Incorporation of deuterium oxide in MCF-7 cells to shed further mechanistic insights into benzo[a]pyrene-induced low-dose effects discriminated by ATR-FTIR spectroscopy

Abstract

This study evaluated the potential of deuteration to enhance the mechanistic information obtainable by biospectroscopy techniques in biological-cell models. These techniques were previously demonstrated to identify low-dose effects (≤nM) induced by test agents; this is of critical interest in terms of developing novel approaches to monitor environmentally-induced cell alterations. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy was coupled with multivariate analysis to characterize a low-dose (10⁻¹⁰ M) compared to a high-dose (10⁻⁶ M) exposure of benzo[a]pyrene (B[a]P) in oestrogen-responsive MCF-7 cells; these results were used as a positive control for spectroscopic detection of B[a]P-induced effects. Deuterium oxide (D₂O) was then applied as part of a fixative solution and/or at low levels incorporated into growth medium prior to ATR-FTIR spectrochemical analysis. The application of D₂O as an alternative solvent in spectroscopy is widespread, but D₂O has never before been applied to biospectroscopic analysis of in vitro toxicology assays. This allowed comparison between deuterated- and typically-derived IR spectra, facilitating significant insights into the effects of deuteration, and suggested that the addition of D₂O to biospectroscopy assays could improve understanding of low-dose effects.