Surfactant Uptake Dynamics in Mammalian Cells Elucidated with Quantitative Coherent Anti-Stokes Raman Scattering Microspectroscopy

et al. (2014) Surfactant Uptake Dynamics in Mammalian Cells Elucidated with Quantitative Coherent Anti- Stokes Raman Scattering Microspectroscopy. PLoS ONE 9(4): e93401. doi:10.1371/journal.pone.0093401 Surfactant Uptake Dynamics in Mammalian Cells Elucidated with Quantitative Coherent Anti-Stokes Raman Scattering Microspectroscopy Masanari Okuno 0 Hideaki Kano 0 Kenkichi Fujii 0 Kotatsu Bito 0 Satoru Naito 0 Philippe Leproux 0 Vincent Couderc 0 Hiro-o Hamaguchi 0 Maria A. Deli, Biological Research Centre of the Hungarian Academy of Sciences, Hungary 0 1 Department of Chemistry, School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan, 2 Safety Science Research Laboratories, Kao Corporation , Haga-Gun, Tochigi , Japan , 3 Analytical Science Research Laboratories, Kao Corporation , Haga-Gun, Tochigi , Japan , 4 Institut de Recherche XLIM, UMR CNRS, Limoges, France, 5 Institute of Molecular Science and Department of Applied Chemistry, National Chiao Tung University , Hsinchu , Taiwan The mechanism of surfactant-induced cell lysis has been studied with quantitative coherent anti-Stokes Raman scattering (CARS) microspectroscopy. The dynamics of surfactant molecules as well as intracellular biomolecules in living Chinese Hamster Lung (CHL) cells has been examined for a low surfactant concentration (0.01 w%). By using an isotope labeled surfactant having CD bonds, surfactant uptake dynamics in living cells has been traced in detail. The simultaneous CARS imaging of the cell itself and the internalized surfactant has shown that the surfactant molecules is first accumulated inside a CHL cell followed by a sudden leak of cytosolic components such as proteins to the outside of the cell. This finding indicates that surfactant uptake occurs prior to the cell lysis, contrary to what has been believed: surface adsorption of surfactant molecules has been thought to occur first with subsequent disruption of cell membranes. Quantitative CARS microspectroscopy enables us to determine the molecular concentration of the surfactant molecules accumulated in a cell. We have also investigated the effect of a drug, nocodazole, on the surfactant uptake dynamics. As a result of the inhibition of tubulin polymerization by nocodazole, the surfactant uptake rate is significantly lowered. This fact suggests that intracellular membrane trafficking contributes to the surfactant uptake mechanism. - Interactions of surfactants with living cells are of considerable interest with regard to their biological functions including cellular toxicity [1]. Understanding their toxicological mode of action is highly important in order to assess and control their safety on human exposure [24]. Previous studies have shown that microorganisms solubilization by surfactants occurs with cell lysis, in which the cell membrane is degraded by surfactants with eventual breakdown of the whole cell [58]. However, the dynamical process of surfactant action in single living cells is still unexplored because of the lack of the mean to visualize surfactant molecules in vivo and in situ. In the present study, we use a recentlyemerging new tool, CARS microspectroscopy [913], which is powerful for studying lipid molecules in living cells. We also use an isotope labeled surfactant (d25-sodium dodecyl sulfate (SDS)) and visualize the dynamics of surfactant molecules in the cell lysis process. Deuterium substitution enables us to selectively trace the SDS molecules among a number of unlabeled biomolecules [10,1416]. d25-SDS gives CD stretch bands in the 2000 2200 cm21 spectral region, which is a window of Raman spectra of unlabeled biomolecules, facilitating its selective detection. Although fluorescence labeling is a powerful technique for tracing the dynamics of lipid molecules in a living cell [1719], introduction of fluorophores may well perturb the physical and chemical properties of the surfactant, such as charge, hydrophobicity, and hydrophilicity. Isotope labeling in vibrational spectroscopy is well established as a unique method for distinguishing the labeled molecule from the others. A great advantage of isotope substitution is the same chemical properties between the labeled and unlabeled species. Recently, we have developed quantitative CARS microspectroscopy [20], which combines multiplex CARS microspectroscopy with the maximum entropy method (MEM) [2123]. The spectral coverage in this method is broad enough (.3000 cm21) to observe all the fundamental vibrational modes including not only the C-H, C-D stretch regions but also the fingerprint region. Thus, quantitative CARS microspectroscopy with deuterium substitution is ideally suited for real-time spectral tracing of cells and the surfactant molecules during the lysis process. Materials and Methods Quantitative CARS microspectroscopy We use a CARS microspectrometer developed in our laboratory. The details of the CARS system are described in File S1 [20]. Sample Chinese Hamster Lung (CHL) cells [24], which are routinely used for toxic evaluation, were used as a sample in the present study. CHL cells were incubated at 37uC under 5% CO2. The culture medium were D-MEM (Dulbeccos modified essential medium, Gibco) supplemented with 10% fetal bovine serum (FBS). Chemicals 2H-substituted sodium dodecyl sulfate (d25-SDS) was used as a surfactant. The culturing media was suspended with d25-SDS solution (0.1 wt% SDS in PBS buffer) so that the final concentration of d25-SDS was approximately 0.01 w%, 0.3 mM. This concentration is too low to be detected by the CARS microspectroscopic system. We found no spectroscopic signature of the CD stretch from the suspended medium. Nocodazole was used as an inhibitor of intracellular membrane trafficking in CHL cells [19]. It inhibits the polymerization of tubulin and subsequent formation of microtubes. Since nocodazole is not soluble in water, it was solved in dimethylsulfoxide. This solution was added to the medium with the final nocodazole concentration of 25 mM. Cells were incubated for 30 min after the addition of nocodazole. Results and Discussion Cell lysis efficiency We first analyzed the cell lysis efficiency of d25- SDS as a model surfactant. Cultured CHL cells were scrape-harvested to microcentrifuge tubes and spin-downed. Then, the supermatants were removed from the solution. Cell pellets were then resuspended to SDS solutions at each concentration of 1, 0.1, 0.01, 0.001 and 0.0001 w% by voltex for 1 min. The suspensions were spindowned and we checked the degree of cell lysis. High concentration of 1,0.1 w% of SDS solution apparently lyses the CHL cells. On the other hand, below 0.001 w% of SDS solution, the cells remain stable as a pellet (Table 1). These results suggest that the 0.01 w% concentration of SDS solution is approximately a threshold of CHL cell lysis and denaturation. Thus, we determined the concentration of SDS (0.01 w%, 0.3 mM) for tracing the lysis process of CHL cells with CARS microspectros (...truncated)