Page 234 - Environmental Nanotechnology Applications and Impacts of Nanomaterials

Principles and Procedures to Assess Nanomaterial Toxicity 219

Toxic oxidative stress can be studied by following changes in mito-
chondrial function or structural integrity. We have demonstrated that
the induction of oxidative stress by ambient UFP or manufactured NP
induces the dissipation of the mitochondrial membrane potential ( m),
large-scale opening of the PTP, and the release of pro-apoptotic factors
[41]. A change in the m can be followed by the use of the cationic dye,
, which is highly concentrated in the negatively charged mito-
DiOC 6
chondrial matrix. Dissipation of the m leads to a decrease in DiOC 6
fluorescence that is also quantifiable by flow cytometry [41]. Apoptosis
can be assessed by detecting annexin V and propidium iodide (PI) flu-
orescence [36, 41]. Annexin V is a calcium-dependent phospholipid bind-
ing protein with high affinity for phosphatidylserine (PS), a membrane
phospholipid component normally localized in the inner layer of the cell
membrane. During early apoptosis, PS translocates to the outer surface
of the cell membrane where this phospholipid becomes accessible to
annexin V binding. At a later stage of apoptosis, the loss of membrane
integrity allows PI to be taken up in the nucleus where its presence can
be followed by flow cytometry. More formal demonstration of mito-
chondrial damage can be accomplished by ultrastructural studies and
confocal microscopy, which are time-consuming and expensive methods
that may be better used as a research tool.

Use of Cellular Assays to Study Other
Responses that Are Relevant to NM Toxicity,
Including Cellular Uptake and Subcellular
Localization
A detailed investigation of NM toxicity should assess NM uptake and
subcellular localization (Figure 6.1). The most common method to visu-
alize NM uptake is transmission electron microscopy (TEM). This
approach is relatively expensive and time-consuming. Fluorescent-
labeled NM can help to determine cellular uptake and localization by
using fluorescent or confocal microscopy. Using fluorescent dyes that
localize in the cell membrane, mitochondria, endoplasmic reticulum,
nucleus, Golgi apparatus, or endocytic vesicles can help to further refine
the subcellular localization of the NM. It is important to demonstrate
that labeling procedures do not alter the size, state of aggregation,
charge, hydrophobicity, or other physico-chemical characteristics of the
NM being tested.

In vivo testing
While in vitro techniques allow one to focus on specific biological and
mechanistic pathways that can be studied under controlled conditions,
the ultimate proof of NM toxicity necessitates the performance of