Assessing the Toxicity of Titanium Dioxide Nanomaterials on Eukaryotic CellsAdrian Cortes, Candace Clark, Mani Tagmount, Chris Vulpe

Department of Nutrition and Toxicology, University of California, BerkeleyAbstract:

Research on nanomaterials has granted modern society an abundance of new applications that may be used across biomedical, optical and electronic fields. The principle behind these findings comes from the peculiar fact that materials shift properties as they are reduced in size. For example, insulating materials such as silicon convert to excellent conductors as nanowires, while chemically inert metals such as gold interact with body proteins when made into nanoparticles. However, there is insufficient research in regards to the toxicity of nanomaterials. This study focuses on TiO2 nanostructures and their effects on in vitro cell cytotoxicity experiments. We exposed carcinomic human alveolar basal epithelial cells (a549), bronchial epithelium cells (BEAS‐2B), and fat head minnow cells (FHM), to TiO2 nanoparticles and nanowires. The aim of this experiment is to determine the half maximal inhibitory concentration and for these cell lines when they’re subjected to TiO2 nanomaterials. Studying these possibly cytotoxic interactions will provide us with more information on ecotoxicology in regards to aquatic life and the health of the general public, and conceivably supply us with a set of precautionary procedures for the handling and disposal of these materials. 

Background:

The field of nanomaterials studies structures that are less than a tenth of amicrometer in size, namely nanoparticles and nanowires, and specifically,TiO2 nanomaterials. TiO2 nanoparticles are of interest because they canpotentially be used in photocatalysis, sensors, and solar cells. Currently, theyare being used in sunscreens for their UV blocking properties.

Methods:

A549, BEAS‐2B, and FHM cells are all cultured in a similar manner usinglaminar flow hoods and precise aseptic technique, taking care to notcontaminate cell lines. Cells are incubated in 75 cm2 flasks, and are grown inDMEM medium +10% fetal bovine serum (FBS) supplemented withglutamine. FBS is a light brown liquid that provides growth factors, lipids, and

Results: Discussion:

The a549 assays results were inconclusive. Absorption values failed toshow a decreasing trend as toxicant concentrations increased. Thissuggests that the A549 cell line was either not sensitive to the doses ofthe nanomaterials we used in this study or there was a decreasedbioavailability of nanomaterials. The reason is the presence of serum in

TiO2 nanowires are still confined to the world of laboratory research, butpossible applications are similar to those of nanoparticles, as in use in solarcells and computer hardware. These may also be used in thin film coatingsand can be assembled into structures for further applications.

Titanium dioxide metal has been known to be an inert substance inbi l i l li ti h b t ti h th

proteins that are necessary for cell growth and division. The FBS that issupplied to the FHM cells was heat inactivated. The BEAS‐2B media (BEBM)is a free serummedium.BEAS‐2B and a549 are incubated in a 37˚C, 5.0% CO2 environment foroptimum growth, while FHM is incubated in 34˚C, 5.0% CO2 environment.

After reaching approximately 80% confluency, cells are split to ensureoptimum growth and constant cell morphology. Our cell lines are adherentcells, so prior to splitting we apply Trypsin (.25% in EDTA) to cleave adhesiveproteins so adherent cells will slide off flask surface.Figure 1; Titanium dioxide nanoparticles/wires

the medium of culture that contains factors that affect thebioavailability of the nanomaterials, therefore providing the cells aprotective effect against such toxicants.

The a549 results may also be attributed to transformed cell morphologyas the a549 cells were cultured in later passages; therefore cellmetabolism rates might vary and cause fluctuations in our absorptionvalues as we’ve observed. Perhaps, we have also allowed our incubatedcultures to overgrow and become overly confluent; this also transformscell metabolism inasmuch that these conditions depletes our mediumof growth factors and increases cellular waste production, forcing thecells to adapt to a slo er metabolism in order to maintain the abilit to

Figure 7; Fathead Minnow cell toxicity to Titanium dioxide nanoparticles for24‐hour exposure.Our half maximal inhibitory concentration for this experiment came out atapproximately 200 ug/ml. The curve represents % survival at eachconcentration

biological applications; however, substance properties change as thesubstance decreases in size. Since they are extremely small in proportion,these nanomaterials are able to permeate through skin and interact withcells through surface chemistry; possibly causing changes in cell morphologyand cell death in the process. Additionally, nanoparticles can becomeaerosolized and suspended in air currents, while runoff water fromindustrial plants may also contain nanowires once they are used in real‐lifeapplications. This is a serious concern for ecosystems where an abundanceof these materials may be present.There is insufficient information regarding the toxicity of thesenanomaterials to both aquatic life and human health.

Cells viability is assessed using a hemocytometer (Improved Neubauer type).Cells are subsequently seeded at 2,000,000/cm2 and incubated according tocell line; cells are plated in 96 well plates at 15 000 cells per 200 microliters a

cells to adapt to a slower metabolism in order to maintain the ability toproliferate in a stressful environment.

Likewise, LDH assays for both a549 and FHM did not show a clear trendin a decrease in cell viability. Perhaps the concentrations used in oursetup were not toxic enough to damage the cell membrane and causeLDH release into the surrounding medium.

Our results indicate that Fathead minnow cells are more sensitive toTiO2 nanomaterials than that of a549 in vitro. Moreover, our assaysindicate that nanowires are generally more toxic to both a549 and FHMcells at 24 hours exposure

Figure 5; Hemocytometer counting grid, size comparison.

This is why we’ve chosen 2 lung cell lines (a549 and BEAS‐2B) and 1 fish cellline (FHM) to assess the toxicity of these materials on these modelorganisms.

Assessing Toxicity:

cell line; cells are plated in 96‐well plates at 15,000 cells per 200 microliters awell and incubated according to cell line.

Plates are incubated for 24 hours to allow cell adherence to wells.Shortly thereafter, we run 24‐hour exposures on our plates with TiO2

nanoparticles and nanowires with increasing concentrations.

Nanoparticles/Nanowires: (in increasing concentration) 10 ug/ml, 25 ug/ml,50 ug/ml, 100 ug/ml, 200 ug/ml

cells at 24 hours exposure.

We can see that aquatic ecosystems possibly are at higher risk of tragicdamage from chronic exposure to these nanostructures. However, thisrequires further experimentation in order for such a statement to bemade certain. Unfortunately, this research is beyond the scope of thisparticular project, therefore I was did not conduct research on BEAS‐2B,another class of human epithelial lung cell.

Future research:‐Repeat these experiments, use higher concentrations of toxicant, andlonger period of exposures (e.g., 48 and 72 hours), this will allow us to

Figure 2; a549 cell line, BEAS‐2B cell line

Figure 8; Fathead Minnow cell toxicity to Titanium dioxide nanowires for 24‐hour exposure.Our half maximal inhibitory concentration for this experiment came out atapproximately 10 ug/ml.

g y

The MTT assay is based on the reduction of yellow tetrazolium salt (MTT)into purple formazan (water in‐soluble) dye by viable cells. The amount offormazan product is correlated to the amount of viable cells in the givensample. Fundamentally, this assay measures mitochondrial health and is anindicator for cell viability in in vitro screening tests. Half of 96‐well plate is setup without cells in order to subtract background

g p p ( g , ),accurately determine the IC50 (half maximal inhibitory concentrations).We may also run exposures on cells cultured in earlier passages to avoidtransformed cell morphology.

‐Use high‐content screening assays (i.e., a cell imaging technique thatsimultaneously measures several parameters of cell health orcytotoxicity) in order to further understand the mode of actions of thetoxicity of TiO2 nanomaterials for in vitro experiments on our specificcell lines.

‐Furthermore, we would also need to conduct more exposures on en

Figure 3; MTT assay mechanics

Figure 6; MTT assay plate setup Figure 7; LDH assay plate setup

Figure 9; a549 cell toxicity to Titanium dioxide nanoparticles for 24‐hour exposure. At least 89% of cells in sample plate survived, cell activity remains well above 50% for this cell line. 

Lactate dehydrogenase (LDH) is an enzyme present in all mammalian cells.The cell membrane is impermeable to LDH under normal conditions; butwhen a cell undergoes lysis or is damaged, the enzyme is released into thet ll l fl id Th LDH i b d th i f t t li

p p ginterference due to TiO2 nanomaterials during absorption readings.

After 24‐hour exposure, 50 uL of cell growth medium are taken from eachwell using a multichannel pipette and transferred to fresh plates.

5 mg/ml MTT in PBS is applied to source plate and incubated at 37˚C for 2hours. Plate is subsequently aspirated of old medium and formazan productis resuspended in 100 uL DMSO per well. Absorption is read with CELLmax190 spectrophotometer at 560nmwith a 670nm background.

2nd plate is treated with substrate (for LDH activity) and allowed to sit for 30

vivo experiments; half maximal inhibitory concentrations change as youscale up organism size, and this is crucial to developing sound disposalprotocols for these possibly cytotoxic materials.

Figure 4; LDH assay mechanics

Figure 10; a549 cell toxicity to Titanium dioxide nanowires for 24‐hour exposure. At least 90% of cells in sample plate survived, cell activity remains

References:Ahmad, A., Awan, Hameed, Aziz Salman. Synthesis and Applications of TiO2 Nanoparticles.  Pakistan Engineering Congress, 70th Annual Session Proceedings. (2009).Allen, Matthew, Rushtan, Neil. Use of the Cytotox 96™ Assay in Routine Biocompatibility testing In Vitro. Promega Notes Magazine. (1994).Castranova, Vincent, Kommineni, C, et al. Pulmonary response to intratracheal instillation of ultrafine versus fine titanium dioxide: role of particle surface area. BioMed Central. (2008).Hamilton, Raymond, Wu, Nianqiang, et al. Particle length‐dependent titanium dioxide nanomaterials toxicity and bioactivity. Part Fibre Toxicol. (2009).

extracellular fluid. The LDH assay is based on the conversion of tetrazoliumsalts, catalyzed by the LDH in extracellular fluid, to red formazan product.Fundamentally, this assay is an indicator of cell membrane integrity in invitro screening tests.

minutes before stop solution is applied. Absorption is subsequently read at490nm.

LC50 and IC50 are computed using a statistics program (Microsoft Excel) withabsorption values. Experiments are repeated to ensure reproducibility ofresults.

exposure. At least 90% of cells in sample plate survived, cell activity remains well above 50% for this cell line. Hiran, Seishiro. A current overview of health effect research on nanoparticles. Environ Health Prev Med. 

(2009).

Acknowledgements:I would like to thank CSE and Seti Sidharta for their support in getting me into the ELP program.

I would also like to acknowledge and thank my ELP cohort, the ELP program and its coordinators, and NSF for their funding.

Lastly, I would like to thank Candace Clark and Mani Tagmount for their support and guidance in this research project; andChris Vulpe for granting me the wonderful opportunity of conducting my research in his lab.