Quartz crystal microbalance measurements of mitochondrial depolarization predicting chemically induced toxicity of vascular cells and macrophages.

Quartz crystal microbalances (QCMs) measure mass on the nanogram (ng) scale. We built novel QCMs as toxicity biosensors incorporating living cells. Human endothelial cells or canine macrophages were equilibrated on QCM crystal surfaces until stable oscillation frequencies occurred. Vehicle or sodium azide (NaN3) (25-100 mM) was added to these QCMs while continuously collecting crystal oscillation frequency data. At these doses, NaN3 alters mitochondrial membrane permeability and causes mitochondrial swelling and intrinsic apoptosis. Our studies demonstrated no frequency change in QCMs with untreated cells or without cells but NaN3. If NaN3 was added to either cell type within QCMs, 5 to 8 min later increases in oscillation frequency (Δf) occurred (400-1600 Hz) that correlated with dose. All frequency changes reverted to baseline by 15 min. In parallel, during the first 30 min, no change in cell or nuclear areas, or in actin or microtubule distributions, was detected. Yet, mitochondrial size and membrane permeability increased significantly during, but not after, 5 to 8 min. Viability studies confirmed dose-dependent toxicity that was predicted and proportionate to the 5- to 8-min Δf. These studies confirm that cell-based QCMs can detect early events in intrinsic apoptosis and reveal unique kinetic information about events occurring within subcellular structures in response to toxins.

Understanding and correcting for carbon nanotube interferences with a commercial LDH cytotoxicity assay.

The lactate dehydrogenase (LDH) assay accurately quantifies cytotoxicity of chemicals via the measurement of LDH released from damaged cells. In the assay, LDH catalyzes formation of a reporter chromophore that can be quantified spectrophotometrically at its 490 nm peak, a standard assay, and related to the released LDH concentration. However, certain engineered nanomaterials have been reported to produce aberrant values, resulting in inaccurate assessment of toxicity as measured by LDH levels in media. We studied this effect spectroscopically by measuring unexpected changes in the complete visible spectrum of the product chromophore resulting from using either purified LDH or LDH from lysed cells in the presence of varying concentrations of single walled carbon nanotubes (SWCNTs) or carbon nanohorns (SWCNH-oxs). Basically, at constant LDH concentrations, the 490 nm product peak decreased with increasing carbon nanotube concentration, while the 580 nm peak increased to a lesser extent and the maximum absorbing wavelength increased. The product chromophore spectrum was altered in different ways by potential interactions with a number of components in the reaction mixture including: BSA, LDH, SWCNTs, SWCNT-oxs, or various combinations of these species. We propose to improve the accuracy of the LDH assay when evaluated in the presence of varying concentrations of these carbon nanostructures by use of both the 490 and 580 nm peak absorbances combined via regression analysis. Our results indicate that molecular probes of cytotoxicity must be assessed individually for accuracy in the presence of engineered nanomaterials.

A living cell quartz crystal microbalance biosensor for continuous monitoring of cytotoxic responses of macrophages to single-walled carbon nanotubes.

BACKGROUND: Numerous engineered nanomaterials (ENMs) exist and new ENMs are being developed. A challenge to nanotoxicology and environmental health and safety is evaluating toxicity of ENMs before they become widely utilized. Cellular assays remain the predominant test platform yet these methods are limited by using discrete time endpoints and reliance on organic dyes, vulnerable to interference from ENMs. Label-free, continuous, rapid response systems with biologically meaningful endpoints are needed. We have developed a device to detect and monitor in real time responses of living cells to ENMs. The device, a living cell quartz crystal microbalance biosensor (QCMB), uses macrophages adherent to a quartz crystal. The communal response of macrophages to treatments is monitored continuously as changes in crystal oscillation frequency (Δf). We report the ability of this QCMB to distinguish benign from toxic exposures and reveal unique kinetic information about cellular responses to varying doses of single-walled carbon nanotubes (SWCNTs). RESULTS: We analyzed macrophage responses to additions of Zymosan A, polystyrene beads (PBs) (benign substances) or SWCNT (3-150 µg/ml) in the QCMB over 18 hrs. In parallel, toxicity was monitored over 24/48 hrs using conventional viability assays and histological stains to detect apoptosis. In the QCMB, a stable unchanging oscillation frequency occurred when cells alone, Zymosan A alone, PBs alone or SWCNTs without cells at the highest dose alone were used. With living cells in the QCMB, when Zymosan A, PBs or SWCNTs were added, a significant decrease in frequency occurred from 1-6 hrs. For SWCNTs, this Δf was dose-dependent. From 6-18 hrs, benign substances or low dose SWCNT (3-30 µg/ml) treatments showed a reversal of the decrease of oscillation frequency, returning to or exceeding pre-treatment levels. Cell recovery was confirmed in conventional assays. The lag time to see the Δf reversal in QCMB plots was linearly SWCNT-dose dependent. Lastly, the frequency never reversed at high dose SWCNT (100-150 µg/ml), and apoptosis/necrosis was documented in conventional 24 and 48 hr-assays. CONCLUSION: These data suggest that the new QCMB detects and provides unique information about peak, sub-lethal and toxic exposures of living cells to ENMs before they are detected using conventional cell assays.