Assessment of Carbon- and Metal-Based Nanoparticle DNA Damage with Microfluidic Electrophoretic Separation Technology.
In this study, we examined the feasibility of extracting DNA from whole cell lysates exposed to nanoparticles using two different methodologies for evaluation of fragmentation with microfluidic electrophoretic separation. Human lung macrophages were exposed to five different carbon- and metal-based nanoparticles at two different time points (2 h, 24 h) and two different doses (5 µg/ml, 100 µg/ml). The primary difference in the banding patterns after 2 h of nanoparticle exposure is more DNA fragmentation at the higher NP concentration when examining cells exposed to nanoparticles of the same composition. However, higher doses of carbon and silver nanoparticles at both short and long dosing periods can contribute to erroneous or incomplete data with this technique. Also comparing DNA isolation methodologies, we recommend the centrifugation extraction technique, which provides more consistent banding patterns in the control samples compared to the spooling technique. Here we demonstrate that multi-walled carbon nanotubes, 15 nm silver nanoparticles and the positive control cadmium oxide cause similar DNA fragmentation at the short time point of 2 h with the centrifugation extraction technique. Therefore, the results of these studies contribute to elucidating the relationship between nanoparticle physicochemical properties and DNA fragmentation results while providing the pros and cons of altering the DNA isolation methodology. Overall, this technique provides a high throughput way to analyze subcellular alterations in DNA profiles of cells exposed to nanomaterials to aid in understanding the consequences of exposure and mechanistic effects. Future studies in microfluidic electrophoretic separation technologies should be investigated to determine the utility of protein or other assays applicable to cellular systems exposed to nanoparticles.
Amanda M
AM
Schrand
Thomas
T
Powell
Tiffany
T
Robertson
Saber M
SM
Hussain
D002460:Cell Line; D002469:Cell Separation; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004249:DNA Damage / Q000235:genetics*; D004586:Electrophoresis / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006801:Humans; D056656:Lab-On-A-Chip Devices; D008264:Macrophages / Q000187:drug effects / Q000502:physiology; D008422:Materials Testing / Q000295:instrumentation; D053768:Metal Nanoparticles / Q000633:toxicity*; D037742:Nanotubes, Carbon / Q000633:toxicity*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D018675:Toxicity Tests / Q000295:instrumentation
D037742:Nanotubes, Carbon; D004247:DNA