Page 423 - Environmental Nanotechnology Applications and Impacts of Nanomaterials

406 Potential Impacts of Nanomaterials

suitable for use in cell culture because of this interference with via-
bility and cytokine assays (Monteiro-Riviere and Inman, 2006). These
studies with the neutral red (NR) assay, which is a widely utilized via-
bility test in cell culture experiments, showed that carbon can adsorb
viability marker dyes such as NR (3-amino-7-dimethylamino-2
methylphenazine hydrochloride) from cell culture media, interfering
with the absorption spectra causing false readings. Interference
studies with the NR assay protocol using different CB sources (Fisher,
Cabot, and Degussa varieties) in the absence of human epidermal
keratinocytes demonstrated a false negative signal indicating the
presence of viable cells. CB was found to adsorb NR dye and gener-
ate a signal in the assay suggesting high cell viability when in fact
cells were not even present. Similar studies were also shown for the
MTT (3-[4,5-dimethyl-2-thiazol]-2,5-diphenyl-2H-tetrazolium bromide)
viability assay. MTT is a colorimetric metabolic assay based on mito-
chondrial dye conversion to assess viability. Tetrazolium salt is used to
assess the activity of various dehydrogenase enzymes where the tetra-
zolium ring is cleaved in active mitochondria, demonstrating the pres-
ence of living cells. In the presence of CB, the tetrazolium dye desolved
from the cells is adsorbed by the CB, thereby reducing the absorbance
reading and giving a false positive signal for reduced viability and thus
cytotoxicity.
These studies have shown how carbon adsorbents may interfere with
in vitro cytotoxicity assays. CB may also adsorb the constituents of the
grow media as well as proteins and growth factors, thereby preventing
the cells from receiving their proper nutrients and growth factors. CB
may adsorb other soluble components that could alter pH and cell via-
bility. Ultrafine CB may act as an adsorbent that could potentially bind
compounds during the manufacturing process (Monteiro-Riviere and
Inman, 2006). The type of CB used in a study and its characterization
and composition is extremely important. Their rational use as particu-
late controls in inhalational studies may not carry over into other organ
or exposure systems.
Table 11.1 lists a number of reports on carbon-based nanomaterials
toxicity.

Titanium Dioxides

Titanium dioxide (TiO 2 ) particles larger than 100 nm are generally con-
sidered to be biologically inert in both humans and animals (Bernard
et al., 1990; Chen and Fayerweather, 1988; Hart and Hesterberg, 1998;
Lindenschmidt et al., 1990; Ophus et al., 1979). Thus, they have been
widely used as a food colorant (Lomer et al., 2002) and as a white pig-
ment (Nordman et al., 1986) in sunscreens and in cosmetic creams