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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5885_Библиотеки_им_академика_М_И_Перельмана
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increasing their toxicity or reducing their therapeutic effect, depending on the
flavonoid structure. The flavanone present in grapefruit juice, Naringenin,
when co-administered with calcium channel blockers (CCBs) like verapamil
and nitrendipine, impairs the metabolism of CCBs mediated due to inhibition
of intestinal CYP3A4 by naringenin. The naringenin can also produce lethal
and teratogenic effects as observed in the amphibian embryo toxicity test. The
dose of 10 mg/l exerted 100% malformations causing the death of 30% of the
abnormal embryos. The major abnormalities observed were decreased body
size, axial curves, microcephaly, abdominal edema, underdeveloped gills, and
delayed development (Galati and O’brien, 2004). Likewise, Curcumin can
inhibit the activity of the drug-metabolizing enzymes CYP450, glutathioneS-transferase, and UDP-glucuronosyltransferase, leading to increased plasma
concentration of concomitantly administered drug and hence its toxicity
(Burgos-Morón et al., 2010).
Another important polyphenol, Curcumin, obtained from the plant Curcuma
longa, has demonstrated substantial safety and efcacy in the prevention and
treatment of several human ailments. Curcumin has anti-inammatory, anti-
microbial, anticancer, thrombosuppressive, hepatoprotective, hypoglycemic,
antiarthritic, and antioxidant activities. It is therefore not surprising to note
that curcumin is widely available as a dietary supplement and is a popular
subject in the ongoing clinical trials (Russo et al., 2010). However, there is
accumulating evidence that has raised concern on the safety and effectiveness of curcumin. Several reports have demonstrated that curcumin may
cause a dose and time-dependent induction of chromosome aberrations and
DNA damage in both in vitro and in vivo conditions. The National Toxicology
Program (USA), in 1993, published an extensive report on the toxicity and
carcinogenicity of the organic extract of turmeric oleoresin containing
79–85% curcumin. The long term toxicity studies carried out for 3 months
and 2 years on the rodents fed with diets containing varied concentrations of
turmeric oleoresin demonstrated possible toxic and carcinogenic effects of
turmeric oleoresin. In the rats, increased incidences of ulcers, hyperplasia, and
inammation of the forestomach, cecum, and colon was observed. The female
mice developed thyroid gland follicular cell hyperplasia. Carcinogenic activity
evident with increased incidences of clitoral gland adenomas, hepatocellular
adenomas in female mice, and carcinomas of the small intestine was also
observed in all the species tested. The probable mechanism involved in the
carcinogenesis process is the generation of ROS, such as, superoxide anion
and hydrogen peroxide. The ROS generation involves a series of chemical
reaction, called Michael addition, wherein, 2 α,β-unsaturated ketones in the
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chemical structure of curcumin reacts covalently with exposed thiol groups of
cysteine residues of the proteins. Curcumin at low concentrations works as an
antioxidant, however, at higher concentrations, increases the cellular levels of
ROS. Curcumin was also found to be an active iron chelator which induced
a state of overt iron deciency anemia in mice fed with an iron-decient diet
(Burgos-Morón et al., 2010; Program, 1993).
Resveratrol, a compound found in grapes, mulberries, and peanuts, has
demonstrated antioxidant, anti-inammatory, antiproliferative, and pro-
apoptotic properties. The dose-limiting toxicity studies were carried out for
determining any potential toxicity associated with resveratrol. A dose of 3000
mg/Kg body weight (BW) for 28 days caused nephrotoxicity in rats, evident
with an enhanced level of serum BUN and creatinine levels, increased kidney
weights, and gross renal pathology changes, also caused anemia due to reduced
erythropoietin synthesis in the kidneys. The dose of 1000 or 300 mg/kg BW/
day did not result in nephrotoxic ndings (Crowell et al., 2004). Some active
constituents of cruciferous vegetables can also produce toxic effects. Akagi
et al. reported that the oral administration of 0.1% phenethyl isothiocyanate
(PEITC) and benzyl isothiocyanate (BITC) in the rat diet-induced continuous
urinary epithelial cell proliferation and simple and papillary or nodular (PN)
hyperplasias, resulting in bladder carcinogenesis in rats (Akagi et al., 2003).
The intracellular ROS generated from the N=C=S group of the isothiocyanates
(ITCs) produces the cytotoxic and genotoxic effects and subsequent oxidative
DNA damage (Russo et al., 2010).
Many ndings have suggested that most of the natural compounds are still
far from reaching the clinical stage due to low solubility, poor oral bioavailability, low stability, as well as possible toxicity at effective dosage. To overcome these challenges, nanotechnology-based approaches have been applied
and found to have shown some desirable output which includes potentiation
of the activity, reducing the side effects as well also reducing the required
dose (Wachtel-Galor and Benzie, 2011). Moreover, bioactive incorporated
nanomedicines have also been used to target individual organs through both
passive and active mechanisms (Allen and Cullis, 2004; Kostarelos, 2003;
Majumder, 2017).
Nanoparticulate systems including polymeric nanoparticles, lipidic nanoparticles, metallic nanoparticles, carbon nanotubes (CNTs), dendrimers,
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nanofibers, quantum dots, etc., are manufactured around the world due
to the advantages offered by them in disease diagnosis and drug delivery
for the management and amelioration of so many diseases (Borel and
Sabliov, 2014). Fabrication of a bioactive loaded nanoparticulate system
helps to achieve a water-soluble and stable drug delivery system owing
to the enhanced surface area. But at the same time, the enhanced surface
area also makes the NMs more reactive in the biological system (Kahru and
Savolainen, 2010; Oberdörster et al., 2005). Nanoscale size reduction allows
the particles to enter the distal biological sections which were not possible
for the larger particles (Vega-Villa et al., 2008). The general mechanism
of NMs induced toxicity is illustrated in Figure 11.1. The nanosize of the
particle can form electronic states in the NPs which undergo electron transfer
reactions resulting in the generation of ROS and free radicals which in turn
induce oxidative stress and disturb the biological electron transfer reactions.
The free radicals can mediate the mutilation of the biomolecules via lipid
peroxidation (LPO), protein destabilization, and damage to the DNA helix.
The oxidative stress enhances the inflammatory process via the upregulation
of NF-KB, kinase, and activator protein (Ahmad et al., 2016). Figure 11.1
shows the possible mechanism of NMs induced toxicity.
Drug delivery systems such as microemulsions, self-micro emulsifying
drug-delivery systems, nanoemulsions, solid lipid nanoparticles (SLNs),
liposomes, etc., are advantageous in terms of selective tissue targeting and
bioavailability enhancement of drugs (Pariser et al., 2011). But due to the
presence of lipid in their structure, these carriers are susceptible to the phagocytic uptake. This interaction of NMs with phagocytes has some immunogenic potential. In addition to this, NMs when interact with lymphocytes and
other cells may develop immune responses (swelling, immunomodulation,
allergy) (Singh and Nalwa, 2007).
NMs induced toxicity is worrisome and cognizance has been taken by the
scientific community in the past few years. To extract the maximum therapeutic
effect of a drug with the help of a nanocarrier, one needs to pay attention towards
the toxicity evaluation of such nanocarriers. The toxicity assessment strategies
among the scientists have also been evolved in last few years. For the toxicity
evaluation, mainly pharmacokinetic parameters such as absorption (Leite-Silva
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et al., 2013), distribution (Balogh et al., 2007; Goel et al., 2009), biochemistry
involved in metabolism and clearance are supposed to be observed. Apart from
these, predictive strategies for the assessment of NMs induced toxicity are also
trending among the scientists (Fischer and Chan, 2007).
The possible mechanisms of nanoparticles (NPs) induced toxicity; NPs
directly generates reactive oxygen species (ROS) by oxidants and free radicals on the NPs
surface. NPs also activate inflammatory cells (macrophages); ROS generation results in
activation of a proinflammatory transcription factor NF-κβ. Reduced clearance of NPs leads
to increased interaction of NP with epithelium. NPs when reaching the nucleus oxidation
and acetylation of histone protein occur resulting in DNA modification; NPs alter the
mitochondrial functions, generates ROS which in turn may lead to cell injury and apoptosis.
NMs induced toxicity has also been estimated in animal models based on
the phenotypic changes of cells/tissues (Guo et al., 2013; Sayes et al., 2007)
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programmed cell death, i.e., apoptosis (Sarhan and Hussein, 2014; Coccini et
al., 2013) inltration and inammation of vital organs (brain, heart, lung, spleen,
and kidney). For example, NMs tend to deposit in Kupffer cells and hepatic
sinusoids and may alter the hepatic metabolism and detoxication process.
The distribution extent of NMs is a function of their physicochemical
properties (particle size, surface charge, chemistry of coating material) and
is responsible for the toxic effects of NMs in vivo. Extent of distribution
is usually estimated after excising the tissues or organs followed by the
physical detection of NMs. The estimation of metallic NMs is comparatively
easier and done by dedicated instruments. For example, a nanochip made
up of gold can be tracked by neutron activation analysis instrument (Balogh
et al., 2007). Similarly, for the estimation of Cadmium and Silicon, the
dedicated instrument is optical emission spectrometry (Guo et al., 2013). In
addition, uorescent or radiolabeled techniques have also been exploited for
the estimation of Nms. Some of the assessment parameters for evaluating
toxicity of nanoparticles are summarized in Table 11.1.
TABLE 11.1 Various Approaches for Evaluating Nanotoxicity in Major Organ Systems
Sl.
System Studied Assessment Parameter References
No.
1. Hepatic system Quantitative analysis of functional changes
was assessed by serum enzymology of
aspartate aminotransferase, c-glutamyl
transferase, and alkaline phosphatase,
alanine aminotransferase.
2. Renal system Cell nuclear antigen measurement was used
for the cell proliferation study. Detection
of pathological changes is determined by
using various markers like fibrotic and
mesenchymal markers which transform
the growth factor-β1, interferon-6, type I
collagen, fibronectin, and vimentin.
3. Gastrointestinal
system
4. Pulmonary
system
Gastrointestinal microvilli, epithelial
atrophy, and the mast cell count in the
stomach were analyzed by the histological
assay.
Lactate dehydrogenase (LDH) assay for
biochemical examination was performed
on Bronchoalveolar lavage (BAL) fluid for
determining pneumonocyte injury.
Pan et al., 2012;
Yamagishi et al.,
2013; Cho et al.,
2009
Coccini et al.,
2013; Gross et
al., 2015; Pan et
al., 2012
Lin et al., 2014;
Han et al., 2012;
Wang et al.,
2013
Paranjpe
and MüllerGoymann, 2014;
Zhang et al.,
2002
TABLE 11.1 (Continued)
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5. Cardiovascular The cardiac injury was assessed by Zhang et al.,
system using serum markers such as creatine 2015; Laverman
kinase-MB, troponin-T, and myoglobin. et al., 2001;
Venous tolerance was indicated by edema, Fornaguera et
purple lump, blood clot, and eye witness. al., 2015
Analysis of oxidative stress was done
by using biomarkers such as LPO, ROS,
and Antioxidant enzymes such as GPx,
catalase, and SOD
6. Nervous system After i.v administration, brain uptake was Win-Shwe and
determined by calculating the ratio between Fujimaki, 2011;
the concentration in brain and in blood Liu et al., 2014;
plasma, for impairment in brain, glutamate Blasi et al., 2013
uptake was analyzed and for determining
acute toxicity, histology of brain was
performed.
7. Immune and Assessment of toxicity was done by Li et al., 2016;
reproductive assessing variation in immune cytokine, Layali et al.,
system molecules, and immune organs as well 2016; Ren et al.,
as in hematological systems. Female 2016
reproductive organ functioning was
evaluated, histopathology was performed.
Some reports for NPs suggest the effect the
offspring development due to crossing of
placenta blood barrier in rats. For the male
reproductive system, toxicity was evaluated
by changes in sperm parameters, sexual
hormones, i.e., testosterone, and testicular
tissue structure.
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An immunohistochemistry study was performed as a part of a toxico-
logical study for determining any inammation of hepatic cells and liver
brosis (Pan et al., 2012). Degradation of renal glomerulus a parameter for
renal toxicity was identied by histopathological study (Coccini et al., 2013).
For determining the degree of injury of the glomerular ltration membrane
(Petrica et al., 2015) various parameters, such as glomerular ltration rate,
urine protein, total proteins, albumin, hematuria, and creatinine ratio were
analyzed (Gandhi et al., 2013).
Oral route of administration is the most widely used mode of adminis-
tration but the drawback is bioavailability due to the rst-pass metabolism.
Polymeric based or lipid-based nano-sized carriers (Pridgen et al., 2014)
increases the bioavailability by intercepting the acidic and enzymatic inactivation and degradation and facilitates the GIT transportation.
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Analysis of short term (Ma-Hock et al., 2009) and long term toxicity
(Bermudez et al., 2004) for pulmonary system by collecting the bronchoalveolar lavage (BAL) uid for biochemical assessment like lactate dehydrogenase (LDH) assay (Paranjpe and Müller-Goymann, 2014). The total
number of neutrophils obtained from BAL indicates the inammation level
(Zhang et al., 2002).
The amount of drug crossing through the blood-brain barrier can be
analyzed by radiography methods such as PET and CT systems (Frigell et
al., 2013). Acute toxicity can be determined by brain histology examina-
tion (LM and TEM) and uorescence imaging (Blasi et al., 2013). The
nerve injury can be assessed by electrophysiological and behavioral
studies. Animal models for the behavioral study show clinical symptoms
such as convulsions, diarrhea, lethargy, salivation, nausea, etc., (Pradhan
et al., 2014).
Teratogenicity induced by NMs is the main toxicity related to the reproductive system. Phytomedicines loaded nanoparticulate systems and excipients must be screened to avoid such functional abnormalities. It has been
observed experimentally that NMs can slow down the embryo developmental
processes. It has been suggested through various modern-day experimental
studies that nanoparticles can have an effect on the developmental process
which in turn influences the reproductive functions apart from other physiological functions. The toxicity estimation in the reproductive system takes
a longer time as the reproductive system is somewhat different from the
other systems which is differentiated into male and female. For the male
reproductive system, the testicular histological structure, sexual hormones
(e.g., testosterone), parameters of epididymal sperm, and the nanoparticles
concentration harbored in the serum and testis need to be evaluated (Li et
al., 2016; Layali et al., 2016). For the female reproductive system toxicity
study in different sexual hormone (e.g., follicle-stimulating hormone, LH
(luteinizing hormone and estradiol) levels need to be measured in the serum
(Saunders et al., 2015) along with the functions of major organs like ovary,
uterus, and vaginal tract. For a detailed investigation, the histopathological
study should be performed, especially for organs like uterus, placenta, testes,
etc. This investigation should be performed for both parental as well as
offspring (Mozaffari et al., 2015).
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Immunogenic activity and related toxicity are mainly due to the surface
properties of NMs (Dobrovolskaia et al., 2016). The entire immune system
is intricated, hence the extent of toxicity produced by NMs is not easy to
determine. In that case, changes in the hematological system, inflammatory
markers (cytokines) and immune organs, etc., are supposed to be observed.
For example, estimation of NMs induced immunotoxicity can be done by
observing the generation and discharge of tumor necrosis factor (TNF) and
Interleukins (Wang et al., 2016; Baron et al., 2016). In addition, one of the
main immune organs, the spleen is usually examined for any histological
changes and for this purpose, staining of the spleen with the help of hematoxylin and eosin dye has to be done (Wang et al., 2016).
There has been a growing concern about the potentially hazardous effects of
NMs on the health of human beings. For that purpose, a great deal of effort
was made to investigate the potentially hazardous effects of NMs, including
genotoxicity. NMs with genotoxic effects can affect various small cellular
components such as DNA or RNA through a different mechanism of actions.
The nanoscale size and higher surface area when they focus on low solubility
particles, their ability to generate ROS is the main area of concern for the
Genotoxicity (Wang et al., 2016).
Furthermore, reduced particle size and higher surface area collectively
generate more ROS as compared to larger particles (Brown et al., 2000)
and hence may produce more genotoxicity. Type of target in the phases of
the cell cycle, mutagen, DNA replication, and methods of DNA repairment
collectively develop a genotoxic effect. For example, NMs may alter the
strands of DNA in G1-phase, may negatively affect the formation of spindle
bers in phase G2. In addition, NMs on getting the right to permeate through
the nuclear membrane may also affect the chromatin during interphase or
mitosis. Examples of NMs induced ROS are; singlet oxygen, superoxide,
hydrogen peroxide, hydroxyl radicals (Fubini and Hubbard, 2003).
Leukocytes (mainly neutrophil) rush during inammation may generate
ROS which in turn damages the DNA and ultimately may produce secondary
genotoxicity. Mammalian cells and bacteria are mainly used to identify
the genetic mutation but the later is comparatively less suitable as the
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cell membrane composition of bacteria differs from the mammalian cell
membrane. For the assessment of secondary genotoxicity, in vivo models
with similar end-points are selected (Driscoll et al., 1997).
The cutting edge nanotechnological advancement in the field of medicine
has many advantages as it helps in early disease diagnosis, personalized or
customized treatment, and better disease amelioration at lower cost. However,
the potential risk assessment and risk minimization associated with the nanotechnology-based drug and delivery system are quintessentially important. The
term “risk” can be defined as the potential “hazard” that can be produced on
“exposure” to certain NMs. The risk assessment includes hazard identification,
hazard characterization, and exposure assessment, and risk characterization.
The prime objective of risk assessment and nanosafety studies is to
produce a nanomedicine with minimum risk of toxicity. In that context,
the utmost important thing is to nd and authenticate some alternate and
new approaches. For this purpose, European initiatives such as “Safety-bydesign” have been taken to interpret the acute in vitro toxicity results for the
prediction of long term toxicity in animal models (Accomasso et al., 2018).
Before starting an in vitro toxicity study of a NM, in silico study is
recommended which includes quantitative nanostructure-activity relationships (QNAR), categorization, or grouping of NMs and read-across. As
the name suggests, QNAR is a relationship developed between a chemical
structure with certain physicochemical properties and a specically determined biological activity. Categorization of NM can be done on the basis of
commercial viability/production volume, materials used (such as Polymer,
Lipid, Metal, Carbon, etc.), morphology (shape and size), mechanism of
action, etc., (Lynch et al., 2014). Categorization of NM based on chemical
properties is particularly important as their structural similarity likely
to produce toxicity with similar pattern. Read-across technique helps in
predicting the end-point of a test nanomaterial by using information of same
end-point previously obtained from other NMs.
High throughput screening (HTS) and high content screening (HCS) are
the automated tools used for the estimation of in vitro toxicity related to NMs.
HTS is mainly a type of automated assay and usually focuses on a separate
biological mechanism or biochemical alteration. HTS-based screening of
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test NMs do not observe the whole phenotypical changes in cell. HCS is also
an automated screening tool and mainly uses uorescence and microscopic
images for the toxicity analysis. Contrary to HTS, it is used to estimate
multiple changes in the phenotype of similar cell population (Godwin et al.,
2015) The HTS based in vitro assays are comparatively simple, less time
consuming and less expensive than complicated animal model experiments.
However, it cannot be used solely for the in vitro toxicity study due to the
poor in vitro-in vivo correlation (IVIC); as a small cell cannot be a representative of an animal with complex body structure.
Small animals such as mice, rats, workhorse, zebrash, etc., are the gold
standard animals for the in vivo toxicity screening of NMs. In such animals,
it is also important to understand the fate of NM at the cellular level, tissue
level, and organ level. Hence, a right approach for the toxicity testing of
NMs should include in silico, in vitro, and in vivo study for the sufcient
IVIC and proper risk assessment.
When a NM comes in contact of biological media, they tend to undergo
transformation such as aggregation or chemical degradation (dissolution,
corrosion, and oxidation) and this transformation ultimately may produce
toxicity. Hence, physicochemical characterization of developed NMs is
required to scrutinize for the risk minimization (Bian et al., 2011). Coating
of FDA approved biocompatible polymers (PEG or PVP) on such NMs may
help improving their stability in biological media.
Early risk assessment and risk minimization strategies are prerequisite
for the successful development of a nanomedicine and regulation from
governing bodies on hazardous NMs must be welcome for the welfare of
human community, ecosystem, and environment (Duncan and Gaspar, 2011).
Emerging technologies create an atypical, often complex, and fundamentally
political problem for global governance due to the persistent uncertainty
that surrounds their potential risks. The regulation of any new technology
is crucial to establish its safety and effectiveness. Nanomedicine has
demonstrated significant potential in providing better health outcomes and
is rapidly shifting from bench to bedside, closer to the realm of practical
applicability. The global nanomedicine market is likely to reach $344.0
billion by 2024 (Agrahari and Hiremath, 2017). The rapid shift and the huge
market share in the near future necessitate the examination of the adequacy
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