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In vitro methods for Nanotoxicity Assessment: Advantages and Applications

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Archives of Applied Science Research, 2011, 3 (2):389-403

(http://scholarsresearchlibrary.com/archive.html)

ISSN 0975-508X
CODEN (USA) AASRC9

In vitro methods for Nanotoxicity Assessment: Advantages and Applications

Poonam Takhar and Sheefali Mahant

MM College of Pharmacy, MM University, Mullana-Ambala, Haryana, India

___________________________________________________________________________

ABSTRACT

Nanotechnology is the production of materials at atomic and molecular level and is expected
to open some new avenues to fight and prevent diseases. It leads to improvement in biology,
biotechnology, medicine and healthcare by uncovering the structure and function of
biosystems at the nanoscale. The size of nanomaterials is similar to that of the most
biological molecules and structures; therefore, nanomaterials can be useful for both in vivo
and in vitro biomedical research and applications. Due to the expected growth in this field
and new materials being employed, there is a call for safety and exposure risks. Hence, for
improved characterization and reliable toxicity assessments, toxicological studies of
nanosystems are growing at exponential rates annually. For these reasons, screening assays
are needed to assess the chemical and physical properties of nanomaterials. Lacking the
proper interactions of nanostructures with the biological systems, it is unclear whether the
exposure could produce harmful biological responses. Deploying these materials in vivo has
even more challenges. So, in vitro methods are commonly used for toxicity assessment of
nanoparticles. Nanoparticle risk assessment can be done with existing cytotoxicity methods,
or with the development of new test systems with new standards for a general in vitro toxicity
testing of nanoparticles. An altogether different approach is required for nanoparticle
characterization and for bioassays, in order to validate their properties in physiology. The
present review focuses on the various in vitro methods of nanotoxicity assessment and the
advantages offered by them. The article also sheds some light on the applications of these
methods.

Keywords: Nanotechnology, Nanotoxicity, Nanomaterials, In vitro methods.

___________________________________________________________________________

INTRODUCTION

Nanotechnology is the technique through which structures with size ranging between 1 and
100 nm are developed, which imparts them unique properties [1].Owing to their unique
properties at this size level, there is a rapid expansion of nanotechnology in scientific,
technical and commercial field. The new and unique applications offered by nanotechnology
in diverse areas have made it so popular, that it is being applied today in almost all aspects of
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daily life. A number of products having nanosize elements are available in the market with
still new more to come [2]. As a result, there is an increasing demand for raw nanomaterials,
which can range from nanosized metals and metal oxides to carbon nanotubes for fulfilling
the growing needs of the market. [3,4]. In view of an increase in manufacturing and
consumer utilization of nanoparticles, there is a release of these materials into the
environment, eco-system, water [5] and food supplies, and the other routes of non-voluntary
entry into the human body [6]. According to conservative estimates [7], more than 800
consumer products containing nanoparticles or nanofibers are already in the market, and a
number of others are still to come. According to “The Nanotechnology Consumer Products
Inventory” [8], the most common material mentioned in the product descriptions was carbon
(29 products), which included fullerenes and nanotubes. Silver was the second most
referenced (25 products), followed by silica [14], titanium dioxide [7], zinc oxide [7], and
cerium oxide [1].

With the growth of nanomaterials in scientific field as well as in technical field, there is an
increasing exposure of nanomaterials to humans, together with the distinct properties like
complex interactions, possible bioaccumulation, unique chemistry and physical parameters.
All of these properties mandate development and validation of accurate nanodevice and
materials characterization protocols, which are capable of predicting toxic as well as
hazardous reactions. These methods must reliably predict and assess the possible outcomes of
effects, from benefits to possible risks, and health hazards associated with exposure to
nanomaterials, as they become more widespread in manufacturing and medicine. The inter-
agency National Toxicology Program classifies the new entity with its data along with their
possible risks associated with the entity. After that the entity is interrogated by a set of tests
which are basically designed to characterize a given risk, and also to characterize the
mechanisms for related outcomes [9].With the ongoing commercialization of nanotechnology
products, human exposure to nanoparticles will dramatically increase, and an evaluation of
their potential toxicity is essential. A number of manufactured nanoparticles have recently
been shown to cause adverse effects in vitro and in vivo [10–12]. The nanomaterials have
some unusual physiochemical properties due to their small size, chemical composition,
surface structure, solubility, shape, and aggregation [13] .Owing to the lack of understanding
of the size, shape, composition and aggregation-dependent interactions of nanostructures with
biological systems [14], it is not confirmed whether the exposure of humans, animals, insects
and plants to engineered nanostructures could produce harmful biological responses [15, 16].
Hence, a new sub-discipline of nanotechnology called nanotoxicology has emerged.

Nanomaterials characterization is important since nanoparticles might interact with assay

components or interfere with detection systems, resulting in unreliable data [17]. There are a
number of different approaches that can be taken to assess the toxic effects of inhaled
complex mixtures, including air pollution particles. These include epidemiology studies,
human clinical studies, animal studies, and in vitro studies. Each of these approaches has its
own strengths and advantages. Various studies suggest that in vitro nanotoxicity data can
reduce the testing of animals by identifying an appropriate starting dose for in vivo studies,
and a limited amount of toxic waste is produced [18]. .In vitro methods can be used to
estimate toxicokinetic parameters and target organ toxicity, thereby, increasing the
predictions of toxicity, and reducing animal use for some tests under controlled testing
conditions [19]. However, many of the necessary in vitro methods for this program have not
yet been developed. Other methods have not been evaluated for reliability and relevance, and
their usefulness and limitations for generating information to meet regulatory requirements
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for acute toxicity testing have not been assessed. Risk assessment of complex mixtures is the
most accurate and defensible, when as many of these approaches as possible, can be used in
an integrated manner to address a specific question [20].

This review, briefly reflects on the utility and advantages of various in vitro assays in
nanotoxicology, provides an overview of currently used in vitro cytotoxicity methods, and
furthermore, it discusses general applications of in vitro methods that may provide new
approaches to nanoparticle risk assessment. These methods are specifically discriminatory to
nanoscale properties, sizes or physical states, and many do not report sensitive information
about the nanomaterial behaviours in biological systems. These assays are important in
characterizing nanomaterial applications in biotechnology, ecosystems, agri- and aqua-
culture, biomedical applications and toxicity screening.

Figure: 1 Role of in vitro studies in pharmacology and toxicology studies

Merits of in vitro systems:

In vitro toxicological assessment is an important tool for nanotoxicology. The various merits
of these systems are as follows:-

• These systems are performed under controlled testing conditions in a particular

environment.
• There is reduction in systemic effects by using these systems.
• Reduction of variability between experiments.
• The same dose range can be tested in a variety of test systems (cells and tissues).
• Time-dependent studies can be performed and samples taken.
• Testing methods are fast and cheap.
• Very small amount of test material is required.
• Limited amount of toxic waste is produced.
• In vitro methods can be performed using human cells and tissues.
• Transgenic cells carrying human genes can be used.
• Reduction of testing in animals [21].

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Need for acute toxicity testing
Internationally, the most common use of acute systemic toxicity data is to provide a basis for
hazard classification and the labelling of chemicals for their manufacture, transport, and use
(Organisation for Economic Cooperation and Development, 1999a). The OECD
guidelines set out how governments expect companies to behave. They offer a basic outline
for corporate codes of conduct on how to deal with socially relevant issues. Other potential
uses for acute toxicity testing data include:

Establish dosing levels for repeated-dose toxicity studies;

Generate information on the specific organs affected;

Provide information related to the mode of toxic action;

Aid in the diagnosis and treatment of toxic reactions;

Provide information for comparison of toxicity and dose response among substances in a
specific chemical or product class;

Aid in the standardization of biological products;

Aid in judging the consequences of exposures in the workplace, home, or from accidental
release, and serve as a standard for evaluating alternatives to animal testing.

Figure: 2 Factors responsible for toxicity due to nanoparticles

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General in vitro methods for nanotoxicity assessment

1) Cell viability assay:

A) Proliferative assay:- These are the mainly metabolic assays which include:-

Tetrazolium salts assay, which measures the viability of a cell population relative to control,
untreated cells [22]. Cells are treated with particulates for various times before addition of
soluble yellow tetrazolium salts such as MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3
carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium, inner salt) or MTT (3-(4, 5-
dimethylthiazol-2- yl)-2, 5-diphenyltetrazolium bromide) for 2-4 hr at 37°C. During this
process, viable cells with active respiratory mitochondrial activity bioreduce MTS or MTT
into an insoluble purple formazan product, via mitochondrial succinic dehydrogenases, which
is subsequently solubilized by dimethyl sulfoxide (DMSO) or detergent, and quantitated on a
visible light spectrophotometer[23,24]. Data are represented as optical density (OD)/control
group. This technique has many advantages when compared to other toxicity assays because
it requires minimal physical manipulation of the model cells and yields quick, reproducible
results, requiring simple optical density acquisition [25]. However, this assay has a number of
drawbacks such as, certain human cell lines are inefficient at processing the tetrazolium salt
reagents, and the requirement of DMSO to solubilize the formazan product generated by
reduction of the tetrazolium salts is problematic. In addition, it exposes the laboratory
personnel to potentially hazardous amounts of solvent [26]. As a result, a number of
modifications have been established, including the use of the tetrazolium derivative XTT
(2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium
hydroxide), which is metabolized to a water soluble formazan product, thereby, eliminating
the solubilization step required with MTS or MTT [26-28].

Alamar Blue has been relatively recently applied to nanotoxicological studies by assaying
cellular redox potential. AlamarBlue is a proven cell viability indicator that uses the natural
reducing power of living cells to convert resazurin to the fluorescent molecule, resorufin. The
active ingredient of alamar blue (resazurin) is a nontoxic, cell permeable compound that is
blue in color and virtually non-fluorescent. Upon entering the cells, resazurin is reduced to
resorufin, which produces very bright red fluorescence. Viable cells continuously convert
resazurin to resorufin, thereby, generating a quantitative measure of viability—and
cytotoxicity [29]. The redox indicator is non-toxic to cells, users and the environment. It also
produces a clear, stable and distinct change, which is easy to interpret.

Incorporation of [3H] thymidine into the DNA (deoxy ribonucleic acid) is a sensitive
measurement of cell proliferation. The use of [3H] thymidine is complicated due to in vitro
toxicity and expensive radioactive material, and requires special training and facilities.
Moreover, this technique often requires a lengthy incubation period (24-48 hr) with [3H]
thymidine [30]. This method has been used to demonstrate the ability of nitric oxide-releasing
nanofiber gels to inhibit vascular smooth muscle cell proliferation in vitro [31].

Cologenic assays: Interactions between nanomaterials and probe molecules can be avoided
altogether through the use of cologenic assays [32, 33] .The cologenic assay allow studying
the effectiveness of specific agents on the survival and proliferation of cells. After plating at a
very low density, cells are allowed to grow until colonies are observed, and then, cells can
either be pre-treated with particulates of interest, or treated following plating. It is assumed
that each colony originates from a single plated cell which can be stained with crystal violet
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or nuclear stains, where colonies of highly proliferating cells are counted by visual
inspection.

B) Apoptosis assay: - Apoptosis, a form of programmed cell death have been used
extensively during nanotoxicological research, and include inspection of morphological
changes, comprising various assays which are as follows:-

DNA laddering, the oldest DNA damage assay technique, characterizes this fragmentation
by isolating and fluorescently labeling DNA from cells exposed to a potential toxicant in
culture. DNA damage is then detected by gel electrophoresis.

Caspase assays are based on the measurement of zymogen processing to an active enzyme
and proteolytic activity [34]. As soon as Caspase-3 is activated, cell death is inevitable.
Activated Caspase-3 can be detected by measuring the cleavage of a Caspase-3 substrate
linked to a chromophore or fluorophore that absorbs or emits light when separated from the
substrate [35].

The Comet Assay, also called single cell gel electrophoresis is a sensitive and rapid
technique for quantifying and analyzing DNA damage in individual cells. Individual cells are
embedded in a thin agarose gel on a microscope slide. All cellular proteins are then removed
from the cells by lysing. The DNA is allowed to unwind under alkaline/neutral conditions
and then DNA undergoes electrophoresis, allowing the broken DNA fragments or damaged
DNA to migrate away from the nucleus. After staining with a DNA-specific fluorescent dye
such as ethidium bromide or propidium iodide, the gel is read for amount of fluorescence in
head and tail, and the length of tail. The extent of DNA liberated from the head of the comet
is directly proportional to the amount of DNA damage [36].

TUNEL assay, which derives its name Terminal deoxynucleotidyl transferase dUTP(deoxy
uridine triphoshate)nick end labeling relies on double-strand breakage, like the damage
necessary for DNA fragmentation during apoptosis. TUNELassay is based on incorporation
of biotinylated nucleotides conjugated to bromodeoxyuridine (BrdU) at the 3’ OH ends of the
DNA fragments that form during apoptosis. This detection system utilizes a biotin conjugated
anti-BrdU antibody and streptavidin-horseradish peroxidase [37].

Annexin V which is regularly used to detect apoptotic cells [38] binds strongly to
phosphatidylserine in a calcium-dependent manner [39]. Phosphatidylserine is normally
excluded from the extracellular side of the plasma membrane [40], but flips between the inner
and the outer side upon the onset of apoptosis [41]. Fluorescently labelled Annexin V can,
therefore, be used to detect apoptotic cells.

C) Necrosis assays:-This includes following assays:-

The Neutral red uptake cytotoxicity assay procedure is a cell viability assay based on the
ability of viable cells to incorporate and bind neutral red, a weak cationic supravital dye that
readily penetrates cell membranes by non-ionic diffusion, and predominately accumulates
intracellularly in lysosomes, with lysosomal fragility and other changes that gradually
become irreversible [42]. Cytotoxicity is expressed as a concentration dependent reduction of
the uptake of neutral red after chemical exposure, thus, providing a sensitive, integrated
signal of both cell integrity and growth inhibition.

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In trypan blue assay cells are treated with agents, trypsinized, and subsequently stained with
trypan blue, a diazo dye, which is taken up by dead cells, but excluded by viable cells.
Unstained cells reflect the total number of viable cells recovered from a given dish. This
method is advantageous because it conveys the actual number of viable cells, and increases or
decreases in comparison to control, untreated cells.

LDH is a soluble cytosolic enzyme which serves as an indicator of lytic cell death. The
colorimetric lactate dehydrogenase (LDH) assay which is based on the oxidation of the
yellow tetrazolium salt, INT, to a red formazan, has a long tradition in the clinic to evaluate
tissue or cell damage [43]. As significant amounts of LDH are released from the cytosol upon
cellular necrosis, LDH activity is measured in the cell culture supernatant.

2) Oxidative Stress Assay:-

Oxidative stress is defined as excess formation and/or insufficient removal of highly reactive
molecules, due to the disturbance in the oxidative balance by engineered nanoparticle,s such
as reactive oxygen species (ROS), and reactive nitrogen species (RNS). ROS include free
radicals such as superoxide (•O2-), hydroxyl (•OH), peroxyl (•RO2), hydroxyperoxyl
(•HRO2-), as well as, non-radical species such as hydrogen peroxide (H2O2) and
hydrochlorous acid (HOCl). RNS include free radicals like nitric oxide (•NO) and nitrogen
dioxide (•NO2), as well as, non-radicals such as peroxynitrite (ONOO-), nitrousoxide
(HNO2) and alkyl peroxynitrates (RONOO). The generation of abnormally large
concentrations of ROS and RNS can have many toxicological implications, by reaction with
proteins, lipids or nucleic acids, leading to abnormal cellular function [44].

In 2, 7-dichlorofluorescin (DCFH) assay, the dye is obtained as a diacetate precursor, which

is cleaved by high pH to make the non-fluorescent product DCFH [45]. The presence of ROS
converts DCFH to a fluorescent product, 2, 7-dichlorofluorescein, which can be measured by
fluorimetry.

Electroparamagnetic resonance (EPR) is also a technique that has been widely used to
assess nanoparticles and particle- induced ROS generation. The use of specific spin traps or
probes in combination with specific reagents can allow for the quantification, as well as,
specific identification of the free radical species generated. For EPR detection of radicals, an
adduct-forming, spin-trapping agent (5,5-dimethyl-lpyrroline N-oxide, DMPO) for hydroxide
(OH-) or superoxide (O2-) radicals or a radical-consuming spin probe (4-hydroxy-2,2,6,6-
tetramethylpiperidine- 1-oxyl) are introduced into the culture or nanoparticle solution, for a
set amount of time, after which the entire supernatant is collected, vortexed, and analyzed on
an EPR spectrometer[46,47].

Lipid peroxidation is the oxidative degradation of cell membranes initiated by the presence
of ROS, and is most commonly measured by assaying the presence of malondialdehyde or
other thiobarbituric acid reactive substances [48-50]. This assay has been used extensively to
demonstrate the ability of a variety of nanomaterials to elicit lipid peroxidation in multiple
cell types, such as: fullerenes in human dermal fibroblasts (HDF) and human liver carcinoma
(HepG2) cells [49].

The plasmid assay has been used to assess ROS production [51]. In this assay, unwinding
and linearization of a coiled bacterial DNA plasmid is used to estimate free radical and/or

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ROS exposure. This technique is not particularly sensitive, and may be subject to DNA
binding to the nanoparticle surface.

Oxidative stress acts by alterations in superoxide dismutase or glutathione production.

Increases or decreases in these responses can be interpreted as an evidence for oxidative
stress, as the cell either compensates for increased stress by upregulating the production of
antioxidants, or the exhaustion of cellular stores of superoxide disumutase (SOD), or
glutathione (GSH) by oxidation from RNS or ROS. GSH is an essential antioxidant that is
oxidized during oxidative stress to form a GSH-GSH disulfide between two GSH molecules
yielding GSSG. The most quantitative assessment monitors the ratio of GSH and its disulfide
oxidative product GSSG using HPLC [50], but chromatographic separation steps are time-
consuming and allow for auto-oxidation, leading to over-estimation in the amount of GSSG.
For this reason, combined GSH and GSSG have been assayed instead, during the
nanotoxicology studies to date, using 5, 50-dithio-bis(2-nitrobenzoic acid) (DTNB)[52].The
total GSH concentration is determined by the colorimetric detection of 5-thio-2-nitrobenzoic
acid after reaction of DTNB with GSH. SOD activity is determined indirectly via the
inhibition of superoxide oxidation of a colored substrate, nitro blue tetrazolium, where
superoxide is generated via exogenous xanthine-xanthine oxidase[53].

3) Inflammatory Assay:-
Enzyme-linked immunosorbent assay (ELISA), is a biochemical technique used mainly in
immunology to detect the presence of an antibody or an antigen in a sample. In ELISA, an
unknown amount of antigen is affixed to a surface, and then a specific antibody is applied
over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and
in the final step a substance is added, that the enzyme can convert to some detectable signal,
most commonly a colour change in a chemical substrate. The most commonly tested human
and murine inflammatory markers are the chemokine Interleukin-8 (IL-8), followed by TNF-
α and IL-6[54].

Current in vitro methods used in nanotoxicity assessment and their advantages:

As with any other man-made materials, both in vitro and in vivo studies on biological effects
of nanoparticles should be performed. Presently, in absence of any clear guideline(s) by the
regulatory agencies on the testing/evaluation of nanoparticulate materials, in vitro studies
(using established cell lines and primary cells derived from target tissues) become extremely
relevant and important. These in vitro model systems could provide a rapid and effective
means to access nanoparticles for a number of toxicological endpoints, allow development of
mechanism-driven evaluations, and provide refined information on how nanoparticles interact
with human cells in many ways. In fact, elaborate in vivo studies on experimental laboratory
animals are mandatory before any clinical trials especially involving human subjects.
Nevertheless, in vitro methods with their advantages are preferred and conducted prior to
animal experimentation and clinical trials. Assessment of defined toxicity endpoints by in
vitro methods is more rapid and economical, as compared to, animal models. Complexity of
selection of appropriate animal models or the human body is not a problem with in vitro test
system, and the metabolic activity of standardized cell lines has often not been
comprehensively characterized.

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Table 1: In vitro methods and their advantages

Advantages Example Used for

Assay Detection Purpose of assay effect nanoparticles Refernce
Principle
Tetrazolium mitochondrial Cell 1)Real time assay 1)Increased Silver [57]
salts (MTT, activity is viability/cell results using low cytotoxicity of nanoparticles [58]
MTS, XTT, determined growth cell numbers thiolated gelatine
WST) colorimetricaly (Cell 2)Provides simple nanoparticles carbon [59][60]
and by visible metabolic method for designed to release nanoparticles [27]
light activity) estimation of live their contents in a Fullerenes [61][26]
spectrometer cell number in reducing
order to assess rate environment[49]
of cell 2) Long circulating
proliferation and monensin
to screen nanoparticles
cytotoxic agents (LMNP) were shown
[55] . to potentiate the in
3)Non radioactive vitro cytotoxic effects
4)Inexpensive of anti-My9, a ricin-
based immunotoxin,
in HL-60 sensitive
(500x potentiation)
and resistant (5x
potentiation) human
tumour cell lines
[56].
Neutral red Colorimetric Cell 1) Quantitative The neutral red Carbon [28]
assay detection of viability estimation of the uptake (NRU) in nanotubes, [64]
intact lysosomes (Lysosomal number of viable NIH3T3 mouse Silver, [57]
and detected via activity) cells in a culture. fibroblasts is the only molybdenum,
fluorescence or 2) One of the most validated in vitro aluminum,
absorption used cytotoxicity method for toxicity iron oxide and
measurement. tests with many testing [15] and has titanium
biomedical and been incorporated dioxide
environmental into the REACH nanoparticles
applications [62]. (Registration,
Evaluation,
Authorisation and
Restriction of
Chemical
substances)for the in
vitro toxicity
assessment of
chemicals[63].
Lactate Detection of Cell Reliability, speed 1) Nanoparticles Carbon [26]
dehydrogenase LDH release viability and simple containing different nanoparticles
(LDH) colorimetrically evaluation metal/metal oxide ZnO (zinc [66]
groups have recently oxide)
been analyzed by the nanoparticles
LDH assay for their Fullerenes [67]
toxic effects on rat Iron Oxide [65]
liver BRL3A cells nanoparticles
[65].
2) LDH release
studies were
conducted on human
lung epithelial
(16HBE14o) cells
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treated with
nanoparticles
consisting of porcine
gelatin and human
serum albumin
Trypan blue Detected either Cell 1)It conveys the 1) Cytotoxicity of Gold [70]
colorimetrically viability/cell actual number of crocidolite asbestos nanoparticles
or fluorescently growth viable cells and as well as other SWCNT [71]
increases (cell minerals including (single-walled
proliferation) or talc and glass beads carbon
decreases on a TERT-1 nanotubes)
(cytotoxicity) in immortalized,
comparison to contact-inhibited
control, untreated human mesothelial
cells cell line, LP9/
TERT-1[68].
2) Poly (lactic) acid
nanoparticles (PLA)
for gene delivery in
human and bovine
retinal pigment
epithelial cells, do
not reduce cell
viability at
concentrations up to
4 mg/ml PLA [69].
Colony Detected Proliferative 1)Reliable 1)Cytotoxicity in Carbon based [73]
formation microscopically capacity determination of A549 cells exposed nanomaterials
Assay or by scanner the number of to medium depleted
cells required to by two types of
distinguish SWCNT in order to
between a cluster determine if these
of cells and a carbonaceous
colony nanoparticles are
2) It enables rapid capable of reducing
and accurate the availability of
enumeration of medium
colony number components[72]
and is more
suitable for higher
throughput
compound
assessment than
current
microscope based
methods.
3) This approach
determines colony
number through
the application of
a volume
algorithm and
permits the
differentiation of
cytostatic effects
Caspase-3 Fluorimetric Apoptosis 1)Easy, fast and Nanoscale Silver [57][58]
activity detection of more convenient HAP(hydroxy, when nanoparticles [77]
Caspase-3 2)Potent, cell administered to
activity permeable and human gastric cancer
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non-toxic cells (SGC-7901) at
fluorochrome 100 µg/ml for 12-48
inhibitor hr, caused release of
3)A direct cytochrome c and
measure of activation of
apoptosis caspases-3 and -9
expressed as the [74]. Finally, it has
number of active been demonstrated
caspase enzymes that both CeO2 (5-40
present in the cell µg/ml) and TiO2 (5-
4)No need for cell 40 µg/ml)
lysis no membrane nanoparticles trigger
permeabilization the activation of
caspase-3 in Beas-2B
cells following 24 hr
of exposure [75, 76].

Applications
1) Novel application of an in vitro technique to the detection and quantification of botulinum
neurotoxin antibodies e.g detection of Clostridium botulinum [BoNT] neutralising antibodies
is currently achieved using the mouse lethality assay [MLA] [96].
2) In vitro techniques are used for the assessment of neurotoxicity [97].
3) Attempts are being made to use this technique to establish new varieties from chimeric
tissues e.g rooted cuttings of Chrysanthemum morifolium cv. Maghi, a small flowered, late
blooming cultivar, were treated with different doses of gamma rays. Somatic mutations in
flower colour (light mauve, white, light yellow and dark yellow) and chlorophyll variegation
in leaves were detected as chimeras in treated populations [98].
4) In vitro methods are used to select highly susceptible individuals among common squirrel
monkeys (Saimiri sciureus) to bacterial lipopolysaccharides by using peripheral whole blood
[99].
5) In vitro techniques are used to forage germplasm [100].
6) Applications of in vitro methods to Eucalyptus germplasm conservation [101].
7) A potential diagnostic application of magnetization transfer contrast: an in vitro NMR
study of excised human thyroid tissues [102].
8) Application of in vitro methods for selection of Lactobacillus casei strains as potential
probiotics[103].
9) In vitro models are also used for Antioxidant Activity Evaluation [104].
10) In vitro methods are also used to determine dermal corrosivity of chemicals [105].
12) In vitro methods can also be applied for detecting cell-mediated immunity in man [106].
13) In vitro methods used to assess the nutritive value of leaf protein concentrate [107].

CONCLUSION

Nanotechnology is the manipulation of structures at molecular level. Owing to its vast

growth in every field, be it biotechnology, agriculture or commercial field, it is necessary to
study its chemical and physical properties, and characterize these nanomaterials according to
them. Due to diverse nature of nanotechnology, there are significant challenges in the
interpretation, validation and correlation of cell and tissue toxicity data collected for
nanomaterials. Advances in nanotoxicology will come from developing a valid set of reliable
toxicity tests and nanomaterial characterization protocols for application to variety of
nanomaterials that have been produced, and the even greater variety that is yet to come. The
unique challenges in nanotoxicity assessments lie in addressing the current lack of
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appropriate tools to directly observe and interrogate nanomaterials in complex biological
systems. Specifically, materials aggregation, physical, and chemical reactivity are nearly
impossible to understand currently. Significantly, pharmacological dose–response
relationships are complicated by time- and condition-dependent nanomaterial chemical and
physical states. Acute versus chronic nanomaterial exposure effects and hazards are,
therefore, difficult to monitor. Hence, multiple different measurement techniques must be
adapted, carefully assessed for validity, and applied to complex nanomaterial systems.
Nanomaterial toxicities in biological systems present unique and complex problems. Hence,
in vitro methods are commonly used to determine nanotoxicity. These methods are
advantageous as they minimize the need for animal testing and can be performed under
controlled testing conditions.

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