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Environmental exposure of toxicants
Organic chemicals and pollutants are found increasing in our environment (water, air,
soil and food) persistently over the past many decades. Many of them are well known
to be toxic to living organisms. Individuals are being exposed to these potentially
toxicants throughout their life, beginning with starting of life during fertilization of
the egg resulting in to zygote. Embryo, developing fetus and newly born children are
exposed to toxicants through the mother either by direct exposure or lactation. The
toxic effects of such toxicants depend on the concentration of the chemicals, route of
exposure, duration of exposure and the stage of exposure during the life cycle of an
organism. A vast number of toxicants have been shown to induce permanent disorders
in organisms by acting as neurotoxicants.
Developmental toxicity
Birth defects are defined as abnormal development of the fetus involving
malformations, growth retardations, functional disorders that lead to lifelong
impairment of humans and ultimately to death. Every year, approximately 6% of
children are born with serious birth defects worldwide, and reason behind these birth
defects is often unknown. The contribution of chemical exposure leading to adverse
effects including pre-postnatal death is well documented (O'Rahilly et al., 2001).
Various developmental toxicants are usually suspected to be normal toxicants in
humans since the variations among species are high (Schardein et al., 2000) and
underlying mode of actions are often unknown. This could lead to misinterpretations
of the risk of chemicals as happened with Thalidomide which was perceived as a
harmless sedative drug that was given to pregnant women in the late 1950s and early
1960s. Thalidomide was strongly teratogenic when given during critical periods of
embryo development, and caused malformations in more than 7000 born children
(Gilbert et al., 2006). Developmental toxicants produce abnormalities only during
certain periods of gestation (Schardein et al., 2000; Carlson et al., 2009). In other
words, some stages of development are more vulnerable to toxic agents than others.
The developmental stage with maximal susceptibility is between the third and eighth
weeks (the embryonic period), because most organogenesis occurs in this stages of
development and interference with the processes may lead to malformations. Any
abnormalities arising during the third to the ninth month of gestation (the fetal period)
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tend to be functional. In general, it is considered that some chemicals harmful to
development may cause their effects at the molecular level at an early stage, although
the effects may be recognized only at later phase e.g. childhood cancer, DNT. To
understand mechanistic insight of developmental toxicants, it is necessary to explore
all the events starting from exposure to the occurrence of the developmental defect
(NRC, 2000). This includes understanding of kinetics of toxicants (ADME),
metabolic fate, molecular interaction, consequences of the molecular interactions in a
cellular/developmental process. Human development is extremely complex process,
and is still not understood in detail. Due to continuous development, the toxicants can
interact at numerous points with an important molecular component, or even more,
with several points further complicating the understanding of the mode of actions of
developmental toxicants.
Vulnerability of the developing brain
Several important cellular process are involved in the development of the central
nervous system viz., precursor cell differentiation, migration of neuronal and glial
cells, differentiation, neuritogenesis, synaptogenesis, programmed cell death,
establishment of neuronal network connectivity, formation and maturation of blood-
brain barrier. In human, formation of neurons and their migration start from six weeks
of gestation period. In general, neurons generated during embryonic development,
migrate to a new location where starts growing and specialized. Interference at any
steps of these cellular processes could lead to lifelong developmental brain
disabilities.
It is well documented that the developing brain in fetuses and children is more
vulnerable to toxic agents than the adult brain even at low exposure level that are
usually not harmful to mature brain (Tilson, 2000; Bal Price et al., 2011). This is
partly due to the fact that the adult brain is well protected by the blood brain barrier
and developing blood-brain barrier is not efficient enough to restrict the entry of
xenobiotics from maternal environment. This might also be due to detoxification
mechanisms and metabolic functionality in immature brain (Tilson, 2000; Stringari et
al., 2008; Bridges et al., 2009). The placental barrier protects the fetus and blood-milk
barrier protects the infants. Many environmental toxicants viz., metals, low-molecular
weight and lipophilic compounds are reported to cross these barriers (Sakamoto et al.,
2004; Andrieux et al., 2009; Powers et al., 2010; Powers et al., 2011; Menezes et al.,
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2011; Nielsen et al., 2011; Liu et al., 2011; Hoekman et al., 2011). Since, the
development of brain is complex process which involves several different important
events, e.g. proliferation, migration and differentiation of cells in strictly controlled
time frames and therefore, each event creates different windows of vulnerability to
xenobiotic exposure (Rice and Barone, 2000; Rodier et al., 1994). Furthermore, the
brain consists of many different cell types like neuronal, glial and endothelial cells
which play specific functions. Although, each cell type is produced at a specific time
during the development and is therefore, susceptible to environmental chemicals.
Some events take place during a very short time period and interference by chemicals
during these stages could lead to serious consequences. The vulnerability of the
developing brain depends on whether a toxicant reaches the target and the period of
exposure. Before or after an organ is developed it is in general less sensitive to
environmental perturbation than during development.
Developmental neurotoxicity guidelines
There are many guidelines developed for making general framework to assess
developmental neurotoxicity (DNT) of broad range of pesticides, insecticides, food
additives, cosmetics, industrial chemicals, nanoparticles with flexibility in the specific
methodology. In 1991, the US Environmental Protection Agency (EPA) issued the
first guideline for DNT (US EPA DNT Guideline 870.6300) that was revised and
published in 1998 (US EPA, 1998a & b). The guidelines were framed upon an
extensive scientific database including between laboratory validation studies (Makris
et al., 2009; Tsuji et al., 2012). The Organization for Economic Co-operation and
Development (OECD) started developing the OECD test guideline 426 using the US
EPA guideline as a template in 1995. Further, it was finalized and adopted by the
OECD council in 2007 (OECD, 2007). However, limited number of environmental
chemicals has been tested according to these guidelines so far. Preclinical
developmental neurotoxicity assessment for human pharmaceuticals are based on the
guideline ICH S7A (ICH, 2000) from the European Medicines Agency (EMEA) and
the US Food and Drug Administration (FDA).
Environmental exposure of potential developmental neurotoxicants
There are large amount of literature on public domain reporting the susceptibility of
early developing human nervous system towards many toxicants and there are full of
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evidences to support the long lasting neurological disabilities following chemical
exposure during development (Rodier et al., 1994; Rice and Barone, 2000; Johri et al.,
2008). Therefore, developing brain cells are at higher risk against environmental
exposure of xenobiotics (Weiss et al., 2004). There are various chemicals known to be
toxic for the adult nervous system and these are of particular concern regarding their
potential to cause DNT effects after early-life exposure (NRC, 2000). These factors
include certain industrial chemicals, pesticides, tobacco smoke, alcohol and certain
drugs as well as maternal stress (Tayebati et al., 2009). If any injury or toxic effects
caused during the developmental of immature fetal nervous system, the effects may
be result in permanent disabilities and likely to be lasting lifelong (Julvez et al., 2007;
Talge et al., 2007; Slotkin and Seidler, 2010). Because of a growing recognition of an
apparent increase in the incidence of developmental disabilities, considerable
attention has been focused on the effects of exposures to environmental pollutants,
including organophosphate and chlorinated pesticides (Slotkin and Seidler, 2010).
Moreover, there is increasing evidence in the literature that chemical exposures can
disrupt neurodevelopment, transient alteration in neurochemicals balance. These
changes can be estimated only by behavioral alterations under challenging test
conditions at later stage (Icenogle et al., 2004). Occupational medicines have
traditionally been studied for their neurotoxic effects and other adverse consequences
in the workers (Liu et al., 2011; Pubill et al., 2011). Literature is full for neurotoxic
effects of metals like lead and mercury, organochlorides, many organophosphorous
pesticides, solvents and other industrial chemicals to adult nervous system (Slotkin
and Seidler, 2009a, b; Kashyap et al., 2010; Kashyap et al., 2011). Assessment of
neurodevelopment may require many years of follow-up and is therefore even more
difficult to document. Improved insight into developmental neurotoxicity is crucial,
because these effects may occur at exposure levels that are lower than existing
occupational exposure limits (Grandjean et al., 2006a). Our study is mainly focused
on developmental neurotoxicity of pesticide which is discussed hereunder….
Developmental neurotoxic pesticides
The effect of many pesticide on human brain particularly immature developing brain
are of great concern since, many pesticides are developed to target the nervous system
of different organisms. The acute neurotoxicity of pesticides is well known from
occupational exposure studies, poisoning events and suicide data. Furthermore, DNT
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effects such as reduced short-term memory, hand-eye coordination, drawing ability
and visuo-spatial deficits have been observed in several studies (Ruckart et al., 2004;
Grandjean et al., 2006b). Insecticides are especially known to induce neurotoxic
effects. They are divided into several different classes and the most widely used are
organophosphates and organochlorides that usually cause acetylcholinesterase
inhibition. Inhibition of this enzyme causes accumulation of the neurotransmitter
acetylcholine at the cholinergic synapses leading to overstimulation of cholinergic
receptors that result in various effects in the brain. Developmental neurotoxicity may
be caused by similar mechanisms, which may lead to more permanent effects, as
acetylcholine has crucial functions during brain development (Eskenazi et al., 2007).
Developing fetus and growing children are more sensitive to the acute toxicity of
these inhibitors than adults, possibly due to lower metabolic capabilities (Thullbery et
al., 2005; Costa et al., 2006). In addition, the adverse effects on the developing brain
could also be mediated by additional mechanisms, such as damage to DNA and RNA
synthesis (Crumpton et al., 2000a, b), deregulation of signal transduction pathways
(Ehrich et al., 1995; Song et al., 1997), oxidative stress (Crumpton et al., 2000b) and
astroglial cell proliferation (Garcia et al., 2001; Guizzetti et al., 2005). Many workers
have been reported the negative health effects including developmental neurotoxicity
of chlorinated pesticide in human and other species (Hein et al., 2010; Cole et al.,
2011; Sledge et al., 2011; Slotkin et al., 2011). Rauh et al. (2011) studied the
relationship between prenatal chlorinated pesticide exposure and neurodevelopment
among cohort children at age 7 years. They measured prenatal chlorinated pesticide
exposure using umbilical cord blood plasma (picograms/ gram plasma), and 7-year
neurodevelopment using the Wechsler Intelligence Scales for Children (WISC-IV)
and reported full-scale IQ declined by 1.4% and working memory declined by 2.8%.
There are more than 600 pesticides registered on the market, including insecticides,
fungicides and rodenticides, and several of these are produced in high volumes
(Grandjean and Landrigan, 2006). Even though the uses of many pesticides are
restricted and an increase in DNT testing is demanded. The general lack of DNT data
for agricultural chemicals is of particular concern because of their widespread use and
ubiquitous exposure (Whyatt et al., 2004).
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Monocrotophos (MCP; Neurotoxic organophosphorous pesticide)
Monocrotophos is a systemic insecticide and acaricide belonging to the vinyl
phosphate group. It controls pests on a variety of crops, such as cotton, rice, and
sugarcane. It is widely used pesticide to control a wide spectrum of chewing, sucking
and boring insects (aphids, caterpillars, Helicoverpa spp, moths, budworm, scale and
stem borer, as well as locusts). Monocrotophos is out of patent and therefore, has
become an easily affordable pesticide. Its low cost and many possible applications
have kept up high demand in India despite growing evidence of its negative impact on
human health. Monocrotophos can be absorbed following ingestion, inhalation and
skin contact. When inhaled, it affects the respiratory system and may trigger bloody
or runny nose, coughing, chest discomfort, difficulty breathing or shortness of breath
and wheezing due to constriction or excess fluid in the bronchial tubes. Skin contact
with organophosphates may cause localized sweating and involuntary muscle
contractions. Eye contact will cause pain, tears, pupil constriction and blurred vision.
Following exposure by any route, other systemic effects may begin within a few
minutes or be delayed for up to 12 hours. These may include pallor, nausea, vomiting,
diarrhea, abdominal cramps, headache, dizziness, eye pain, blurred vision,
constriction or dilation of the pupils, tears, salivation, sweating and confusion. Severe
poisoning will affect the central nervous system, producing lack of coordination,
slurred speech, loss of reflexes, weakness, fatigue, involuntary muscle contractions,
twitching, tremors of the tongue or eyelids, and eventually paralysis of the body
extremities and the respiratory muscles. In severe cases there may also be involuntary
defecation or urination, psychosis, irregular heartbeat, unconsciousness, convulsions
and coma. Respiratory failure or cardiac arrest may cause death (WHO, 2009).
Monocrotophos is an organophosphorous compound that inhibits acetylcholinesterase.
It is highly toxic by all routes of exposure. Monocrotophos can be absorbed following
ingestion, inhalation and skin contact. The acute oral lethal dose (LD50) for rats is 14
mg/kg. According to WHO, the ingestion of 120 mg monocrotophos can be fatal to
humans. In the WHO 2004 edition of the Recommended Classification of Pesticides
by Hazard and the guidelines to Classification 9, monocrotophos is classified in the
WHO Class Ib. i.e. as a highly hazardous pesticide (WHO, 2009).
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Alternative in vitro models for DNT evaluation
In vitro models of the nervous are being used for many years in basic research and
provide an important powerful tool for functional studies at both cellular and
molecular levels (Silva et al., 2006; Costa et al., 2011; Zhang et al., 2012). The major
advantage of in vitro cell-based models is the ability to reproduce, discrete stages of
brain development and maturation in a simplified way. The reduced complexity as
compared to in vivo makes it easier to detect changes in key cellular developmental
processes, such as proliferation, migration and differentiation. These processes are
well understood at the cellular and molecular levels and are surprisingly similar to
those in vivo. In general, the level of similarity to the in vivo situation is directly
related to the complexity of the in vitro model, starting from simple neuronal/glial cell
lines, primary neuronal/ glial cells, to more complex models such as monolayer of
primary mixed neuronal cultures or three-dimensional models.
Cell lines: Cell lines are simplest alternative model which can be maintained in
culture for a long period of time. Neuronal cell lines normally originate from a single
common ancestor cell and are often derived from tumors e.g. phaeochromocytomas
(adrenal medullary tumor) (Greene and Tischler, 1976) and neuroblastomas (Augusti-
Tocco and Sato, 1969). There are several neuronal cell lines commercially available
(James and Wood, 1992) and many of them have been used for DNT studies
(Jameson et al., 2006; Lau et al., 2006; Radio and Mundy, 2008; Slotkin and Seidler,
2009a & b). The cell lines are relatively easy to maintain and some of them can be
indefinitely propagated. Moreover, they provide a homogenous population of cells
that can divide rapidly and be continuously subcultured to provide large numbers of
cells in a short period of time. The cells can normally be differentiated into neuronal-
like cells by the addition of various inducers and growth factors into the medium. This
makes it possible to control the onset of development and adjust the experimental
design to the needs of the DNT study. These advantages make cell lines suitable for
high throughput screening of many neurotoxicants.
Among various cell lines, one of the most widely used neuronal cell line is the PC12,
derived from rat pheochromocytoma cells of the adrenal gland (Greene and Tischler,
1976). Undifferentiated PC12 cells resemble immature adrenal chromaffin cells that
are polygonal in shape (Tischler and Greene, 1978). In the presence of nerve growth
factors (NGF), the cells differentiate into neuronal-like cells with the properties of
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mature sympathetic neurons, such as electrical excitability, secretion of
neurotransmitters (dopamine, noradrenalin and acetylcholine), expression of
cholinergic and ionotropic N-methyl D-aspartate glutamate (NMDA) receptors (Fujita
et al., 1989; Casado et al., 1996). The PC12 cell line has been widely used in different
neurobiological (Greene and Tischler, 1976; Radio et al., 2008 & 2010) and
toxicological studies involving mechanisms of toxicity i.e. apoptosis, mitochondrial
dysfunction, disturbance in neurotransmitter synthesis and vesicle release (Gartlon et
al., 2006; van Vliet et al., 2007; Siddiqui et. al., 2008; Kashyap et. al., 2010 & 2011).
Another most commonly used neuronal cell lines are neuroblastomas. Many
neuronal cell lines have been derived from tumors arising from immature nerve cells
(neuroblastomas) in both rodents and humans. There are several different
neuroblastoma cell lines, such as rat B50, mouse NB2a and N2a, human IMR-32, SH-
SY5Y and SK-N-SH, that can be induced to differentiate into neuronal like cells with
the addition of diverse inducing factors, such as retinoic acid, dibutyryl cAMP and
nerve growth factors, or by reduced serum content (Augusti-Tocco and Sato, 1969;
Greene and Tischler, 1976; Ross and Biedler, 1985). Depending on the cell line, the
differentiated cells can express cholinergic, adrenergic and/ or dopaminergic markers
and therefore, been widely used to investigate neurotoxicity of various
organophosphate pesticides (Ehrich et al., 1995). The neuroblastoma cell lines have
been used in several DNT studies evaluating different key processes, such as neuronal
migration (Miller et al., 2006) and differentiation including network elaboration
(Hong et al., 2003; Radio and Mundy, 2008) and synaptogenesis (Chamniansawat and
Chongthammakun, 2009).
It should be kept in mind that these cells are usually immortalized and do not retained
many of the complex properties that can be seen in the original primary cells. Many
properties might be lost or differ from the in vivo situation.
Primary cell cultures
Neuronal primary cultures are generally harvested either from the peripheral nervous
system (PNS) or CNS of a living organism and can be maintained in culture for at
least 24 hours (Aschner and Syversen, 2004). The advantages of primary cultures are
that many developmental key processes can be largely followed and functional
capacity of neuronal/ glial cells can be maintained. Moreover, processes that play
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important roles for normal and abnormal cell development and maturation can be
addressed, such as regional specificity, expression of receptors and neurotransmitters,
and neuronal-glial interactions. Primary neuronal cultures consist predominantly of
post-mitotic neurons which do not proliferate, resulting in limited life span.
Consequently, the cultures always need to be freshly isolated and may not be fully
suitable for high throughput screening. Neuronal primary cultures from the PNS and
CNS can be derived from many different regions, such as superior cervical ganglion,
dorsal ganglion, hippocampus, cortex, cerebellum, midbrain or sub-ventricular zone.
Primary cultures of neuronal/ glial cells have now been well established in mice and
rats for neurotoxicity. However, primary cultures of human neuronal/ glial cells are
mainly hampered due to non-availability of human developing brain tissues even
mature brain tissues which is ethically and socially unacceptable.
In vitro human stem cells; DNT model
Human stem cells
In recent years, stem cells have been the subject of increasing scientific interest
because of their utility in numerous biomedical applications. Stem cells are capable of
renewing and can be cultured for long time in an undifferentiated state, giving rise to
more specialized cell types such as cardiomyocytes (Shim et. al., 2004), hepatocytes
(Chen et. al., 2012), bone (Sun et. al., 2012), cartilage (Sun et. al., 2012), pancreatic
islet (Zanini et. al., 2012) and nerve cells (McGuckin et al., 2004) under the influence
of specific growth condition.. Therefore, stem cells are an important new tool for
developing unique, in vitro model systems to test environmental chemicals and drugs
to predict toxicity in humans (Buzanska et al., 2009). The unique feature of
differentiation make them as a boon in improving current treatment strategies of
various diseases and providing functional tissues to transplant against diseased
tissues. However, using stem cells therapies as a regenerative medicine and clinical
applications is still under research but showed very promising potential in pre-clinical
and some clinical trials (Harris and Juriloff, 2007; Andre et al., 2012; Harding et al.,
2012). Based on sources, stem cells are classified into embryonic stem cells which are
derived from the inner cell mass of the blastocyst (Tamm et al., 2006; Leist et al.,
2008; Stummann et al., 2009; Xiao Guan et al., 2012); adult stem cells such as the
stem cells in the bone marrow (Scott and Reijo, 2008; Ning et al., 2009); neural stem
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cells (NSCs) isolated from CNS (Tamm et al., 2006; Coecke et al., 2007; Leist et al.,
2008; Breier et al., 2009; Moors et al., 2009; Stummann et al., 2009; De Filippis and
Delia, 2011) and umbilical cord blood stem cells (UCBSCs) can be viewed as neither
embryonic nor adult stem cells since they are isolated nine months after fertilization
and possess differences to both kinds of stem cells, therefore it can be listed as a third
source of stem cells as fetal stem cells (Buzanska et al., 2002; McGuckin et al., 2006;
Kucia et al., 2007; McGuckin and Forraz, 2008).
Human umbilical cord blood stem cells
Umbilical cord blood is considered as one of the most abundant and richest source of
stem cells (McGuckin and Forraz, 2008; Ali and Bahbahani, 2010). Unlike,
embryonic stem cell, use of cord blood stem cells is non-controversial as it is
generally discarded material after the birth of child. In addition, the non-invasive
collection make it comparatively wonderful tool for regenerative medicine (Watt and
Contreras, 2005; McGuckin et al., 2006; Ballen et al., 2008). Umbilical cord blood
stem cells have been thought to possess higher proliferating capacity due to longer
telomeres than other somatic stem (Pipes et al., 2006; Slatter and Gennery, 2006).
Moreover, umbilical cord blood can be cryopreserved for longer period that may be
used in transplantations. There are number of cord blood banks throughout the world
including India (Watt and Contreras, 2005; McGuckin et al., 2006; Lee et al., 2007;
Solves et al., 2008). Transplantations of umbilical cord blood have lower risk of graft-
versus-host diseases in compare to bone marrow due to MHC-II compatibility (Slatter
and Gennery, 2006; Mochizuki et al., 2008; Ringden et al., 2008) demonstrating
superiority of umbilical cord blood in clinical applications. Although, the average
sample size of cord blood unit is considered small and generally restricted for single
adult transplantation but the immature status of cord blood HLA allowed successful
combination of multiple unrelated cord blood units for allogenic transplantations
(Ringden et al., 2008; Ali and Bahbahani, 2010). After the transfection with necessary
pluripotent genes, umbilical cord blood stem cells have been shown their ability to
produce induced pluripotent stem cells (iPS) (Giorgetti et al., 2010; Takenaka et al.,
2010) which behaves like embryonic stem cells. Takenaka and coauthors (2010) have
successfully reprogrammed CD34+ cord blood cells into iPS cells after viral
transfection of Oct-4, Sox-2, Klf-4 and c-Myc while repressing P53
gene via RNA
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silencing. Therefore, the umbilical cord blood stem cells can be viewed as the stem
cells source of choice for clinical and non-clinical research applications.
Recently, human umbilical cord blood derived neural stem cell (HUCB-NSC) line has
been established, which have the potential for a stable growth rate, the ability for self-
renewal and differentiating into neurons, astrocytes and oligodendrocytes (Buzanska
et al., 2002 & 2009) after receiving adequate stimulation by different
neuromorphogens, such as retinoic acid, brain-derived neurotrophic factor or di-
butyryl cAMP. The advantages of this model are that it is derived from human cord
blood and can be maintained in culture at different developmental stages depending
on the culturing conditions (Buzanska et al., 2005, 2006 & 2009). The differentiating
neuronal cells could serve as an important tool for DNT as the whole range of
neurodevelopment processes, such as proliferation, migration, differentiation,
synaptogenesis and apoptosis, can be studied (Buzanska et al., 2005).
Hematopoietic stem cells (HSCs)
Till and McCulloch (1960) showed for the first time the presence of HSCs in bone
marrow of mice. HSCs are rare pluripotent cells present in the bone marrow and cord
blood which provide life-long hematopoiesis. Mixed populations consisting of both
HSCs and progenitor populations are referred as hematopoietic stem and progenitor
cells (HSPC). According to the classical model of hematopoiesis, the hematopoietic
differentiation hierarchy is divided in two parts at the level of the common
lymphoid progenitor, precursor of all lymphoid cells, and the common myeloid
progenitor, giving rise to myeloid and erythroid cells (Weissman et al., 2008). Today,
vast knowledge has accumulated on how hematopoiesis proceeds from a HSC through
intermediate progenitors, producing different lineages of mature blood cells. Initially,
the extrinsic factors (growth factors, stromal cells, extracellular matrix) affecting
hematopoiesis were main point of focus for research. But presently the emphases are
being given to explore intracellular events that regulate hematopoiesis, mainly at the
transcriptional level. It has been shown that cord blood HSC can be selectively
induced into specific hematopoietic lineages in vitro including erythroid,
megakaryocytic and monocytic lineages following the addition of growth factors and
cytokines (Felli et al., 2010).
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HSC markers
Based on cell surface markers and the degree of self-renewal, HSC have been divided
into three subpopulations: long-term HSC (LT-HSC), short-term HSC (ST-HSC), and
multipotent progenitors (MPP) (Morrison and Weissman, 1994). LT-HSCs give rise
to ST-HSC which in turn gives rise to MPP irreversibly. Umbilical cord blood has
been reported to be richest source of HSCs which can be characterized by their
differential expression of hematopoietic antigens CD133, CD34, Thy and CD45
(McGuckin et al., 2003 & 2007). CD34+ cells have been isolated from umbilical cord
blood, fetal liver, fetal bone marrow, adult bone marrow and peripheral blood. Human
CD34 marker is detected in very early stem cells but its expression gradually
decreases as the stem cells differentiate. The CD34 molecule is a highly glycosylated
type I transmembrane glycoprotein of 385 amino acids. The cytoplasmic domain
contains two sites for protein kinase C phosphorylation and tyrosine phosphorylation
(Simmons et al., 1992). Although, the function of CD34 is not well known, CD34
molecule promotes the adhesive interactions of hematopoietic cells with the stromal
microenvironment. The CD34 molecule is not the only marker that researchers have
used to characterize and purify. Several studies have also shown CD133 (human
homolog of the prominin transmembrane glycoprotein) as a HSC marker being co-
expressed on CD34+
cells (Miraglia et al., 1997). It has been demonstrated that
purified CD34+
cells possess high cloning efficiency that are capable of repopulating
in irradiated NOD/SCID mice (Bonanno et al., 2004). Another marker found to be
highly expressed on HSC is the vascular endothelial growth factor receptor 2
(VEGFR2, also known as KDR or Flk-1). Flk-1+
cells are enriched in primitive
CD34+CD38
-CD90
+CD117
low
cells, while lineage-committed progenitor cells are
CD34+Flk-1
-
(Ziegler et al., 1999). Another way of characterizing HSC is to focus on
conserved function rather than expression of cell surface markers since their
expression can be altered either with the progression of cell cycle or during ex vivo
culture. Using this strategy, it has been shown that lineage depleted umbilical cord
blood cell fractions with high aldehyde dehydrogenase (ALDH) activity are enriched
in cells with primitive HSCs phenotype (CD3+CD38
-) (Hess et al., 2004). Similar
strategy based on the ability of HSCs to efflux the fluorescent dye Hoechst 33342 has
been used to obtain HSCs cell populations (Goodell et al., 1996). The immature
umbilical cord blood stem cells have been shown to express pluripotency markers
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such as Oct-4, Sox-2 and Nanog, which are usually expressed in pluripotent
embryonic stem cells (Kucia et al., 2007; McGuckin et al., 2008; Orkin et al., 2008).
Maintenance of pluripotency and self renewal
Pluripotency, self renewal and differentiation of stem cell is a tightly regulated
process where decisions have to be made on whether the HSC should self-renew,
proliferate, differentiate or enter the apoptotic pathway. The self-renewal of
pluripotent stem cells is regulated by a transcriptional network involving Oct4, Sox2
and Nanog (Jaenisch and Young, 2008). The POU domain transcription factor Oct4 is
critical for the pluripotency of cultured stem cells (Nichols et al., 1998; Niwa et al.,
2000). The SRY-related HMG-box transcription factor Sox2 is also required for the
maintenance of pluripotency in the embryo and in stem cells in culture (Avilion et al.,
2003; Masui et al., 2007). Sox2 cooperates with Oct4 to activate the expression of a
number of genes that regulate pluripotency (Masui et al., 2007). The homeodomain
protein Nanog is required for the maintenance of pluripotency (Mitsui et al., 2003).
Overexpression of Nanog can bypass the requirement of leukemia inhibitory factor
(LIF) in maintaining pluripotency and it appears to stabilize the pluripotent state
(Chambers et al., 2007). These three factors form the core of a transcriptional
regulatory network that promotes the expression of genes that maintain pluripotency
rather than induce differentiation (Fujikura et al., 2002; Niwa et al., 2005). Ectopic
expression of Oct4 together with various combinations of other transcription factors
including Sox2 and Nanog can reprogram differentiated mouse and human cells into
ES-like induced pluripotent stem (iPS) cells (Takahashi and Yamanaka, 2006; Okita
et al., 2007; Wernig et al., 2007; Yu et al., 2007; Park et al., 2008). To maintain the
pluripotency, the Oct4-Sox2-Nanog network needs to be fine-tuned by positive and
negative regulation, as slight hyper- or hypo-activation of some of these factors can
disrupt pluripotency (Niwa et al., 2000). These three transcription factors interact
physically with each other, and co-occupy regulatory regions in many target genes co-
ordinately regulating the pluripotent state (Boyer et al., 2005; Wang et al., 2006;
Masui et al., 2007). Oct4, Sox2, and Nanog also regulate their own expression as well
as each other’s expression, forming a positive feedback circuit that maintains
pluripotency.
There are also other critical transcription factors beyond Oct4, Nanog and Sox2. The
zinc finger DNA-binding protein Ronin is necessary and sufficient for maintaining
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stem cell pluripotency (Dejosez et al., 2008). Upon induction of differentiation, Ronin
(Dejosez et al., 2008) and Nanog (Fujita et al., 2008) are inactivated by caspase-3
proteolysis to allow differentiation. LIF is a key factor that blocks the differentiation
and its receptor consists of a heterodimer of LIFR and gp130 which activates the
JAK/STAT3 pathway (Niwa et al., 1998). The targets of JAK/STAT3 pathway are
largely unknown but have been suggested to include c-MYC, a known promoter of
pluripotency (Cartwright et al., 2005; Takahashi and Yamanaka, 2006). Bone
morphogenetic proteins (BMPs) also required for maintaining pluripotency that signal
through SMAD proteins to promote the expression of Inhibitor of differentiation (ID)
transcriptional regulators (Ying et al., 2003). LIF/JAK/STAT3 and BMP/ID signalling
pathways work together to prevent the differentiation of stem cells in culture by
inhibiting the consequences of Mitogen-Activated Protein Kinases (MAPKs) pathway
signalling, which tends to promote differentiation (Ying et al., 2008). These studies
demonstrate that the inhibition of differentiation is a key mechanism to promote self-
renewal. Regulatory networks act in a concerted manner in pluripotent stem cells to
do this at the level of signal transduction, chromatin structure, and transcriptional
regulation. Transcription factors are the final targets of many signalling pathways
and several transcription factors have been shown to have important HSC
regulatory functions. c-MYC and n-MYC are both involved in HSC self-renewal and
genetic ablation of these genes leads to reduced HSC function (Wilson et al., 2004;
Laurenti et al., 2008).The hox family of transcription factors also includes several
members with known HSC regulatory activity, most notably HOXA9 and HOXB4
(Brun et al., 2004). Control over the cell cycle is another main point of intrinsic HSC
regulation. Proliferating cells sequentially go through the different phases of the cell
cycle: growth and preparation of the chromosomes for replication (G1), DNA
synthesis (S), additional growth and preparation for cell division (G2) and mitosis
(M). In addition, cells can exit the cell cycle and stay quiescent in the G0 phase.
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20
Figure 2.1. The regulation of pluripotency. Stem cell self-renewal is maintained by the
Oct4-Sox2-Nanog transcriptional regulatory network, which forms a positive feedback loop
that negatively regulates the expression of differentiation promoting genes. Polycomb family
(PcG) proteins aid in this process by suppressing the expression of genes associated with
differentiation. Leukemia inhibitory factor (LIF) and bone morphogenetic protein (BMP)
signaling suppress differentiation by inhibiting MAPK pathway signaling, which is activated
by autocrine fibroblast growth factor (FGF) signaling. The figure is reproduced from the
review article of He et al., 2009.
Ex vivo expansion of HSCs
There are many reports on expansion of HSC on a larger scale without losing their
self-renewal ability for safe and transplantation free from feeder cells, serum proteins,
or microbial agents (Lu et al., 2010). In order to determine optimal conditions for in
vitro expansion of HSCs, researchers have adjusted permutation of various parameters
to increase the number of stem cells. It is known that stem cells in the bone marrow
are found in niches created by non-stem cells, and that these stem cells will remain
undifferentiated and appear immortal as long as they do not leave the niche.
Therefore, the signaling pathways occurring in this niche are important to understand
and examine. The manipulation of these signaling pathways for genes such as Notch,
HOX-B4, and Wnt (Aggarwal et al., 2010) have shown some positive results for ex
vivo expansion. Others have shown that over expression of genes, such as SALL4
(Aguila et al., 2011) have the capacity to substantially increase the number of
HSCs/HPCs in vitro. TAT-SALL4 fusion protein (SALL4 protein fused with the
TAT cell-penetrating peptide) has also been shown to rapidly expand CD34+CD38
-
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21
and CD34+CD38
+ cells ex vivo (Aguila et al., 2011). The transcription factor SALL4
is a member of the SALL gene family and has been reported to play an essential role
in maintaining pluripotency through interaction with Oct4 and Nanog (Yang et al.,
2010 & 2011). Other experimental trials have tried to expand HSCs/HPCs with aryl
hydrocarbon receptors, chelators, stromal coculture (Aguila et al., 2011). Notch
proteins are important for the survival, self-renewal, and lineage determination of
stem cells (Milner et al., 1994). There are four Notch receptors (Notch1, Notch2,
Notch3, and Notch4), five ligands (Jagged-1, Jagged-2, Delta-like-1, Delta-like-2,
Delta-like-3, and Delta-like-4), and several modifier proteins (Aggarwal et al., 2010)
that constitute the Notch signaling network in vertebrates. It has been demonstrated
that manipulating the signaling pathway for the Notch gene plays a role in HSC/HPC
growth and expansion. Several studies have found that this signaling network can
augment HSCs/HPCs in vitro and lead to a 100-fold increase in CD34+ precursors.
The homeobox gene family member HOXB4 has been shown to be a regulator of
hematopoietic differentiation (Jackson et al., 2012). It is currently the most
investigated transcription factor for its potential to increase the self-renewal properties
and expansion of HSCs. Human UCB CD34+ cells treated with HOXB4 fusion
proteins have resulted in a 2.5-fold increase in long-term repopulating cells compared
to uncultured controls (Jackson et al., 2012). Utilizing a more stable form of this
protein may prove to be one effective strategy for ex vivo expansion in the future
(Jackson et al., 2012). Wnt proteins are secreted morphogen that are essential for
basic developmental processes, such as progenitor-cell proliferation, cell-fate
specification, and the control of asymmetric cell division in various tissues (Bejsovec
et al., 2005). Wnt has been found to stimulate in vitro expansion of HSCs/HPCs (Ge
et al., 2010). Therefore, these findings may suggest a possible new venue for
investigating mechanisms of stem cell self-renewal and achieving clinically
significant expansion of human HSCs.
Neural plasticity of cord blood stem cells
The formation of the nervous system in vivo is an integrated series of complex
developmental steps, which initiates during embryogenesis but continues during
postnatal development. Previously, it was assumed that neurons are incapable of self-
repair and regeneration after a neuronal injuries or disorder. Recent reports and
discoveries proved that the some areas of brain possess a limited self-repair and
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22
regeneration capability due to the presence of neural stem cells (Basak and Taylor,
2008). The discoveries on these neural stem cells attracted the interest of scientists for
using such types of cells in nervous tissues repair and in the treatment of neuronal
disorders. In terms of specific neurological applications, it was demonstrated that
embryonic stem-like stem cells in cord blood could expand in culture condition
supplemented with thrombopoietin, flt-3 ligand, and c-kit ligand and differentiated
into neuronal cell exhibiting neural morphology and expression of neuronal markers
(GFAP, nestin, musashi-1, nectin, synaptophysin, GFAP, NMDA and GABA
receptors) (McGuckin et al., 2004; Haquea et al., 2012; Visana et al., 2012). Jang et
al. (2004) showed that purified CB CD133+ stem cells upon exposure to retinoic acid
differentiated into neuronal and glial cells that expressed neuronal markers (tubulin β-
III, NSE, NeuN, MAP2) and the astrocyte-specific marker GFAP. Further,
hematopoietic stem cells found in CB were studied extensively that could become
neural-like cells in culture (Kogler et al., 2004; Chen et al., 2005; Buzanska et. al.,
2006; Habich et al., 2006; Jurga et al., 2009; Cho et al., 2012). The multipotent non-
hematopoietic stem cells, isolated by culture in a serum free, growth factor
supplemented medium (SCF+FLT+FGF), were observed to express the stem cell
markers Oct-4 and Nanog, the early tissue developmental markers nestin, desmin,
GFAP and cfab1; and were capable of differentiating into bone, muscle, neural, blood
and endothelial cells after exposure to specialized differentiation media (Mitsui et al.,
2003; Takahashi and Yamanaka et al., 2006). Sanchez-Ramos et al. (2001) have
demonstrated that the culture of mononuclear fraction of HUCB in a proliferating
medium supplemented with all-trans-retinoic acid (RA) and nerve growth factor
(NGF) promoted the expression Musashi-1 and TUJ-1 and GFAP. Likewise, Ha et al.
(2001) have shown that HUCB cultured in β-mercaptoethanol differentiated into
neural phenotype as determined by expression of neural nuclear antigen (NeuN),
neurofilament and MAP2. In another study, a different HUCB cell fraction that is
positive for both CD34 and the leukocyte marker CD45 was isolated by Buzanska et
al. (2002) by means of magnetic cell sorting. These cells upon culture in DMEM and
hEGF started expressing nestin. Following exposure to retinoic acid and BDNF, these
cells were found immuno-responsive for TUJ1, MAP2, GFAP, and Gal-C
(galactocerebroside) oligodendrocyte marker. After culture of CB MSC, expressing
SH2, CD13, CD29 and ASMA, in neurogenic differentiation medium, both
immunofluorescence and RT-PCR analyses indicated elevated expression of Tuj1,
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23
TrkA, GFAP and CNPases neural markers. In order to move closer towards
regenerative medicine clinical applications, different populations of stem cells in
umbilical cord blood stem cells have been identified and have shown
electrophysiological properties similar to primary neurons (Jurga et al., 2009). Now,
the protocols have been well established for the differentiation of stem cells into
neural precursor cells (Lee et al., 2010) as well as several differentiated cells such as
motor neurons (Lee et al., 2007), glia cells (Lee et al., 2010), dopaminergic neurons
(Zeng et al., 2006; Iacovitti et al., 2007), and cerebellar cells (Erceg et al., 2010).
Being able to generate functional neurons from stem cells in-vitro is a very important
step towards moving into clinical and neural therapeutic applications as well as to
study the process of neuronal differentiation or developmental neurotoxicity.
Metabolomics to advance DNT testing
Metabolomics is the systematic study of the unique biochemical fingerprints that
cellular processes leave behind which provides mechanistic insight into cellular
physiology. This approach is a promising technique for getting data on toxicity
(Griffin and Bollard, 2004; Craig et al., 2006; van Vliet et al., 2008), disease
processes (Lindon et al., 2004), aging (Wang et al., 2007) and drug development
(Lindon et al., 2007). However, a recent study has applied metabolomics using an in
vitro approach for neurotoxicity evaluation with promising results (van Vliet et al.,
2008), which suggests that metabolomics could also be valuable tool for DNT testing.
Stem cell technology now came up with versatile applications in the field of
regenerative medicine, pharmacology and toxicology including pre-screening of
various environmental chemicals and drugs for possible developmental neurotoxicity.
To understand the cellular and molecular mechanism of neurotoxicity, it is first step to
understand the metabolic fate of environmental chemicals/ drugs reaching to the brain
and metabolic capability of brain cells. Thus, in the present study, we have chosen
xenobiotic metabolizing enzymes cytochrome P450s as potential endpoints to assess
developmental neurotoxicity and xenobiotic metabolic capability of developing
neuronal cells.
Xenobiotic metabolism
To understand why the CNS is so vulnerable to xenobiotic disturbance, it is necessary
to have some basic understanding of mechanism of xenobiotic metabolism during
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24
brain development. Animals have evolved with complex set of biochemical
machinery to facilitate their own metabolic processes and to defend their system from
foreign chemicals. During evolution they have encountered different array of
chemicals, which are of foreign origin and new for their biochemical machinery.
These foreign chemicals are termed as “xenobiotics” and defense machinery evolved
to metabolize them was termed as “xenobiotic metabolizing enzymes”. Adult humans,
growing children and developing fetus are being exposed to a multitude of
xenobiotics which come in contact by invading our ecosystem directly or through
mother. Most of these xenobiotics have high chemical stability and accumulates at
different levels in food chain through which humans are exposed.
Metabolism of foreign compounds to polar hydrophilic metabolites is an important
prerequisite for detoxification and elimination of xenobiotics from the body. In
general, it results in detoxification but in some cases, xenobiotics can be bio-activated
into reactive toxic intermediates that may cause toxicity (Katagi et al., 2010). Since,
the body is not familiar with the chemical natures of the great variety of possible
xenobiotics; it must have a wide range of different enzyme activities that can catalyze
a huge range of chemical reactions, including isoenzymes which can recognize
diverse chemical structures. Among them, cytochrome P450 enzymes (CYPs) play a
crucial role and constitute a superfamily of enzymes involved in the oxidative
metabolism of both endogenous and exogenous substrates. The major component of
human defense system consists of biotransformation enzymes of Phase I, Phase II,
Phase III pathways and various orphan nuclear receptors to regulate the induction of
xenobiotic metabolizing enzymes and transporters genes in response to environmental
chemicals and drugs (Rushmore and Kong, 2002; Wang and Le Cluyse, 2003; Xu et
al., 2005; Zhang et al., 2009). Before elimination from the body, xenobiotics undergo
biotransformation which involves two major pathways called Phase I or
functionalization or detoxification and Phase II or derivativization or conjugation
(Chin and Kong, 2002; Rushmore and Kong, 2002). Along with these two pathways,
recently third pathway known as Phase III has been recognized which consists of
xenobiotic transporters and is involved in the elimination of xenobiotics (Lee et al.,
2011; Aleksunes et al., 2012). A unique feature of few members of these pathways is
their increased expression upon exposure of xenobiotics (Xu et al., 2005). Increase in
expression of members of all these pathways is largely by the participation of
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25
multigene family of receptors which include several members of the steroid/nuclear
receptor superfamily viz., constitutive androstane receptor (CAR), pregnane-X-
receptor (PXR) (Aleksunes et al., 2012) as well as the aryl hydrocarbon receptor
(AHR), a highly conserved member of the basic-helix-loop-helix (bHLH)-Per-ARNT-
Sim (PAS) gene superfamily of transcription factors (Kewley et al., 2004; Aleksunes
et al., 2012; Chen et al., 2012).
The major enzymes involved in phase I metabolism are cytochrome P450 (CYP)
enzymes which is heme-thiolate containing proteins. Phase I reactions involves direct
enzyme mediated changes of molecules, like oxidation, reduction and hydrolytic
cleavages. With the help of reducing equivalents from NADPH or NADH, CYPs
catalyze monooxygenase reactions of lipophilic compounds and allow subsequent use
of the attached group as a reactive group for phase II enzymes. As a consequence of
this step, reactive molecules which may be more toxic than the parent molecule are
produced. The major phase II xenobiotic metabolizing enzymes are glutathione S-
transferase (GST), sulfotransferases, UPD-glycosyl transferases (UDPGT), N-acetyl
transferases (NAT) and various methyltransferases (Bock et al., 2011; Golka et al.,
2012). Phase II reactions consist mainly of glucuronidation, sulfation, and attachment
of glutathione, methylation, N-acetylation, or conjugation with amino acids. Phase II
reactions help in elimination of reactive intermediates of phase I reactions which if
not further metabolized by phase II reactions, may cause damage to proteins and other
tissue macromolecules within the cell (DuTeaux et al., 2003).
Cytochrome P450s (CYPs)
The human body has a remarkable system of enzymes for eliminating the myriad of
chemicals that it encounters. The enzymes involved in the biotransformation of
therapeutic drugs and other xenobiotics have received increasing attention over the
past decade since they play an important role in therapeutic drug response and
toxicity. CYPs are membrane bound heme-thiolate proteins which constitute
superfamily (Schleinkofer et al., 2005). Cytochrome P450 were named for their
spectral absorbance peak of their carbon-monoxide-bound species at 450 nm. They
are found in every class of organism, from archaea to mammals, and believed to have
originated from an ancestral gene that existed over 3 billion years ago (Danielson et
al., 2002). CYPs play important role in oxidative, peroxidative and reductive
metabolism of various endobiotics and xenobiotic compounds (Niwa et al., 2009;
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26
Amacher et al., 2010). The enzyme system takes lipid-soluble chemicals as substrates
and converts them to more water-soluble products by insertion of an oxygen atom into
the substrate molecule. Due to these properties they were called nature’s most
versatile biological catalyst (Coon et al., 2005). Based on amino acid homologies, the
CYP superfamily has been classified into several families and subfamilies. The CYP
proteins with 40% or greater sequence identity are included in the same family
(designated by Arabic number), and those with 55% or greater identity in the same
subfamily (designated by a capital letter). Half of the total CYPs are found in families
1-4, which are considered as xenobiotic metabolizing enzymes (Nelson, 2009;
Ferguson et al., 2011).
Cytochrome P450s in brain
In adults, nervous system is protected by the blood-brain barriers and in early
developing fetus by placental barrier which effectively retards the transfer of charged
and large molecular weight compounds from circulation to nervous tissue and vice-
versa (Sykova et al., 2008; Nielsen et al., 2011). However, these barriers do not
provide protection against lipid soluble agents and toxicants that damage blood-brain
barrier. Continuous exposure to lipophilic pesticides present in the food chain results
in their accumulation in brain. Many cytochrome P450 enzymes (CYPs) have tissue-
and cell type-specific expressions and regulations, and the brain expresses its own
unique complement of these enzymes (Miksys and Tyndale, 2004). Brain CYPs are
present in many different subcellular membrane compartments including plasma
membrane, endoplasmic reticulum, Golgi, and mitochondria (Marini et. al., 2007;
Seliskar and Rozman, 2007). Strobel et al. (1989) for the first time reported that the
treatment of rats with phenobarbital (PB), and tricyclic antidepressants markedly
increased the transcripts of brain CYP2B1. Using Reverse transcription-polymerase
chain reaction (RT-PCR), Hodgson et al., (1993) demonstrated expression of
CYP1A1, 2B2, 2D and 2E1 in rat brain. As CYPs are shown to be expressed not only
in brain but also in supporting endothelial cells and blood brain barrier, they could be
playing important role in metabolism of xenobiotics entering the brain (Decleves et
al., 2011; Ghosh et al., 2011). Bioactivation of xenobiotics in situ within the brain
could result in metabolites that cause damage to macromolecules in the brain cells
and/ or bind at different receptor sites. Similar to liver, classical inducers of hepatic
CYPs like PB, MC, ethanol, and phenytoin are also known to induce the expression of
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27
brain CYPs (Upadhya et al., 2002; Miksys and Tyndale, 2004; Johari et al, 2008).
Interestingly, many neuroactive drugs and solvents are metabolized by brain CYPs
and some of them also act as effective inducer of brain CYPs (Yadav et al., 2006;
Meyer et al., 2007 & 2010; Khokhar and Tyndale, 2011).
Like hepatic CYPs, brain CYPs are also expected to have role in metabolism of
xenobiotics and drugs reaching to brain, as brain microsomes have been shown to
metabolize same substrates which are used to assess specific CYP activity in liver
(Tyndale et al., 1999; Voirol et al., 2000; Parmar et al., 2003; Yadav et al., 2006;
Johari et al., 2008; Tiwari et al., 2010). Further studies have also shown that brain
CYPs are involved in metabolism of endogenous compounds like neurotransmitters,
hormones, vitamins, cholesterol (Liu et al., 2004), aromatization of androgens to
estrogens (Roselli et al., 2009) and neurosteroids synthesis (Azcoitia et al., 2011).
Many neurotransmitter and their precursors were found to modulates the activity of
CYPs (Gervasini et al., 2007). Moreover, studies have shown the involvement of
brain CYPs in metabolism of neurotransmitters like dopamine, noradrenalin, and
serotonin (Bromek et al., 2011). Studies of Ravindranath et al. (1989 & 1990) also
demonstrated CYPs in human and rat brain. Based on immunohistochemical,
immunoblotting and enzymatic studies, similar CYP 2B1/2B2 activity was
demonstrated in human brain and rat liver microsomes. Significantly high
immunoreactivity with anti- CYP2B1/2B2 and anti- CYP1A1/1A2 was reported in
male and female rat brain microsomes. Whereas, distinct sex related differences were
observed in the levels of total CYPs and monooxygenase activities, mediated by the
CYP2B1/2B2 isoenzymes, no sex differences were observed in the CYP enzymes
regulated by CYP1A1/1A2 isoenzymes. Anandatheerthavarada et al. (1990 & 1992)
purified P450 and NADPH CYP reductase to apparent homogeneity from the brain
microsomes of PB treated rats. The activity of these CYPs was reconstituted in vitro
and the immunological characterization of these multiple forms was demonstrated in
rat and human brain. Immunoblotting experiments further demonstrated the
constitutive presence of the PB regulated CYP isoenzymes (CYP2B1/2B2) in rodent
and human brain microsomes (Upadhya et al., 2002). Woodland et al. (2008)
demonstrated the expression, regulation and functional activity of CYP3A4 in rodent
and human brain. Thus, expression of CYPs in brain could contribute significantly to
metabolic capabilities of brain.
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Drug metabolism by CYPs in brain and therapeutic consequences
Metabolism of drugs by CYPs result in the formation of hydrophilic metabolites
easily excreted from the body in biological fluids. It is conceivable to hypothesize
that, if such metabolites are produced in the brain, it would result in the prolonged
presence and lower clearance from the brain. Cerebral metabolism could modulate the
pharmacological response and explain a part of the variability in response to centrally
psychoactive drugs, reflecting inter individual differences in localized brain CYP-
mediated metabolism (Britto et al., 1992). Indeed, in the case of neuroleptics and
antidepressants, poor correlations are often observed between plasma drug
concentrations and their therapeutic effects, suggesting the role of in situ metabolism
as possible modulator of drug response. Metabolism of several drugs, e.g.
debrisoquine, sparteine, dextromethorphan, although low, has been described on brain
microsomes, likely due to CYP2D6 activity (Jolivalt et al., 1995; Tyndale et al.,
1999). Since, CYP2D6 metabolizes a wide variety of centrally acting drugs such as
analgesics, anti-dementia drugs, beta-blockers, tricyclic antidepressants, anti-
psychotics, monoamine oxidase inhibitors and vasodilators (Zanger et al., 2004), it
could be of interest to better understand the capability of this isoform to participate in
drug metabolism in brain. Moreover, minor metabolic pathways of drugs could
potentially produce significant pharmacological or toxicological response, particularly
if they occur at the site of action. For example, a minor metabolic pathway for the
codeine results in the formation of morphine which is the O-demethylated metabolite
produced by CYP2D6 (Chen et al., 1990). The hypothesis of cerebral metabolism of
codeine is reinforced by the fact that codeine increases the pain threshold in extensive
metabolizers but not in poor metabolizers of CYP2D6 (Sindrup et al., 1992). Finally,
alternative splicing was proposed as a way to produce tissue-specific isoforms of
some CYPs (Turman et al., 2006). Indeed, Pai et al. (2004) showed that the CYP2D7
brain-specific protein, corresponding to a functional splice variant, metabolizes
codeine to morphine with greater efficiency than CYP2D6, suggesting the existence
of tissue-specific isoforms, particularly in brain, to mediate selective metabolism and
to generate active drugs (Pai et al, 2004). However, a more recent study did not
confirm the existence of such functional CYP2D7 enzyme in brain (Gaedigk et al.,
2005).
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Heterogeneous expression of CYPs in brain
However, the enzymes are not uniformly distributed among the different regions and
cells of the brain and sex-related differences have also been reported. Moreover, cyto-
architectonic organization and cell functions are extremely variable in brain and,
when compared cell to cell, the levels of CYPs in specific neurons can be as important
as in hepatocytes (Miksys et al., 2000). In general, the distribution of xenobiotic
metabolizing CYPs in the brain is heterogeneous, with expression levels varying
among different brain regions. Within a particular brain region, CYP expression is
usually restricted to specific populations of neuronal and/ or glial cells. In the frontal
cortex of the human brain, CYP2B6 is highly expressed in astrocytes surrounding
cerebral blood vessels in layer I, whereas CYP2D6 can be found predominantly in
pyramidal neurons in layers III-V and in white matter (Miksys et al., 2003; Howard et
al., 2003). In the cerebellum of human non-smokers, CYP2B6 and CYP2D6 are
expressed in neurons within the molecular and granular layers, but are undetectable in
Purkinje cells; however, in human smokers, CYP2B6 and CYP2D6 are highly
expressed in the Purkinje cells of the cerebellum (Miksys et al., 2002; Miksys et al.,
2003). The region and cell-specific expression of CYPs in the brain may provide
some insight into their functional significance and metabolic roles. For instance, the
high expression of CYP2B6 at the blood-brain interface may help to regulate the
penetration of drugs and toxins into the brain (Meyer et al., 2007). Dutheil et al.
(2009) have shown the transcript analysis of all 24 CYPs belonging to families 1
through 3 and CYP46A1, seven ABC transporters, and 14 transcription factor in
human whole brain, cerebellum, dura mater, and in a high number of other brain
regions. Further they showed the selective distribution of a large variety of xenobiotic
transporters and metabolizing enzymes (CYP1B1, CYP2D6, CYP2E1, CYP2J2,
CYP2U1, and CYP46A1) in different cerebral regions of human brain. Upon
induction, CYP2E1 has been reported to generate reactive oxygen species that
represent toxic molecules that have been implicated in the pathogenesis of
neurodegenerative disorders such as Parkinson’s disease. Furthermore, CYP2E1 could
modulate dopamine release and free radical production in agreement with its presence
in the substantia nigra. Total CYP levels in the brain are low, approximately 0.5–2%
of those in the liver (Hedlund et al., 2001). Although, the levels of CYPs in specific
neurons may be comparable to, or even higher than, levels in hepatocytes. For
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30
example, nicotine-induced CYP2B expression in neurons within rat frontal cortex
appeared to exceed levels found in hepatocytes under identical experimental
conditions (Miksys et al., 2009). Differential expression according to the cerebral
region has been described, with the highest CYP content in the brain stem and
cerebellum and the lowest values in the striatum and hippocampus, showing some
degree of similarity with rat brain (Tirumalai et al., 1998; Gilbert et al., 2003). The
CYP sub-cellular localization in brain is extremely diverse. High activity of CYPs has
also been demonstrated in endothelial cells of brain capillaries forming the blood-
brain barrier and in isolated brain microvessels (Ghosh et al., 2010). Localization of
CYPs in blood brain interfaces and in the cirumventricular organs have further
suggested that CYPs may form an enzymatic barrier to protect brain tissue from
harmful compounds (Dauchy et al., 2009). In brain, a major binding protein for
several inhibitors of dopamine transporters was identified as P450 isoenzyme, which
catalyzes 4-hydroxylation of debrisoquine (Hedenmalm et al., 2006).
It is now established that brain CYPs correspond to the functional enzymatic system.
To date, 41 of the 57 human CYPs have been identified in brain, and among them, 20
isoforms (CYP1A1, 1A2, 1B1, 2B6, 2C8, 2D6, 2E1, 3A4, 3A5, 8A1, 11A1, 11B1,
11B2, 17A1, 19A1, 21A2, 26A1, 26B1, 27B1 and 46A1) were found in several brain
localizations. Moreover, a limited number of CYP isoforms have been extensively
studied in human brain: CYP1A1, 1A2, 2B6, 2D6, 2E1 and 46Al; most of these are
largely distributed in brain regions (e.g. cortex, cerebellum, basal ganglia,
hippocampus, substantia nigra, medulla oblongata, pons), but data vary depending on
the reports. The discrepancies between the studies are likely due to the different
techniques used in order to measure the expression and activity of CYPs in brain. The
conflicting results between studies are partly explained by the problems of specificity
and sensitivity of primers or antibodies due to the high degree of sequence homology
between CYPs (Miksys et al., 2002). Brain regions differ in cell types, density and
function and the expression pattern of brain CYPs is also extremely variable (Strobel
et al., 2001). CYP1A1 was predominantly localized in neurons of cerebral cortex,
Purkinje cells and granule cell in dentate gyrus and pyramidal neurons of CA1, CA2
and CA3 subfields of hippocampus and reticular neurons in midbrain. CYP1A1,
CYP1A2, CYP2A6 and CYP2E1 were detected in the mitochondria of different brain
regions such as striatum, thalamus, pons and medulla oblongata (Bhagwat et al.,
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31
2000). CYP1B1 was strongly expressed in the nuclei of a majority of astrocytes and
neurons in human brain cortex (Muskhelishvili et al., 2001). CYP2B6 is also largely
distributed in brain but the basal level of expression is generally low (Miksys et al.,
2004). In the neocortical layer I, this enzyme is present in both neurons and glial cells,
including astrocytes surrounding cerebral blood vessels where this CYP co-localizes
with glial fibrillary acidic protein (Miksys et al., 2003). CYP2D6 is the most
extensively studied enzyme because initially it was found to be associated with
personality traits (Llerena et al., 1993) and in neurological disorders such as
Parkinson’s disease (Mann et al., 2012). Human CYP1A1, 1A2, 2B6 and 2D6,
CYP2E1 protein is found in a number of brain regions with a cell-specific manner.
Immunocytochemical localization revealed the preferential localization of CYP2E1 in
the neuronal cell bodies of specific brain regions such as hippocampus, cortex, basal
ganglia, hypothalamic nuclei and reticular nuclei in the brain stem (Howard et al.,
2003).
CYPs in cultured brain cells
In vivo studies for cell specific CYPs are largely hampered due to the non-availability
of pure homogenous cells of specific region of brain. Hence, tissue culture could help
as an alternative to address this issue. The use of cell cultures has proven to be a
powerful approach to study and elucidate the organ specific expression and activity of
CYPs and associated toxicity/ metabolism. The expression and inducibility of various
brain specific cytochrome P450s have been studied in variety of neuronal/ glial cell
lines of rat, mouse and human following the exposure of environmental chemicals and
drugs (Howard et al., 2003; Kapoor et al., 2006a, b & 2007; Gehlhaus et al., 2007;
Mann and Tyndale, 2010; Ande et al., 2012).
However, the cell lines used in such studies were either of tumor cell lines or
genetically transformed, hence not comparable to the live situations. So, to make the
system more identical towards to natural life situations, the use of primary cultures of
purified population of cells came into consideration.
Astroglial cells play a role in neurotoxicity associated with exposure to various
xenobiotics like phenytoin and also a protective role in the brain (Meyer et al., 2001).
The expression and inducibility of various CYPs have been documented in primary
cultures of rat brain neuronal and glial cells (Kapoor et al., 2006a, b). Studies of
Review of Literature
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Kapoor et al. (2006a, b & 2007) have shown constitutive and inducible expression
(mRNA and protein) and catalytic activity of CYP1A1/1A2, 2B1/2B2 and 2E1 in
primary cultures of rat brain neuronal and glial cells exposed to known inducers of
CYPs viz., 3-methylchlorantherene, phenobarbital and ethanol respectively. Both
neuronal and glial cells were found to have the significant express and activity for
CYP1A1, 2B2 and 2E1 with neurons exhibiting relatively higher levels. Greater
magnitude of induction for CYP2E1 was observed in neuronal cells, whereas glial
cells were found be more sensitive for CYP1A and 2B following known CYP
inducers. The induction of specific CYPs in glial cells is also of significance, as these
cells are thought to be involved in protecting the neurons from environmental insults
and safeguard them from toxicity. The differences in the induction of CYPs in
cultured neuronal and glial cells have indicated the sensitivity differences of these
CYPs, which may help to understand the regional specificity of brain. Kashyap et al.
(2010 & 2011) demonstrated the expression of CYPs in culture PC-12 cell and their
association to generation of ROS and LPO which played role in triggering of caspase
cascade regulated mitochondria mediated apoptosis following the exposure of MCP.
The activation of cytochrome P450s and their interaction with mitochondrial chain
complexes have been suggested in chemical induced apoptosis (Namazi et al., 2009;
Galluzzi et al., 2009). The involvement of CYPs in organophosphates-induced
apoptosis in neuronal cells has also been indicated (Kaur et al., 2007).
But such in vitro systems with primary cultures even have the limitations that they can
give the status of CYPs expression, activity and regulation only for adult brain and/or
adult terminally differentiated neuronal/ glial cells. However, there is a complete
lack of literature on the expression and inducibility of xenobiotic metabolizing CYPs
in the developing brain, because of non-availability of brain tissue at developmental
stages of fetus during gestational periods. Therefore, we have proposed to utilize the
plasticity and pluripotency potential of human cord blood derived hematopoietic stem
cells to understand the xenobiotic metabolizing capabilities in differentiating
neuronal cells at various maturity.