Page 230 - Environmental Nanotechnology Applications and Impacts of Nanomaterials

Principles and Procedures to Assess Nanomaterial Toxicity 215

electrons from DTT to form metastable semiquinones that, in turn, can
transfer these electrons to molecular oxygen to form the superoxide
radical [45]. We have successfully implemented this assay to study the
ability of ambient ultrafine particles, which are collected at different
sites in the Los Angeles Basin, to generate ROS [19].
ROS production by NM can also be quantitated by using the ROS
quencher, furfuryl alcohol (FFA) [41, 46]. Furfuryl alcohol chemically reacts
with ROS with a reaction rate constant three orders of magnitude higher
than the rate of physical quenching. Because FFA functions as an ROS
quencher, the quantities of ROS that are being produced by the particles
can be measured as the decrease in dissolved oxygen, corrected for an
appropriate blank sample. Results are plotted as the log of the ratio of the
instantaneous to initial concentrations of oxygen measured over time [41].
Unfortunately this indirect method of ROS measurement does not dis-
tinguish between individual oxygen radicals [41, 46]. This can be accom-
plished by ESR or nanobiosensor technology.
All things considered, cell-free systems reflect the intrinsic abilities of
NM to generate oxygen radicals. It is important to remark, however,
that these tests do not automatically lead to cellular ROS production or
the ability of the NM to induce oxidant injury or toxicity. For that to
happen, the material needs to be taken up by the target cell and must
overcome the antioxidant defenses that are capable of removing ROS or
restoring redox equilibrium [41]. It is also possible that coating of the NM
surface with biological components such as proteins may passivate the
material surface, leading to decreased ROS generation. Some materials
may lack the ability to generate ROS but could do so biologically due to
their ability to perturb mitochondrial electron transduction, induce the
assembly of NADPH oxidase, or engage metabolic pathways that lead to
ROS production [41]. Thus, although useful for predicting the intrinsic
capabilities of NM to produce ROS, the acellular tests need to be inter-
preted in the context of the biological response outcome.

Predictive In Vitro Toxicological Assays
in Tissue Culture Cells that Are Premised
On the Hierarchical Oxidative Stress Paradigm

In vitro assays for NM toxicity should consider the portal-of-entry as well
as possible systemic cellular targets (Table 6.4). Different NM may
necessitate different test strategies depending on the exposure risk.
With this sense of physiological relevance in mind, we can select cell
type, dosage, and endpoints according to the demands of the situation.
For instance, if a particular NM is found in skin-care products, it would
be more logical to study its effects on keratinocytes. If, on the other
hand, the NM is being produced as singlet particles that can be easily