tri-organotin(IV) carboxylates (0.1 mug/ml, 1.0 mug/ml or 10.0 mug/ml) were markedly cytotoxic as determined by the clonogenic assay following treatment of MCF-7 cells with >/= 1.0 mug/ml. Our results demonstrate that ATR-FTIR spectroscopy can be applied to detect molecular alterations induced by organotin(IV) compounds at sub-cytotoxic and sub-genotoxic concentrations. This biophysical approach points to a novel means of assessing risk associated with environmental contaminants.PACS codes: 87.15.-v, 87.17.-d, 87.18.-h.

No MeSH data available.


Related in: MedlinePlus