Environmental Engineering Reference
In-Depth Information
Live/dead viability assays.
E. coli
and
B. subtilis
cultures grown to logarithmic phase in M9 medium and
B. subtilis
in
minimal medium, respectively, are treated with different concentrations (i.e., 50, 100, and 150 mg/l) of nanoparticles.
Following exposure, the impact on bacterial membrane integrity is assessed using a live/dead BacLight bacterial viability
kit. To quantify the relative numbers of live and dead cells, the relative fluorescence intensities are measured using a fluo-
rescence plate reader (excitation at 485 nm, emission at 525 and 625 nm).
Monitoring superoxide production.
Superoxide production upon exposure of bacterial suspensions to various concentrations
of nanoparticles is monitored by following the absorbance at 470 nm due to the reduction of 100 μM 2,3-bis(2-methoxy-
4-nitro-5-sulfophenyl)-2
H
-tetrazolium-5-carboxanilide (xTT) to xTT-formazan by superoxide (O
2
−
) [37, 38].
Microarray hybridization and analysis.
For microarray experiments, an overnight
E. coli
culture is used to inoculate 250 ml
flasks containing 100 ml of prewarmed M9 medium to an Od600 of approximately 0.1 and incubated at 37°C with
shaking at 200 rpm until the mid-log phase (Od600, ~0.5). Cultures are treated with either prewarmed nanoparticles
(100 mg/l) or Milli-Q water. After 1 h, cells are harvested by rapid centrifugation (5000 ×
g
, 2 min at 4°C) and snap-
freezing in liquid N
2
. Three separate controls and three experimental cultures are examined for each condition. Total
cellular ribonucleic acid (RNA) is isolated as described by Brown and Pelletier: the cells are first resuspended in TE
(Tris 10 mM-EdTA 1 mM, pH 7.6) buffer and incubated with 1 mg/ml of lysozyme to lyse the cells [39, 40]. Purified,
fluorescently labeled cdNA is hybridized to
E. coli
K-12 gene expression 4-by-72 K arrays (or other microarrays) using
a Nimblegen hybridization system. Microarrays are washed according to the array manufacturer's procedure. Briefly,
microarray mixers are removed in 42°C Nimblegen wash buffer I and then washed manually in room-temperature buffers:
wash buffer I for 2 min, wash buffer II for 1 min, and wash buffer III for 15 s. Microarrays are dried for 80 s using a Maui
wash system and then scanned and the images quantified. Microarray data are normalized using the Lowess normaliza-
tion algorithm, and an analysis of variance (ANOvA) is performed to determine significant differences in gene expression
levels between conditions and time points using the false discovery rate testing method (
P
< 0.01).
Cyanobacteria and green algae models.
These microorganisms have been also used to determine the toxicity of an NM due
to their ecological position at the base of the aquatic food chain and their essential role in nutrient cycling and oxygen
production. Cyanobacteria constitute a phylum of bacteria that obtain their energy through plant-like photosynthesis.
They are the most widespread primary producers in the marine food chain and are crucial in many other habitats including
freshwater bodies, saline lakes, and biological soil crust.
For example, the toxicity of nano-CeO
2
suspension was determined by monitoring the growth inhibition of the green alga
Pseudokirchneriella subcapitata
and by determining the constitutive luminescence inhibition of the recombinant biolumines-
cent cyanobacterium
Anabaena
CPB4337. The bioassays using the bioluminescent cyanobacterium
Anabaena
CPB4337 are
based on the inhibition of constitutive luminescence caused by the presence of toxins [41].
30.2.3
In Vitro
assays
In order to evaluate the biological activity and/or toxicity of NMs, some alternatives have been explored to determine the effect
of a particle upon a living organism. Conventional
in vitro
analyses and cell-based assays were performed to obtain an estimate
that could mimic the
in vivo
physiologic environment of a living being, and thus determine their possible biological risk in case
the material is toxic. To determine the metabolic state of a group of cells we must consider a concept known as cell viability,
which indicates the potential of this group of cells to proliferate and grow. A normal cell population must be metabolically active
in culture, which must indicate that all their functions are normal. In toxicology, there are many ways to determine cell viability,
from simple dye exclusions to the use of sophisticated instruments. In nanotoxicology, the same techniques have been explored
that have been used in toxicology to evaluate the effect of an NM when a cell population is exposed; nevertheless, these studies
have not resulted in the creation of standards that could be useful to most of the new NMs released into the environment [28].
Besides,
in vitro
analyses are very popular because of their established methodologies, low costs, broad number of replicates,
small setups, safety and efficacy, and a few ethical issues. The important advantage of
in vitro
testing of NMs is that it is a solu-
tion for the replacement or reduction of laboratory animals, reducing at the same time the uncertainty caused due to the vari-
ability between individuals [42]. As with other man-made materials such as cosmetics and drugs,
in vitro
evaluation of NMs
needs to be performed because of the increase in nanostructured materials and nanoparticles that are released into the environ-
ment. Table 30.1 summarizes the most popular
in vitro
analyses employed in nanotoxicology, some of them validated by the
OECd, the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECvAM), and the National
Institutes of Health via the Nanotechnology Characterization Laboratory (NCL-NIH). Many of them include common dye exclu-
sions, indirect determinations of metabolic disruptions, microscopy analyses, and cell viability determinations by impedance.
