Reviewof

Literature

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6

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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19

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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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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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

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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.