1 23

NeuroMolecular Medicine ISSN 1535-1084 Neuromol MedDOI 10.1007/s12017-013-8243-0

3-Methylcholanthrene InducesNeurotoxicity in Developing NeuronsDerived from Human CD34+Thy1+ StemCells by Activation of Aryl HydrocarbonReceptorAbhishek K. Singh, MahendraP. Kashyap, Vivek Kumar, VinayK. Tripathi, Dharmendra K. Yadav,Firoz Khan, Sadaf Jahan, et al.

1 23

Your article is protected by copyright and all

rights are held exclusively by Springer Science

+Business Media New York. This e-offprint is

for personal use only and shall not be self-

archived in electronic repositories. If you wish

to self-archive your article, please use the

accepted manuscript version for posting on

your own website. You may further deposit

the accepted manuscript version in any

repository, provided it is only made publicly

available 12 months after official publication

or later and provided acknowledgement is

given to the original source of publication

and a link is inserted to the published article

on Springer's website. The link must be

accompanied by the following text: "The final

publication is available at link.springer.com”.

ORIGINAL PAPER

3-Methylcholanthrene Induces Neurotoxicity in DevelopingNeurons Derived from Human CD34+Thy1+ Stem Cellsby Activation of Aryl Hydrocarbon Receptor

Abhishek K. Singh • Mahendra P. Kashyap • Vivek Kumar •

Vinay K. Tripathi • Dharmendra K. Yadav • Firoz Khan •

Sadaf Jahan • Vinay K. Khanna • Sanjay Yadav • Aditya B. Pant

Received: 26 February 2013 / Accepted: 26 June 2013

� Springer Science+Business Media New York 2013

Abstract Developing neurons, derived from the human

umbilical cord blood stem cells (hUCBSCs), were investi-

gated for their stage-specific responses against 3-methyl-

cholanthrene (MC), a well-known polycyclic aromatic

hydrocarbon. Three-dimensional (3D) molecular docking

demonstrates the strong hydrogen bonding and hydrophobic

interactions of MC with amino acids of aryl hydrocarbon

receptor (AHR) and aryl hydrocarbon receptor nuclear

translocator (ARNT) within 4 A and subsequent inhibition of

cAMP response element-binding protein (CREB), a-amino-

3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)

and N-methyl-D-aspartate (NMDA) receptors. Protein–pro-

tein docking also confirms that induced levels of AHR inhibit

the neurogenesis-related transcription factor (CREB) with

maximum docking scores. In concurrence with in silico data,

MC exposure significantly up regulates the expression and

activity of AHR, CYP1A1 and glutathione S-transferase P1-

1 (GSTP1-1) and down regulates the expression of CREB,

AMPA and NMDA receptors in hUCBSC-derived neuronal

cells at various maturity (0, 2, 4, 8 days of differentiation).

MC-mediated significant down regulation in the expression

of stage-specific neuronal markers (Nestin, neural cell

adhesion molecule–NCAM, synaptophysin–SYP, CREB,

AMPA and N-methyl-D-aspartate receptor subunit 2A–

NR2A) was also noticed in cells all through the differentia-

tion. Data identify the possible interference of MC in neu-

ronal transmission and neurogenesis.

Keywords Aryl hydrocarbon receptor � Developmental

neurotoxicity � Human cord blood stem cells � Molecular

docking � 3-Methylcholanthrene

Introduction

It is reported that the developing brain is more vulnerable

to drugs and chemicals even at concentrations that are

usually safe to mature developed brain (Bal-Price et al.

2010). One among the reasons suggested is the non-avail-

ability of blood-brain barrier (BBB) in the developing brain

(Tilson 2000). In addition, placental barriers are also

insufficient to restrict maternal exposure to developing

fetuses (Bal-Price et al. 2010). Brain development is a

complex process that involves various strictly controlled

steps, viz. proliferation, differentiation and migration of

neuronal precursor cells, neuritogenesis, formation of

neural network, receptors and apoptosis. Environmental

chemicals may interfere at any step of brain development.

The chemicals shown to interact with brain tissues are

polycyclic aromatic hydrocarbons (PAHs), pesticides,

cigarette smoke, alcohol, etc. (Tayebati et al. 2009). Due to

lipophilicity, these chemicals can cross placenta and

immature BBB and hence affect the developing brain cells

(Liu et al. 2011; Nielsen et al. 2011). Any such injury to the

developing brain cells may lead the long-lasting disability

(Slotkin and Seidler 2010; Talge et al. 2007). The role of

A. K. Singh � M. P. Kashyap � V. Kumar �V. K. Tripathi � S. Jahan � V. K. Khanna � S. Yadav �A. B. Pant (&)

In Vitro Toxicology Laboratory, CSIR-Indian Institute

of Toxicology Research, Post Box: 80, MG Marg,

Lucknow 226001, India

e-mail: abpant@rediffmail.com; abpant@yahoo.com

D. K. Yadav � F. Khan

Metabolic & Structural Biology Division, CSIR-Central Institute

of Medicinal and Aromatic Plants, Lucknow 226015, India

123

Neuromol Med

DOI 10.1007/s12017-013-8243-0

Author's personal copy

cytochrome P450s (CYPs) is well reported in the metab-

olism of toxicants reaching the brain (Ferguson and Tyn-

dale 2011; Nebert and Russell 2002). However, the

reactive intermediates produced by CYPs have also been

reported to induce deleterious effects to brain cells by

binding to the receptors, ion channels and macromolecules

(Wells et al. 2010). CYPs are also reported to play a key

role in the metabolism of endogenous fatty acids, steroids,

hormones, cholesterols, vitamins and neurotransmitters

(Liu et al. 2004).

The fate of xenobiotics and their neurotoxic effects in

developing fetus brain has not been investigated in detail,

mainly due to the non-availability of developing human

fetal brain tissue and cells. Therefore, most of the mecha-

nistic studies have been carried out using mature and ter-

minally differentiated cultured cells of neuronal origin, and

for neurotoxicity assessment only (Grandjean and Landri-

gan 2006; Schreiber et al. 2010). Besides that, neural

progenitor cells have also been used to study the effect of

chemicals on proliferation (Breier et al. 2008). We, as

others, have used PC12 cells, a rat pheochromocytoma cell

line, in our investigation, to study the effect of these

chemicals on neurite outgrowth (Radio et al. 2008; Sidd-

iqui et al. 2008). Recently, we have been able to success-

fully demonstrate the complete neuronal differentiation of

human hematopoietic stem cells derived from umbilical

cord blood. We have also been able to confirm the xeno-

biotic metabolizing capabilities of these human stem cell-

derived neuronal cells at various stages of development

(Singh et al. 2012).

The present investigations were carried out to study the

applicability of developing neuronal cells derived from

human umbilical cord blood stem cells as a prescreening

tool to assess the drug- and chemical-induced develop-

mental neurotoxicity. 3-Methylcholanthrene (MC), an

environmental contaminant polycyclic aromatic hydrocar-

bon, is usually generated by the incomplete combustion of

organic compounds at high temperature. MC, a known

inducer of CYP1A1, was used as a test compound in the

study, as the exposure of human subjects to this chemical is

very common. Naruse et al., 2002 have already shown MC-

mediated prevention in the proliferation and differentiation

of osteoblasts under both in vitro and in vivo conditions.

The selection of MC for the study was also based on the

existing literature, which says that the exposure of PAHs to

developing brain in fetuses is quite common through

maternal cigarette smoking during pregnancy (Polanska

et al. 2009; Rodgman et al. 2000). It is also reported that

maternal cigarette smoking and consumption of alcohol

during pregnancy results in reduced intelligence quotient

(Batty et al. 2006), psychiatric disorders and behavioral

problems (Grandjean and Landrigan 2006; Thapar et al.

2003) in the offspring.

Materials and Methods

In Silico Studies

Protein Structure Homology Modeling of Aryl

Hydrocarbon Receptor (AHR)

The three-dimensional (3D) structure of cytochrome P450

(CYP) regulator receptor AHR can be experimentally

determined by radiographic (X-ray) crystallography or by

nuclear magnetic resonance (NMR) spectroscopy. As no

experimental structure is available in any protein data

bank, a homology model was developed by us. Homology

modeling represents a starting point for the biologist

involved in structure–function relationships studies. In the

homology modeling study, the distance geometry, simu-

lated annealing and energy minimization approaches were

used to build the 3D model of AHR protein by employing

GENO3D, an automatic web server for protein structure

modeling (http://geno3d-pbil.ibcp.fr). Query protein

sequence of AHR protein with length of 318 amino acids

was retrieved from GenBank database (NCBI, USA).

Homologous template protein sequences with known 3D

structure were identified through Basic Local Alignment

Search Tool (BLAST) program. Template-query protein

sequence validation was done by multiple sequence

alignment method through CLUSTALW, while the statis-

tical verification of generated model was evaluated by

PROCHECK, and model comparisons in three-dimensional

form were performed by RasMol-3D viewer. Finally, after

structural and energy minimization evaluation, Model_9

was identified as more accurate than others with maximum

homology to the known 3D structure. Though this com-

puter-generated molecular model cannot act as a substitute

for a crystal structure, it can support the investigation of the

structure–function relationships.

Molecular Modeling and Energy Minimization Parameters

The molecular modeling of compound, viz. MC, was per-

formed with CYP regulators AHR (aryl hydrocarbon

receptor), ARNT (aryl hydrocarbon receptor nuclear

translocator), neurogenesis-related transcription factor

CREB (cAMP response element-binding protein), and

neurotransmission receptors; AMPA receptor (a-amino-3-

hydroxy-5-methyl-4-isoxazolepropionic acid receptor) and

NMDA receptor (N-methyl-D-aspartate receptor) by using

Sybyl X 1.3 molecular modeling and drug discovery soft-

ware (www.tripos.com; Tripos International, USA). The

molecular docking, construction, geometry optimization

and energy minimization of MC were carried out using the

Sybyl X 1.3. All the molecules were initially designed in

Sybyl. However, the molecular construction, geometry

Neuromol Med

123

Author's personal copy

optimization and energy minimization process were done

by using HP XW4600 workstation configured with Intel

Core 2 Duo E8400 (3.2 GHz) processor and 6 GB of

RAM, running the Red Hat� Enterprise Linux 4.0 (32-bit

compatible) operating system (Silicon Graphics Inc.,

Mountain View, CA, USA). The Tripos force field with a

distance-dependent dielectric and Powell gradient algo-

rithm with a convergence criterion of 0.001 kcal mol-1

were used for optimization. Partial atomic charges were

assessed by using Gasteiger–Huckel method. Two-dimen-

sional structures were converted to 3D structures using the

program Concord v4.0, and the maximum number of iter-

ations performed in the minimization was set to 2000.

Further, the geometry optimization was done through

MOPAC-6 package using the semiempirical PM3 Hamil-

tonian method (Kalani et al. 2011).

3-Methylcholanthrene Protein Molecular Docking Studies

To find the possible bioactive conformations of MC-

interacting targets, the Sybyl X 1.3 interfaced with Surflex-

Dock program (Tripos International, USA) was operated to

dock the compound into the active site of AHR (model_9,

designed by us) and known targets, viz. ARNT (PDB ID:

2A24), CREB (PDB ID: 1DH3), NMDA receptor (PDB ID:

2A5S) and AMPA receptor (PDB ID: 3KG2). Program

automatically docks ligand into binding pocket of target

proteins by using protomol-based algorithm and empiri-

cally produced scoring function. The protomol is a very

important and necessary factor for docking algorithm and

works as a computational representation of the proposed

ligand that interacts with binding site. Surflex-Dock’s

scoring function has several factors that play an important

role in the ligand–receptor interaction, in terms of hydro-

phobic, polar, repulsive, entropic and solvation, which is a

worldwide well-established and recognized method. The

most standard docking protocols have ligand flexibility into

the docking process, while the protein is counted as a rigid

structure. In the present study, molecular docking involved

several steps, viz. the first step involved importing the

protein structure into Surflex and addition of hydrogen

atoms, and generating protomol using a ligand-based

strategy. During the second step, two parameters, first

called protomol_bloat (which determines how far the site

should extend from a potential ligand) and the second

called protomol_threshold (which determines deepness of

the atomic probes, used to define the protomol penetration

into the protein), were specified to form the appropriate

binding pocket. Therefore, protomol_bloat and proto-

mol_threshold were set to 0 and 0.50, respectively. In a

reasonable binding pocket, the compound was docked into

the binding pocket and 20 possible active docking con-

formations with different scores were obtained. During the

docking process, all of the other parameters were assigned

their default values. Surflex-Dock total scores, expressed in

log10 (Kd) units to represent binding affinities, were

applied to estimate the ligand–receptor interactions of

selected molecule (MC). Docking simulation-based muta-

tion studies were carried out to further confirm the binding

affinity of MC to these proteins. Two conserved amino

acids (lysine—Lys; and arginine—Arg) were found in all

the binding sites within 4A radii, and therefore, these two

residues were selected for mutation studies. Less conserved

residues within docked binding sites were replaced with

either of Lys or Arg, and docking was performed for each

mutant. Following the identical protocol, numbers of

mutational dockings were performed for each mutant.

Protein–Protein Molecular Docking Studies

Protein–protein docking was performed by ZDOCK v2.5

module of Discovery Studio (DS; Accelrys, USA) (Gabb

et al. 1997; Katchalski-Katzir et al. 1992). The terms

‘receptor’ and ‘ligand’ refer to the two input proteins, with

the receptor generally being the larger protein or known to

function as a receptor in vivo (e.g., an antibody in an

antigen–antibody interaction). ZDOCK docking score

contains pairwise shape complementarity (PSC) scoring

function of each docking pose. In the present study, Pose-1

showed the best and highest ZDOCK docking score and

was thus selected for further studies.

In vitro Validation Studies

Reagents and Consumables

All the chemicals, reagents and kits used in this study were

purchased from Stem Cell Technologies, Vancouver, BC,

Canada and Sigma St. Louis, USA, unless otherwise stated.

All cytokines and growth factors such as recombinant

human nerve growth factor [rhNGF], basic fibroblast

growth factor [rhbFGF], thrombopoietin [rhTPO], stem cell

factor [rhSCF] and fetal liver tyrosine kinase 3 ligand

[rhFLT-3 ligand] were purchased from Pepro-Tech (Rocky

Hill, NY). All the antibodies were procured from Chem-

icon International, USA, and Abcam, CA, USA. Culture

wares and plastic wares were procured from Nunc, Den-

mark, and Corning Incorporated, NY. Autoclaved Milli-Q

water was used in all the experiments.

Ethical Clearance for Collection and Transportation

of Human Tissues

The entire study was carried out by following the protocols

and procedures approved by Institutional Human Ethical

Committees of Indian Institute of Toxicology Research

Neuromol Med

123

Author's personal copy

(IITR), Lucknow, India, and CSM Medical University, Luc-

know, India. A written informed consent of parents was

obtained prior to collecting the blood from the umbilical cord.

Isolation and Purification of Human Cord Blood

Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) were purified from col-

lected human umbilical cord blood following the protocols

described earlier (Singh et al. 2012). In brief, 23 cord blood

samples (*40 ml/cord) were collected from the cord vein

in a sterile container having an anticoagulant citrate dex-

trose buffer and immediately transported to the Indian

Institute of Toxicology Research (IITR), Lucknow, India,

for further processing. Blood was diluted in the ratio of 1:1

with Dulbecco’s phosphate-buffered saline (DPBS) with-

out Ca2? and Mg2? at pH 7.5 (Stem Cell Technologies).

Subsequently, cord blood mononuclear cells were segre-

gated by negative immunodepletion of CD3?, CD14?,

CD19?, CD38?, CD66b? cells, followed by FicollpaqueTM

(1.077 g/cm3, Stem Cell Technologies) density gradient

centrifugation (7009g for 30 min). CD34? hematopoietic

stem cells were isolated from mononuclear (1 9 108 cells/

ml) cells using automated robotic and magnetic cell sepa-

rator RoboSepTM (Stem Cell Technologies, catalog no.

20000) and EasySepTM human cord blood CD34 positive

selection kit (Stem Cell Technologies, catalog no. 18056)

as per the manufacturer’s instructions.

Undifferentiated Proliferation and Culture

of Hematopoietic Stem Cells

Freshly isolated hematopoietic stem cells were cultured in a

plastic 25-cm2 ultralow-attachment culture flask (Corning

Incorporated, USA) at a density of 1 9 105 cells/ml in serum

containing myelocult medium (Stem Cell Technologies

#H5150) supplemented with hydrocortisone (10-6M) and

other growth factors, viz. rhbFGF (50 ng/ml), rhSCF (25 ng/

ml), rhTPO (25 ng/ml) and rhFLT-3 ligand (10 ng/ml)

(PeproTech, Rocky Hill, NY). Cells were maintained in a

suspension in a humidified atmosphere at 37 �C and 5 %

carbon dioxide (CO2) (Singh et al. 2012). Half of the medium

was changed twice in a week. Cells were subcultured at the

confluence of 80–90 % in a myelocult medium supple-

mented with hydrocortisone and other growth factors.

Characterization of Purified Population of Hematopoietic

Stem Cells

Following the purification, HSCs were checked for CD34?

and Thy1? markers by flow cytometry to ascertain the

purity of stem cells and colony-forming unit (CFU) assay

to establish lineage-specific plasticity potential.

(a) Flow cytometry: Purity of HSCs was analyzed by

phenotyping of HSC cell surface markers CD34? and

Thy1? using flow cytometry. In brief, stem cells

(1 9 105) were centrifuged at 5009g for 10 min and

then stained with fluorescein isothiocyanate (FITC)-

conjugated anti-CD34 and PE-Texas Red-conjugated

anti-Thy1 antibodies (Stem Cell Technologies) in

dark for 30 min at 4 �C. The cells were then washed

three times with DPBS and analyzed using flow

cytometry (BD FACS Canto II) equipped with BD

FACS Diva, version 6.1.2 software. The data are

presented in percent population of stem cells.

(b) Colony-forming unit assay: HSCs (1 9 106) was

suspended in 0.5 ml myelocult medium and gently

mixed with a 3.5 ml of semisolid methylcellulose

culture medium (MethoCultTM Stem Cell Technolo-

gies; catalog no. 03434), which was supplemented with

recombinant colony-stimulating factors, viz. erythro-

poietin (EPO) and granulocyte–monocyte colony-

stimulating factors (GM-CSF); 1 ml of the mixed cells

was transferred gently into small gridded petri dishes.

The dishes were incubated at 37 �C (95 % oxygen–O2

and 5 % CO2) for 14 days. The CFU were classified

and enumerated based on the morphological recogni-

tion of one or more types of hematopoietic lineage cells

within the colony in situ by inverted light microscopy.

Differentiation of Hematopoietic Stem Cells into Neuronal

Subtype

Hematopoietic stem cells were differentiated into neuronal

subtypes following the protocols described earlier by us

(Singh et al. 2012). In brief, confluent growing cells were

subcultured as per the experimental demand and allowed to

adhere in SynthemaxTM 6-well culture plate and 75-cm2

culture flask (Corning Incorporated, NY). The cells were

then induced to differentiate into neuronal subtype in

serum-free neurobasal medium (Gibco-BRL, USA) sup-

plemented with N2, B27 supplements, rhNGF (50 ng/ml),

rhFGF-basic (50 ng/ml), rhTPO (25 ng/ml), rhSCF (25 ng/

ml) and retinoic acid (10-5M) for a period of up to 8 days.

Differentiation medium was changed every alternate day.

Differentiating cells were observed for morphological

changes and expression (mRNA and protein) of markers of

neuronal development at various points of maturity.

Identification of Non-cytotoxic Doses

of 3-Methylcholanthrene

Prior to the experiments, non-cytotoxic dose of MC

was ascertained using standard endpoint of cytotox-

icity, that is. MTT (3-(4, 5-Dimethylthiazol-2-yl)-2,

Neuromol Med

123

Author's personal copy

5-diphenyltetrazolium bromide) assay (Kashyap et al.

2010). Cytotoxicity analysis was done by exposing the

cells at days 0, 2, 4 and 8 of differentiation. Cells (1 9 104

cells/well) at various stages of maturity (days 0, 2, 4 and 8)

were incubated with a medium containing different con-

centrations of MC (1–10 lM) for 6–72 h and MTT assay

was done. Tetrazolium bromide salt (10 ll/well; 5 mg/ml

of stock in PBS) was added 4 h prior to the completion of

respective incubation periods. Then, the reaction mixture

was carefully taken out and 200 ll of culture grade

dimethylsulphoxide (DMSO) was added to each well by

pipetting up and down several times, until the content was

homogenized. After 10 min, the color was read at 550 nm

using multiwell microplate reader (Synergy HT, Bio-Tek,

USA). The unexposed sets were also run simultaneously

under identical conditions, which served as control.

Exposure of Differentiating Neuronal Cells

to 3-Methylcholanthrene

Cells at various maturities of days 0, 2, 4 and 8 were exposed to

MC (4 lM) for a period of 12 and 24 h for mRNA and protein

expression studies, respectively. The unexposed control sets

were run under identical conditions at every point of matu-

ration. Upon the completion of respective exposures, cells

were harvested and processed for transcriptional, translational

expression and enzymatic activity studies.

RNA Extraction and Real-Time PCR (RT-PCRq)

Transcriptional changes in marker genes associated with

stemness, neuronal development and xenobiotic metabolism

were studied in the experimental and control groups by real-

time PCR using SYBR Green dye. Total RNA was isolated

using Gene Elute mammalian total RNA Miniprep Kit

(Sigma, USA; catalog no. RTN-70). Total RNA (1 lg) was

reverse transcribed into cDNA by SuperScript III first-strand

cDNA synthesis kit (Invitrogen Life Science, USA; catalog

no. 18080-051). Sequences of different primers are given in

Table 1. The specificity of each primer was assessed by

melting curve analysis and no template controls for respective

primers. Quantitative PCR reactions were then carried out

using the ABI PRISM 7900HT Sequence Detection System

(Applied Biosystems, USA). Real-time reactions were carried

out in triplicate wells for each sample. b-actin was used as an

internal control to normalize the data. Exposure-induced

alterations are expressed in relative quantification.

Western Blot Analysis for Markers of Stemness, Neuronal

Cells and Xenobiotic Metabolism

The altered expression of marker proteins of stemness (CD34,

THY1, CD133 and NANOG), neuronal cells (NES, PSA-

NCAM, SYP, CREB, AMPA receptor and NR2A receptor) and

xenobiotic metabolism (AHR, CYP1A1 and GSTP1-1) was

studied in differentiating cells at days 2, 4 and 8 of maturation.

Proteins harvested from different experimental and control

groups were processed for western blot analysis following the

protocol described earlier by us (Kashyap et al. 2010). In brief,

following exposures, cells were scraped, pelleted and lysed

using CelLyticTM M Cell Lysis Reagent (Sigma St. Louis,

USA) in the presence of protein inhibitor cocktail (Sigma St.

Louis, USA). After protein estimation by Bradford’s Reagent

(Fermentas Inc., Maryland, USA), equal amount (40 lg/well)

of denatured proteins was loaded onto 10 % SDS gel and

blotted to polyvinylidene fluoride (PVDF) membrane (Milli-

pore, USA) by wet transfer method using transfer buffer

(25 mM Tris (pH 8.3), 190 mM glycine, 20 % methanol) at

Table 1 Real-time PCR primer sequence for genes of human stem cell markers, cytochrome P450s, their receptor regulators and GSTP1-1

Genes Forward primer Reverse primer Reference

CD34 50-gccctgctggctgtcttg-30 50-gctgcggcgattcatca-30 Primer express 3.0

THY 50-tcaggaaatggcttttccca-30 50-tcctcaatgagatgccataagct-30 (True et al. 2010)

CD133 50-cgtgattttttactacctgg-30 50-gggtggcatgcctgtcatag-30 Primer express 3.0

NANOG 50-aaatctaagaggtggcagaa-30 50-cttctgcgtcacaccattgc-30 Primer express 3.0

NESTIN 50-gaactctcgcttgcagacacct-30 50-tgctggtcctctggtatcccaa-30 (Scobioala et al. 2008)

NCAM 50-gagatcagcgttggagagtcc-30 50-ggagaaccaggagatgtctttatctt-30 (Valentiner et al. 2011)

SYP 50-actcctcgtcagccgaattct-30 50-gcccccatggagtagaggaa-30 Primer express 3.0

CREB 50-ttcagtctccacaagtccaaaca-30 50-cccttcgcttttgggaatc-30 Primer express 3.0

GRIA2 50-tggcatcgcaacacctaaag-30 50-ccttgctcactgagtttcaatactg-30 Primer express 3.0

GRIN2A 50-cacggagagaaacattcggaata-30 50-agactgcggcatcgtagatga-30 Primer express 3.0

AHR 50-cggctgggcaccatga-30 50-ctgggattggctttactgttttct-30 Primer express 3.0

CYP1A1 50-accttccgacactcttccttcg30 50-aagcggatttgtcttggtgaa-30 (Shao et al. 2007)

GSTP1-1 50-gtagtttgcccaaggtcaag-30 50-agccacctgaggggtaag-30 Primer express 3.0

b-actin 50-aaccccaaggccaaccg-30 50-agggatagcacagcctgga-30 (Shao et al. 2007)

Neuromol Med

123

Author's personal copy

250 mA current for 2 h. Nonspecific binding was blocked with

2 % BSA and 3 % non-fat dry milk powder in TBST (20 mM

Tris–HCl (pH 7.4), 137 mM NaCl and 0.1 % Tween 20) for 2 h

at 37 �C. After blocking, the membranes were incubated

overnight at 4 �C with primary antibodies specific for AHR

(1:1000, Abcam, USA), CYP1A1, NESTIN, SYP, PSA-

NCAM, CREB, AMPA, NR2A (1:1000, Chemicon, USA),

GSTP1-1 (1:1000, Calbiochem, USA), b-actin (1:5000, Sigma,

USA) in blocking buffer (pH 7.5). The membrane was then

incubated for 2 h at room temperature with secondary anti-

primary immunoglobulin G (IgG)-conjugated with horseradish

peroxidase (Chemicon, USA). Then, the blots were developed

using Super Signal West Fempto Chemiluminescent Sub-

strateTM (Thermo Fisher Scientific, USA) and Bio Rad Versa

DocTM Imaging System 4000 (Bio-Rad, PA, USA). The den-

sitometry for protein-specific bands was done in Gel Docu-

mentation System (Alpha Innotech, USA) with the help of

Alpha EaseTM FC Stand Alone v.4.0 software.

7-Ethoxyresorufin-O-Deethylase (EROD) for CYP1A1

Catalytic Activity

Microsome Preparation

Following the completions of exposure period (24 h), cells

were harvested from MC exposed and control groups and

processed for microsome preparation following the protocol

described earlier by us (Kapoor et al. 2006). In brief, cells

were scrapped in PBS at 4 �C and pelleted by centrifuging at

5009g for 10 min. The cell pellet was resuspended in

microsomal dilution buffer containing 0.1 % (v/v) glycerol,

0.25 mM protease inhibitors cocktail, 0.01 M EDTA and

0.1 mM dithiothreitol. The cells were then sonicated thrice

at 20 Hz for 10 s each. Following sonication, the cells were

again centrifuged at 9,0009g for 20 min. The supernatant

was then further centrifuged at 105,0009g for 60 min, to

isolate the microsomal fraction. The microsomal pellet thus

obtained was then resuspended in microsomal dilution buf-

fer and protein estimation was done by Bradford’s Reagent

(Fermentas Inc., Maryland, USA).

Enzymatic Activity of CYP1A1

CYP content in microsomes of differentiating neuronal cells

(days 0, 2, 4 and 8), exposed to MC described earlier along

with unexposed control, were assayed by the carbon mon-

oxide difference spectrum after reduction with sodium

dithionite. The activity of 7-ethoxyresorufin-O-deethylase

(EROD) was determined in microsomes by the methods of

Kapoor et al. 2006. The reaction mixture in 1.25 ml con-

tained 50 mM Tris buffer, pH 7.5, 25 mM MgC12,

0.005 mM ethoxyresorufin and suitable amount of micro-

somal enzyme. The reaction was initiated by the addition of

0.5 mM nicotinamide adenine dinucleotide phosphate-oxi-

dase (NADPH) following incubation of reaction mixture at

37 �C for 30 min. After incubation, reactions were termi-

nated by adding 2 ml of methanol and the mixtures were

centrifuged at 2,0009g for 10 min. Levels of resorufin in the

supernatant were measured using a Perkin Elmer LS 55

Luminescence spectrometer at excitation wavelength of

550 nm and emission wavelength of 585 nm.

Suppression of AHR Gene Expression in Stem Cells

by siRNA Transfection

Human cord blood stem cell–derived differentiating neu-

ronal cells (days 2, 4 and 8) were transfected with 100 nM

siRNA using LipofectamineTM 2000 (Invitrogen, USA)

following the manufacturer’s instructions. Transfection

efficiency was recorded more than 60 % through all the

experiments as determined by counting the number of

siGLO Lamin A/C (Dharmacon, USA) positive cells using

fluorescent microscope (Nikon Eclipse 80i equipped with

Nikon DS-Ril 12.7-megapixel camera, Japan). Following

24 h of transfection, differentiating neuronal cells were

exposed to 4 lM of MC. After the completion of exposure

period, the expression (mRNA/protein) of AHR and

selected neuronal markers, viz. nestin, SYP, PSA-NCAM,

CREB, AMPA receptor and NR2A receptor, was measured

all through the differentiation. siRNAs specific to AHR,

control siRNA and siGLO Lamin A/C siRNA were

obtained as siGENOME SMARTpool reagent (Dharmacon,

USA), which contained a pool of four different siRNAs.

Further, the specificity of siRNA was assessed, and it was

ensured that it did not suppress irrelevant genes like b-actin

and suppressed the expression of the relevant gene—AHR.

Statistical Analysis

The results are expressed as mean and standard error of

means (Mean ± SE) for at least three experiments. One-

way analysis of variance (ANOVA) followed by post hoc

Dunnett’s test and Student’s t-test were employed to detect

differences between the groups of treated and control.

Graphpad Prism 3.0 software (Graphpad Software) was

used for all the statistical analyses. The value p \ 0.05 was

considered to be significant.

Results

In Silico Molecular Docking Analysis

of 3-Methylcholanthrene

The aim of the molecular docking study was to elucidate

the possible mechanisms of action of test compound MC in

Neuromol Med

123

Author's personal copy

modulation of the human CYP regulators, AHR and

ARNT; neurogenesis-related transcription factor CREB;

and neurotransmitter receptor, NMDA and AMPA recep-

tors. The results of the molecular docking suggest that

studied compound modulates the activity of these recep-

tors. In the present study, we explored the orientation and

binding affinity (in terms of the total docking score) of MC

toward the selected targets (Fig. 1 and Table 2).

Homology modeling approaches were used to predict

the structure of AHR which was used as a template on the

basis of highest sequence identity. To check the authen-

ticity of predicted 3D structure, models were subjected to

Ramachandran plot analysis. The model_9 was selected

based on highest number of residues present in the allowed

region of Ramachandran plot. The docking results for MC

against AHR (through homology modeling) also showed

high binding affinity docking score indicated by total score

of 4.1343. In docking pose, the chemical nature of binding

site residues within a radius of 4A from bound compound

was basic (polar, hydrophobic and positive charged), that

is, LEU-19, LEU-20, LEU-190, PRO-226 (leucine), PRO-

21, PRO-146 (proline), ARG-191 (arginine); nucleophilic

(polar, hydrophobic), that is, SER-147, SER-152 (serine),

CYS-192 (cysteine); aromatic (hydrophobic), that is, PHE-

22 (phenylalanine); and polar amide, that is, ASN-145

(asparagine); thus, bound compound showed high binding

affinity and strong hydrophobic interaction which may lead

to more stability and activity (Fig. 1a).

The docking results for MC against ARNT (PDB ID:

2A24) showed a high binding affinity docking score indicated

by a total score of 2.419. However, the docking score for

ARNT is lower than that of AHR confirming the lesser binding

of MC to ARNT. In the docking pose, the chemical nature of

binding site residues within a radius of 4A from bound com-

pound was nucleophilic (polar, hydrophobic), that is, THR-85

(threonine); basic (polar, hydrophobic and positive charged),

that is, HIS-12 and HIS-57 (histidine); hydrophobic, that is,

MET-16, MET-53, MET-73 (Methionine), GLY-87 (gly-

cine), ALA-41 (alanine), ILE-101 (isoleucine); aromatic

(hydrophobic), that is, TYR-45, TYR-71 (tyrosine), PHE-8,

PHE-18 (phenylalanine); nucleophilic (polar, hydrophobic),

that is, SER-10, SER-68 (serine), CYS-103 (cysteine); and

polar amide, that is, GLN-86 (glutamine), ASN-105 (aspara-

gine); thus, bound compound showed high binding affinity

and strong hydrophobic interaction which may lead to more

stability and activity (Fig. 1b).

Similarly, the docking results for MC against neuro-

genesis-related transcription factor CREB (PDB ID: 1DH3)

showed high binding affinity docking score indicated by

total score of 5.5392. In docking pose, the chemical nature

of binding site residues within a radius of 4 A from bound

compound were basic (polar, hydrophobic and positive

charged), that is, ARG301 (arginine), LYS-304, LYS-305

(lysine); nucleophilic (polar, hydrophobic), that is, SER-

300 (serine); thus, bound compound showed high binding

affinity and indicates strong hydrophobic interaction which

may lead to more stability and activity (Fig. 1c). The

docking of adenosine-50-triphosphate (ATP) onto CREB

showed high binding affinity with total docking score of

4.5622 and also showed the formation of a three H-bond of

length 2.9, 2.6 and 2.3 A to the hydrophobic nucleophilic

(polar, hydrophobic and positive charged) residues, that is,

ARG-301, LYS-304 and LYS-305. The ATP-CREB pro-

tein docked complex also showed binding site residues

within a radius of 4A of bound ligand such as basic (polar,

hydrophobic and positive charged) residues, for example,

ARG-301 (arginine), LYS-304, LYS-305 (lysine) with

strong and stable hydrophobic interactions (Fig. 1d). Sur-

prisingly, MC share the same residue of amino acids in

binding pocket as in the case of ATP with higher total

docking score of 5.5392. Thus, MC showed competitive

binding to CREB and possibly interfered with the phos-

phorylation of CREB protein which is necessary for its

functionality.

Similarly, the docking results for MC against neuro-

transmitter NMDA receptor (PDB ID: 2A5S) showed high

binding affinity docking score indicated by total score of

2.6179. In docking pose, the chemical nature of binding

site residues within a radius of 4A from bound compound

was basic (polar, hydrophobic and positive charged), that

is, HIS-88 (histidine), LYS-87, LYS-222 (lysine); hydro-

phobic, that is, ALA-17 (alanine), ILE-21 (isoleucine),

LEU-14 (leucine), VAL-197 (valine); aromatic (hydro-

phobic), that is, TYR-214 (tyrosine); and acidic (polar,

negative charged), that is, GLU-15, GLU-16, GLU-198

(Glutamic acid); thus, bound compound showed high

binding affinity and strong hydrophobic interaction which

may lead to more stability and activity (Fig. 1e).

Likewise, the docking results for MC against neuro-

transmitter AMPA receptor (PDB ID: 3KG2) showed high

binding affinity docking score indicated by total score of

5.2795. In docking pose, the chemical nature of binding site

residues within a radius of 4A from bound compound was

acidic (polar, negative charged), that is, GLU-402, GLU-705

(Glutamic acid); nucleophilic (polar, hydrophobic), that is,

THR-707 (threonine); basic (polar, hydrophobic and posi-

tive charged), that is, ARG-485 (arginine); hydrophobic, that

is, MET-708 (Methionine), GLY-451, GLY-653 (glycine),

LEU-479, LEU-650 (leucine), PRO-478 (proline); aromatic

(hydrophobic), that is, TYR-405, TYR-450, TYR-732

(tyrosine); and nucleophilic (polar, hydrophobic), that is,

SER-403, SER-654 (serine); thus, bound compound showed

high binding affinity and strong hydrophobic interaction

which may lead to more stability and activity (Fig. 1f). The

docking reliability between MC and AMPA receptor was

validated by using the known crystallized X-ray structure of

Neuromol Med

123

Author's personal copy

Fig. 1 In silico molecular docking studies elucidating the possible

mechanisms of 3-methylcholanthrene (MC)-induced modulation of

CYP regulators AHR (aryl hydrocarbon receptor), ARNT (aryl

hydrocarbon receptor nuclear translocator), neurogenesis-related

transcription factor CREB (cAMP response element-binding protein)

and neurotransmission receptors AMPA receptor (a-amino-3-

hydroxy-5-methyl-4-isoxazolepropionic acid receptor) and NMDA

receptor (N-methyl-D-aspartate receptor). The docking studies were

carried out using Sybyl X 1.3 molecular modeling and drug discovery

software (www.tripos.com; Tripos International, USA). a MC docked

on to homology modeled AHR (Model_9) with high binding affinity,

as indicated by a total docking score of 4.1343. b MC docked on to

ARNT (PDB ID: 2A24) with high binding affinity, as indicated by a

total docking score of 2.419. c MC docked on to CREB (PDB ID:

1DH3) with high binding affinity, as indicated by a total docking

score of 5.5392. d ATP docked on to CREB (PDB ID: 1DH3) with

high binding affinity, as indicated by a total docking score of 4.5622

compared. e MC docked on to NMDA (NR2A) receptor (PDB ID:

2A5S) with high binding affinity, as indicated by a total docking score

of 2.6179. f MC docked on to AMPA receptor (PDB ID: 3KG2) with

high binding affinity, as indicated by a total docking score of 5.2795

Neuromol Med

123

Author's personal copy

AMPA receptor complexed with {[7-morpholin-4-yl-2,

3-dioxo-6- (trifluoromethyl)-3,4-dihydroquinoxalin-1-

(2H)-yl] methyl} phosphonic acid inhibitor. The co-crys-

tallized inhibitor was re-docked into the binding site of

AMPA receptor with total docking score of 12.7636 and was

selected as the most probable binding conformation. The low

root mean-square deviation (RMSD) of 0.6798 A between

the docked and the crystal conformations indicates the high

reliability of Surflex-Dock software in reproducing the

experimentally observed binding mode for this inhibitor. As

shown in Fig. 2a, redocked molecules were almost in the

same position with co-crystallized at the active site of

inhibitor. This redocking study was carried out to confirm the

proper functionality of our system which served as a positive

control.

Protein–Protein Molecular Interaction

The docking result for AHR (model_9; designed by us)

against neurogenesis transcription factor CREB (PDB ID:

1DH3) showed a high binding affinity docking score

indicated by a ZDOCK score of 25.48 (Fig. 2b). In the

docking pose, the chemical nature of binding site residues

within a radius of 6A from bound surface was hydrophobic

due to glycine (GLY-113, GLY-357), valine (VAL-119,

VAL-166, VAL-167), leucine (LEU-127, LEU-181), iso-

leucine (ILE-128, ILE-174), alanine (ALA-134) and pro-

line (PRO-171, PRO-175, PRO-176, PRO-180). This

binding site also showed hydrophobic chemical nature due

to aromatic residues, viz. phenylalanine (PHE-114, PHE-

136) and tyrosine (TYR-125, TYR-169). The binding site

Table 2 Comparison of docking score and binding site residues of

3-methylcholanthrene (MC) docked on human CYP regulators AHR

and ARNT, neurogenesis transcription factor CREB, neurotransmitter

receptors AMPA and NMDA (NR2A) receptors; protein–protein

docking between homology model of AHR docked on CREB

Compound-protein docking results (Sybyl X 1.3-Tripos International, USA)

S.

No

Compound

(ligand)

Protein (Target receptor) Target

protein and

PDB ID

Total score

(docking score-

Surflex-Dock)

Biding site residues (within 4A)

1. MC Aryl hydrocarbon receptor

(AHR)

Homology

Model_9

4.1343 LEU-19, LEU-20, PRO-21, PHE-22, ASN-145, PRO-

146, SER-147, SER-152, LEU-190, ARG-191, CYS-

192, PRO-226

2. MC Aryl hydrocarbon receptor

nuclear trans locator (ARNT)

2A24 2.419 PHE-8, SER-10, HIS-12, MET-16, PHE-18, ALA-41,

TYR-45, MET-53, MET-73, HIS-57, SER-68, TYR-

71, THR-85, GLN-86, GLY-87, ILE-101, CYS-103,

ASN-105

3. MC cAMP response element-binding

protein (CREB)

1DH3 5.5392 SER-300, ARG301, LYS-304, LYS-305

4. ATP cAMP response element-binding

protein (CREB)

1DH3 4.5622 ARG-301, LYS-304, LYS-305 (number of hydrogen

bond were 3 with length of 2.9, 2.6 and 2.3A)

5. MC N-methyl-D-aspartate receptor

(NMDA)

2A5S 2.6179 LEU-14, GLU-15, GLU-16, ALA-17, ILE-21, LYS-87,

HIS-88, VAL-197, GLU-198, TYR-214, LYS-222

6. MC a-amino-3-hydroxy-5-methyl-4-

isoxazolepropionic acid

receptor (AMPA receptor)

3KG2 5.2795 GLU-402, SER-403, TYR-405, TYR-450, GLY-451,

PRO-478, ARG-485, LEU-479, LEU-650, GLY-653,

SER-654, GLU-705, THR-707, MET-708, TYR-732

Protein–protein docking result (ZDOCK-Accelrys, USA)

S. No. Peptide

(ligand)

Protein (target receptor) Target

protein

PDB ID

Docking

score

Biding site residues (within 6A)

7. AHR cAMP response element-

binding protein (CREB)

1DH3 25.48 GLN-109, GLY-113, PHE-114, GLN-115, ASP-118,

VAL-119, SER-123, TYR-125, GLU-126, LEU-127,

ILE-128, HIS-129, THR-130, GLU-131, ASP-132,

ARG-133, ALA-134, GLU-135, PHE-136, THR-165,

VAL-166, VAL-167, CYS-168, TYR-169, PRO-171,

ASP-172, GLN-173, ILE-174, PRO-175, PRO-176,

GLU-177, ASN-178, SER-179, PRO-180, LEU-181,

MET-182, GLU-183, ASP-354, GLU-355, GLY-357,

THR-358, GLU-359, HIS-360

DC10, DT9, DT8, DG7, DG6, DC5, DT4, DG3, DA2,

DC1 and DA2, DC1, DG1, DT2, DC3, DA4, DG5,

DC6, DC7, DA8, DA9, DG10

Neuromol Med

123

Author's personal copy

also showed nucleophilic chemical nature due to polar

hydrophobic residues, viz. threonine (THR-130, THR-

165), cysteine (CYS-168) and methionine (MET-182). The

binding site also showed basic polar hydrophobic chemical

nature due to positive-charged residues such as arginine

(ARG-133) and histidine (HIS-129, HIS-360). The binding

site also showed polar amide chemical nature due to glu-

tamine (GLN-109, GLN-115, GLN-173), asparagines

(ASN-178, ASP-118, ASP-132, ASP-172, ASP-354) and

glutamic acid (GLU-126, GLU-131, GLU-135, GLU-177,

GLU-183, GLU-355, GLU-359) amino acid residues. The

binding site also showed hydroxylic chemical nature due to

serine (SER-123, SER-179), amino acid residues. More-

over, DNA nucleotide residues, viz. DC10, DT9, DT8,

DG7, DG6, DC5, DT4, DG3, DA2, DC1 and DA2, DC1,

DG1, DT2, DC3, DA4, DG5, DC6, DC7, DA8, DA9,

DG10, were also present at DNA-binding domain in the

vicinity of bound peptide and showed the DNA-binding

site of target protein. Therefore, bound peptide showed a

high binding affinity and strong hydrophobic interaction on

target protein surface which may lead to more stability and

biological activity (Fig. 2b).

Mutation Docking Analysis of 3-Methylcholanthrene

In order to confirm the binding affinity of MC with down-

stream target proteins, docking simulation-based mutation

studies were carried out. In the mutation studies, it was

observed that all mutants were having low binding affinity

with MC, except the N-methyl-D-aspartate (NMDA) recep-

tor (PDB: 2A5S) mutant (Fig. 3a–e, Table 3). When less

conserved residues of low docking score binding sites are

replaced with Lys or Arg, the binding affinity for MC

increases, e.g., N-methyl-D-aspartate receptor (NMDA),

while high docking score binding sites showed no/decrease

in binding affinity, e.g., cAMP response element-binding

protein (CREB). Mutational docking results suggest that

lysine (Lys) residues play an important role in binding

affinity. Less conserved residues were identified on the basis

of their presence in low docking score binding sites and their

absence in high docking score complexes.

In Vitro Studies

Isolation, Purification and Culture of CD34?

Hematopoietic Stem Cells

Following the purification, the frequency of CD34? cells

was found to be 95-115 cells/ml of cord blood. The via-

bility of the HSCs was determined with trypan blue dye

exclusion method, and viability was found ranging from 83

to 92 %. Purified HSCs were cultured in selective myelo-

cult culture medium supplemented with cocktails of growth

factors for 4–6 months. The total cell counts were per-

formed weekly using haemocytometer. The cells grown

under deprivation of growth factors showed a rapid

decrease in cell number during the first 4 weeks, and by the

week 5, no cells were alive. In the long-term cultures

(LTC) containing the cocktails of growth factors, a pro-

gressive, steady increase in cell production could be seen.

After reaching 80–85 % confluency, the cells were pas-

saged for further proliferation.

Fig. 2 a The co-crystallized {[7-morpholin-4-yl-2, 3-dioxo-6- (tri-

fluoromethyl)- 3,4-dihydroquinoxalin-1 (2H)-yl]methyl} phosphonic

acid inhibitor was re-docked into the binding site of AMPA receptor

(PDB: 3KG2) with 0.6798 A of RMSD between docked and

crystallized conformation with total score of 12.7636. This docking

was performed to confirm the proper functionality of docking studies.

b In silico molecular docking studies elucidating the possible protein–

protein interaction between homology modeled AHR and CREB.

Protein–protein docking was performed by ZDOCK v2.5 module of

Discovery Studio (DS; Accelrys, USA). AHR (model_9) docked on to

CREB (PDB ID: 1DH3) showed high binding affinity docking score,

as indicated by a total ZDOCK score of 25.48

Neuromol Med

123

Author's personal copy

Characterization of Human Umbilical Cord Blood Stem

Cells

Purified population of hematopoietic stem cells showed the

cell surface expression of CD34 and Thy1 markers. Fol-

lowing the purification of HSCs, the cells were showing the

expression of surface markers CD34 (93.2 ± 11.73 %

cells) and Thy1 (67.3 ± 6.55 % cells) (Fig. 4a-II and III).

The co-expression of CD34?Thy1? was found to be

69.8 ± 6.23 %. It is evident from dual staining that

69.8 ± 6.23 % population were CD34?Thy1? and rest of

the cells were CD34? only (30.2 ± 2.76 %) (Fig. 4a-IV).

In selective myelocult culture medium supplemented with

10-5M hydrocortisone and various growth factors, the

percent population of CD34?, Thy1? and CD34?Thy1?

was increased with further passages. Further, these cells

Fig. 3 Docking simulation-based mutation studies to compare the

docking score and binding affinity of MC to AHR, ARNT, CREB,

NMDA and AMPA proteins. Two conserved amino acids (lysine and

arginine) residues were selected for mutation studies. Less conserved

residues within docked binding sites were replaced with either of

lysine or arginine, and docking was performed for each mutant

Neuromol Med

123

Author's personal copy

were characterized by clonogenic potential under the

influence of lineage-specific cytokine. HSCs showed the

commitment toward erythrocytes, granulocytes, monocytes

and granulocytes–monocytes lineages and resulted in the

formation of well-differentiated colony of respective lin-

eages, viz. CFU-E, CFU-G, CFU-M and CFU-GM

(Fig. 4b).

Neuronal Differentiation of Human Umbilical Cord Blood

Stem Cells

Morphological Study: hCBSCs were differentiated into

neuronal subtype, and differentiating neuronal cells at

various stages of maturities are highlighted in Fig. 5a.

After the onset of differentiation at day 2, the HSCs started

differentiating into neuronal morphology with clearly dis-

tinct cell body and dendrites. Upon further differentiation,

the neuronal cell bodies were found increasing with mul-

tiple cell processes at day 4. Subsequently, at day 8, more

than 95 % of cells acquired neuronal morphology with

extended neurites and well-arranged neuronal networks.

Study of Stemness Markers: Morphological changes

observed were accompanied by detectable down regulated

expression in transcripts of stemness marker genes. Quan-

titative RT-PCR analysis revealed that the mRNA expres-

sion of stemness marker genes (CD34, THY1, CD133 and

NANOG) was down regulated significantly (p \ 0.001) with

the progress of differentiation. The expressions of CD34,

THY1 and CD133 were found to be significantly decreasing

with the progress of neuronal differentiation, that is,

0.9 ± 0.05, 0.83 ± 0.04, 1.0 ± 0.03-fold and 0.73 ± 0.03,

0.37 ± 0.02, 0.67 ± 0.03-fold on days 2 and 4, respectively,

when compared to undifferentiated cells at day zero. At day 8

of neuronal maturity, the transcriptional expression of CD34,

THY1 and CD133 was not detectable, that is, 0.18 ± 0.012,

0.15 ± 0.012, 0.11 ± 0.013-fold, respectively, while

NANOG showed a biphasic response with increased

expression at day 2 (2.24 ± 0.14) and thereafter at days 4

and 8 reduced to 0.43 ± 0.03-fold and 0.22 ± 0.023-fold of

control (Fig. 5b).

Data of western blot analysis are highlighted in Fig. 5c.

Data showed significant alterations in protein expression of

stemness markers, viz. CD34 (0.29, 0.23 and 0.06-fold of

undifferentiated control), THY1 (0.58, 0.19 and 0.03-fold

of undifferentiated control), CD133 (0.9, 0.49 and 0.02-

fold of undifferentiated control) and NANOG (1.73, 0.13

and 0.11-fold of undifferentiated control) at days 2, 4 and 8

of differentiation, respectively.

Study of Neuronal Markers: The transcriptional changes

in the expression of selected neuronal markers are pre-

sented in Fig 5d. The mRNA expression of neural pro-

genitor marker nestin (NES) recorded significantly higher

at day 2 (13.2 ± 1.52-fold) and thereafter a reduction in

subsequent differentiation at day 4 (8.87 ± 1.03-fold) and

day 8 (2.66 ± 0.32-fold). However, the overall expression

of nestin was higher than the undifferentiated cells. The

expressions of early and late neuronal markers were

increased with progression of differentiation. In general,

the mRNA expression of early neuronal markers (neural

cell adhesion molecules; NCAM and synaptophysin; SYP)

Table 3 Comparison of docking score and binding site residues of 3-methylcholanthrene (MC) docked on virtual designed mutants of human

CYP regulators AHR and ARNT, neurogenesis transcription factor CREB, neurotransmitter receptors AMPA and NMDA (NR2A) receptors

S. No Compound

(ligand)

Protein (Target receptor) Target protein

and PDB ID

Total score (docking

score-Surflex-Dock)

Biding site residues (within 4 A)

1. MC Aryl hydrocarbon receptor

(AHR)

Homology

Model_9

4.1012 LEU-19, LEU-20, PRO-21,

PHE-22, ASN-145, ARG-147,

SER-191, CYS-192, LEU-190,

PRO-226

2. MC Aryl hydrocarbon receptor

nuclear trans locator (ARNT)

2A24 2.0245 LYS-16, HIS-57, ILE-101, GLY-

87, CYS-103, GLN-86, SER-68,

THR-85

3. MC cAMP response element-binding

protein (CREB)

1DH3 3.2566 ARG-301, LYS-305, LYS-304

4. MC N-methyl-D-aspartate receptor

(NMDA)

2A5S 3.0296 LEU-14, GLU-15, ILE-21,

LYS-197, GLU-198, TYR-214,

LYS-222

5. MC a-amino-3-hydroxy-5-methyl-4-

isoxazolepropionic acid receptor

(AMPA receptor)

3KG2 4.1199 LYS-449, GLY-451, ALA-452,

ASN-461, ARG-485, GLY-653,

SER-654, THR-655, THR-686,

LEU-704, GLU-705, LYS-732

(1) At binding site position 147, SER replaced with ARG in virtual designed AHR mutant. (2) At binding site position 16, MET replaced LYS in

virtual designed ARNT mutant. (3) At binding position 300, SER residue replaced with ARG in virtual designed CREB mutant that was not

involved in binding. (4) At binding site position 197, VAL replaced with LYS in virtual designed NMDA mutant. (5) At binding site position

732, TYR replaced with LYS in virtual designed AMPA mutant

Neuromol Med

123

Author's personal copy

was recorded maximum at day 4, while late neuronal

(cAMP response element-binding protein; CREB, a-amino-

3-hydroxy-5-methyl-4-isoxazole propionic acid receptor;

GRIA2 and N-methyl-D-aspartate receptor subtype 2A;

GRIN2A) markers at day 8 of differentiation. The expression

of early neuronal markers, viz. NCAM and SYP increased

significantly at day 2 (5.32 ± 0.46 and 16.73 ± 1.21-fold)

and peaked at day 4 (14.3 ± 1.22 and 23.65 ± 1.69-fold),

respectively, but was reduced by day 8 (10.54 ± 1.12 and

18.09 ± 1.37-fold) when compared with undifferentiated

control cells, though the expression of late neuronal markers

such as CREB, GRIA2 and GRIN2A was increased signifi-

cantly all through differentiation from day 2 (3.5 ± 0.29,

1.7 ± 0.09 and 2.32 ± 0.25-fold, respectively) till day 4

(8.72 ± 0.75, 4.54 ± 0.52 and 17.89 ± 0.93-fold, respec-

tively) and peaked at day 8 (10.32 ± 1.32, 7.83 ± 0.83 and

24.04 ± 1.33-fold, respectively) (Fig. 5d).

Similar trends in the expression of these neuronal

markers at protein level by western blot analysis were also

observed. Data of western blot analysis for translational

changes are highlighted in Fig. 5e. Neural progenitor

marker nestin showed initial increase, that is, 5.24-folds at

day 2 of differentiation, and subsequently down to 3.73 at

day 4, and 1.1-fold of control at day 8 of differentiation.

Markers of early neuronal development such as PSA-

NCAM and SYP showed peak expression by day 4 of

differentiation, that is, 6.22 and 12.1-folds of undifferen-

tiated control, respectively (Fig. 5e). A gradual increase in

the expression of late neuronal marker proteins was

observed all through the differentiation and peaked by day

8, that is., CREB (4.26-fold), AMPAR2 (1.9-fold) and

NMDA receptor NR2A (16.76-fold) of undifferentiated

control, respectively.

Constitutive Expressions of AHR, CYP1A1 and GSTP1-1

in Differentiating Neuronal Cells

Constitutive expression of aryl hydrocarbon receptor

(AHR), cytochrome P4501A1 (CYP1A1) and glutathione

S-transferase (GSTP1-1) in differentiating neuronal cells

derived from human cord blood hCBSCs was measured at

both mRNA (Fig. 6a) and protein (Fig. 6b) level.

Real-time data revealed the significant up regulated

constitutive mRNA expression of CYP1A1 and GSTP1-1

during neuronal differentiation (Fig. 6a). The transcripts of

CYP1A1 and GSTP1-1 recorded a gradual increase from

day 2 (3.82 ± 0.31 and 2.3 ± 0.19-fold) of neuronal dif-

ferentiation to day 4 (8.32 ± 0.77 and 2.52 ± 0.21-fold)

and peaked at day 8 (10.64 ± 0.98 and 3.42 ± 0.34-fold)

when compared to undifferentiated control. The differen-

tiation-induced alteration in mRNA expression of AHR was

insignificant.

Western blot analyses for translational changes have

shown similar trends at transcriptional level (Fig. 6b). The

protein expression of CYP1A1 and GSTP1-1 gradually

Fig. 4 Purification and characterization of human umbilical cord

blood hematopoietic stem cells. a Unstained selected umbilical cord

blood hematopoietic stem cells population (I); cells stained with

FITC-conjugated CD34? (II); and PE-Texas Red-conjugated Thy1?

antibodies (III); cells stained with FITC-conjugated CD34? and PE-

Texas Red-conjugated Thy1? antibodies as a dual labeling for co-

expression of CD34?Thy1? cells. b Characterization of human

hematopoietic stem cells by colony-forming unit (CFU) assay in

semisolid methylcellulose culture medium (MethoCultTM Stem Cell

Technologies, Vancouver, BC, Canada; catalog no. 03434) supple-

mented with specific recombinant colony-stimulating factors. Colo-

nies of erythrocytes (I); colonies of granulocytes (II); colonies of

monocytes (III); and colonies of granulocytes and monocytes (IV).

Microphotographs of colonies were captured at 9200 magnification

using Leica phase contrast microscope equipped with Leica IM50

software (Leica Microsystems)

Neuromol Med

123

Author's personal copy

increased from day 2 (1.33 and 1.18-fold of control) of

neuronal differentiation to day 4 (4.49 and 1.33-fold of

control) and peaked at day 8 (4.62 and 1.97-fold of con-

trol). The expression of AHR was found unaltered all

through the differentiation.

Constitutive Catalytic Activity of CYP1A1

in Differentiating Neuronal Cells

The EROD activity for CYP1A1 showed the increasing

metabolism capability of differentiating neuronal cells. The

Fig. 5 Differentiation of human cord blood hematopoietic stem cells

into neuronal subtypes. a Microphotographs of cultured human cord

blood stem cells and neuronal differentiation. Images were captured at

9200 magnification using Leica phase contrast microscope equipped

with Leica IM50 software (Leica Microsystems). (I) Purified popu-

lation of undifferentiated human HSCs at day 0. (II) Differentiating

neuronal cells at early stage from HSCs at day 2. (III) Differentiating

neuronal cells from HSCs at day 4. (IV) Fully differentiated neuronal

cells derived from HSC at day 8. Bar 50 lm. b Relative quantification

of altered mRNA expression of stemness genes in differentiating

neuronal cells at days 2, 4, and 8 (2D, 4D, and 8D, respectively).

b-actin was used as endogenous control to normalize the data and

differentiation-induced alterations in mRNA expression are expressed

in relative quantity (mean ± SE) compared with the expression in the

cells at day 0 (RQ value = 1). ** p \ 0.001. c Western blot analysis

of expression of marker proteins of stemness all through

differentiation. The values obtained at day 0 were considered basal,

that is, relative quantification in expression at days 2, 4, and 8 (0D,

2D, 4D, and 8D, respectively) was done comparing the values at day

0. b-actin was used as loading control to normalize the data.

d Relative quantification of altered mRNA expression of selected

neuronal marker genes in differentiating neuronal cells at days 2, 4,

and 8 (2D, 4D, and 8D, respectively). b-actin was used as endogenous

control to normalize the data and differentiation-induced alterations in

mRNA expression are expressed in relative quantity (mean ± SE)

compared with the expression in the cells at day 0 (RQ value = 1).

** p \ 0.001. e Western blot analysis of expression of neuronal

marker proteins all through differentiation. The values obtained at day

0 were considered basal, that is, relative quantification in expression

at days 2, 4, and 8 (0D, 2D, 4D, and 8D, respectively) was done

comparing the values at day 0. b-actin was used as loading control to

normalize the data

Neuromol Med

123

Author's personal copy

EROD activity of CYP1A1 is presented in Fig. 6c. The

enzymatic activity of CYP1A1 recorded a gradual increase

from day 2 (20.7 ± 2.4 pmol resorufin/min/mg protein) of

neurogenesis to day 4 (25.9 ± 2.8 pmol resorufin/min/mg

protein) and peaked at day 8 (31.43 ± 3.0 pmol resorufin/

min/mg protein) when compared to day 0 control

(16.3 ± 1.5 pmol resorufin/min/mg protein).

Cytotoxicity Studies of 3-Methylcholanthrene

Non-cytotoxic doses of 3-methylcholanthrene (MC) were

identified in neuronal cells at various time points of

maturity (0, 2, 4 and 8 days) using standard endpoint MTT

by exposing cells to different concentrations (1–12 lM) of

MC for different time intervals (6–72 h). The result high-

lights are summarized in Fig. 7. The severity of cytotoxic

responses was increased with the increase in exposure

period.

In general, there was no significant variation in the

sensitivity of cells against the exposure of MC at days 0, 2,

4 and 8 of differentiation. All the concentrations (1–4 lM)

of MC could not pose any significant cytotoxic responses at

any point of exposure in both the differentiated and

undifferentiated cells, whereas a dose-dependent decrease

in percentage of cell viability was recorded in cells exposed

to 8 and 12 lM of MC. By comparing the results of MTT

assay at different stages of neuronal maturities, it was

observed that the decrease in percent cell viability, against

specific dose of MC, was found more in early differenti-

ating cells at day 2 followed by days 4 and 8. Thus, it was

anticipated that the early stages of differentiation were

found to be more vulnerable to MC than that of mature

well-differentiated cells (Fig. 7a). In order to confirm the

sensitivity of differentiating cells against MC exposure,

IC50 values were also determined using GraphPad Prism

5.0 (GraphPad Software, San Diego, CA). IC50 values

determined using logarithmic nonlinear regression analysis

have shown the more vulnerability of the cells at day 2 of

differentiation than the late differentiating cells, i.e., days 4

and 8 (Fig. 7b).

3-Methylcholanthrene Induced Alterations

in the Expressions (mRNA and protein) of AHR, CYP1A1

and GSTP1-1 in Differentiating Neuronal Cells

In real-time PCR, expression of mRNA in control cells was

compared to expression of mRNA in cells exposed to MC

(4 lM) for 12 h. For western blot analysis, cells were

exposed to MC (4 lM) for 24 h.

The expression of mRNA of AHR, CYP1A1 and GSTP1-1

was induced significantly by MC (4 lM) at all the points

of neuronal maturities, that is, days 2, 4 and 8. The sig-

nificant peak induction for the transcripts of AHR and

CYP1A1 was recorded at day 2 (21.19 ± 1.52 and

25.29 ± 1.71-fold), followed by day 4 (14.79 ± 1.45 and

15.7 ± 1.38-fold), and was minimum at day 8 (9.77 ±

0.77 and 10.46 ± 1.08-fold) of differentiation, when

compared to respective unexposed controls. However, the

Fig. 6 Constitutive expression of aryl hydrocarbon receptor (AHR),

cytochrome P4501A1 (CYP1A1) and GSTP1-1 in differentiating

neuronal cells. a Relative quantification of altered mRNA expression

of AHR, CYP1A1 and GSTP1-1 in differentiating neuronal cells at

days 2, 4, and 8 (2D, 4D, and 8D, respectively). b-actin was used as

endogenous control to normalize the data. The data are expressed in

relative quantity (mean ± SE) compared with the expression at day 0

(RQ value = 1). ** p \ 0.001. b Western blot analysis for relative

quantification of altered protein expression of AHR, CYP1A1 and

GSTP1-1 in differentiating neuronal cells at days 2, 4 and 8 (2D, 4D,

and 8D, respectively). b-actin was used as loading control to

normalize the data. The data are expressed in relative quantity

compared with the expression at day 0. c 7-ethoxyresorufin-O-

deethylase (EROD) activity for catalytic activity of CYP1A1 in

microsomal fraction of differentiating neuronal cells at days 0, 2, 4

and 8 (0D, 2D, 4D and 8D, respectively). ** p \ 0. 001

Neuromol Med

123

Author's personal copy

peak induction of GSTP1-1 was found at day 2 (7.53 ±

0.82-fold) followed by day 8 (5.93 ± 0.61-fold) and day 4

(4.55 ± 0.65-fold) of respective controls (Fig. 8a).

Western blot analyses for translational changes have

shown similar trends at transcriptional level. Western blot

analysis showed that MC exposure induced significant

protein expression of AHR, CYP1A1 and GSTP1-1 at day

2 (5.22, 3.65 and 1.56-fold, respectively, of control), day

4 (3.02, 1.26 and 1.19-fold, respectively, of control) and

day 8 (3.0, 1.19 and 1.34-fold, respectively, of control).

Western blot data revealed that the peak induction in

protein expression of AHR and CYP1A1 was found at

day 2 followed by day 4 and day 8 of neuronal differ-

entiation. GSTP1-1 also showed peak induction at day 2

followed by day 8 and day 4 of differentiation (Fig. 8b).

Xenobiotic metabolism in early stages of neuronal dif-

ferentiation was greater than in fully differentiated neu-

ronal cells.

MC Induced Alterations in Catalytic Activity of CYP1A1

The microsomal fractions prepared from MC (4 lM for

24 h) exposed differentiating neuronal cells (days 2, 4 and 8)

Fig. 7 a Identification of non-

cytotoxic doses of

3-methylcholanthrene (MC) in

undifferentiated stem cells and

differentiating neuronal cells at

various stages of maturation,

i.e., days 0, 2, 4, and 8. Cells

were exposed to MC (1–12 lM)

for 6-72 h. The percent cell

viability was assessed using

MTT assay. Values are

mean ± SE of the data obtained

from three independent

experiments, and in each

experiment, values were taken

from six wells. b Determination

of IC50 values of MC in

hematopoietic stem cell derived

differentiating neuronal cells at

various stages of maturity, i.e.,

0, 2, 4 and 8 days. IC50 values

were determined using

logarithmic nonlinear regression

analysis with the help of

GraphPad Prism 5.0 (GraphPad

Software, San Diego, CA). Cells

at day 2 of differentiation show

more vulnerable to MC

exposure than later stages of

differentiation, i.e., 4 and 8 days

Neuromol Med

123

Author's personal copy

showed significant induction in CYP-dependent EROD

(CYP1A1) activity (Fig. 8c). The trend in the induction of

CYP1A1 activity was fairly correlative with the data of real-

time PCR and western blot analysis. The cells showed peak

induction in the catalytic activity of CYP1A1 at day 2

(47.8 ± 4.7 pmol resorufin/min/mg protein) compared to

unexposed day 2 control (20.7 ± 2.4 pmol resorufin/min/

mg protein) and subsequently decreased in induction at day 4

(37.7 ± 3.52 pmol resorufin/min/mg protein) compared to

unexposed day 4 control (25.88 ± 2.7 pmol resorufin/min/

mg protein) and at day 8 (39.45 ± 4.27 pmol resorufin/min/

mg protein) of differentiation when compared to unexposed

control of day 8 (31.43 ± 3.0 pmol resorufin/min/mg

protein).

Developmental Neurotoxicity Studies

of 3-Methylcholanthrene

Cells receiving MC (4 lm; 12 h for mRNA and 24 h for

protein expression) exposure showed statistically signifi-

cant down regulation in the expression of (mRNA and

protein) all the neuronal marker genes studied.

At transcriptional level, the mRNA expression of NES,

NCAM and CREB was recorded as significantly down

regulated all through the differentiation, following the

exposure of MC with maximum reduction at day 2

(0.37 ± 0.04, 0.29 ± 0.03 and 0.23 ± 0.02-fold, respec-

tively) then at day 4 (0.59 ± 0.03, 0.55 ± 0.04 and

0.78 ± 0.06-fold) and day 8 (0.9 ± 0.04, 0.72 ± 0.05 and

0.88 ± 0.052-fold) of differentiation in comparison with

unexposed respective controls, while the maximum

reduction in the mRNA expression of SYP was recorded at

day 8 (0.3 ± 0.01-fold) followed by day 4 (0.67 ± 0.05-

fold) and minimum inhibition at day 2 (0.83 ± 0.045-fold).

However, the mRNA expression of GRIA2 and GRIN2A

was not detectable at day 2 of differentiation, and MC

down regulated GRIA2 and GRIN2A most significantly at

day 4 (0.07 ± 0.009 and 0.39 ± 0.046-fold, respectively)

and then at day 8 (0.97 ± 0.04 and 0.47 ± 0.07-fold,

respectively) of differentiation in comparison with the

respective unexposed controls (Fig. 9a).

The trend in down regulated protein expression (Fig. 9b)

of selected early and late neuronal markers was fairly

correlated with the data of real-time PCR. Western blot

analysis revealed that MC most significantly down regu-

lated the expression of NES, PSA-NCAM and CREB at

Fig. 8 MC induced altered expression of AHR, CYP1A1 and

GSTP1-1 in differentiating neurons. a MC induced altered expression

in transcripts of AHR, CYP1A1 and GSTP1-1 genes in human HSC-

derived differentiating neuronal cells at days 2, 4 and 8 (2DT, 4DT

and 8DT, respectively). b-actin was used as internal control to

normalize the data. MC-induced alterations in mRNA expression are

expressed in relative quantity compared with respective unexposed

control groups (RQ values of controls = 1). Reliability of specific

products was checked by melting curve analysis as well as by running

the product on 2 % agarose gel. ** p \ 0.001. b MC-induced

alterations in the protein expression of AHR, CYP1A1 and GSTP1-

1 in differentiating neuronal cells at days 0, 2 and 4. b-actin were used

as loading control to normalize the data. MC-induced alterations in

protein expression are expressed in relative quantity compared with

respective unexposed control groups. C2: unexposed control cells at

day 2 of differentiation; T2: differentiating cells at day 2 exposed to

MC (4 lM) for 24 h; C4: unexposed control cells at day 4 of

differentiation; T4: differentiating cells at day 4 exposed to MC

(4 lM) for 24 h; C8: unexposed control cells at day 8 of differen-

tiation; T8: differentiating cells at day 8 exposed to MC (4 lM) for

24 h. c MC induced 7-ethoxyresorufin-O-deethylase (EROD) activity

for catalytic activity of CYP1A1 in microsomal fraction of differen-

tiating neuronal cells at days 2, 4 and 8 of differentiation (2D, 4D and

8D, respectively). * p \ 0.05, ** p \ 0. 001

b

Neuromol Med

123

Author's personal copy

day 2 (0.65, 0.26 and 0.12-fold, respectively) followed by

day 4 (0.73, 0.69 and 0.78-fold, respectively) and day 8

(0.82, 0.87 and 0.83-fold, respectively) of differentiation in

comparison with respective controls. However, the maxi-

mum down regulation in SYP protein expression was

recorded at day 8 (0.39-fold) of differentiation. The

reduction in protein expression of AMPAR2 and NR2A

was most severe. The protein expressions of AMPAR2 and

NR2A were not detectable in either the control or the

exposed cells at day 2 of neuronal differentiation. MC

significantly inhibited the expression of AMPAR2 and

NR2A at day 4 (0.32 and 0.29-fold, respectively) and then

at day 8 (0.98 and 0.5-fold, respectively) in comparison

with the respective unexposed controls (Fig. 9b).

AHR-Dependent Repression of Neuronal Markers

in Human Stem Cells Derived Differentiating Neuronal

Cells

The results of AHR-dependent alterations in the expres-

sion (mRNA and proteins) of neuronal markers in human

differentiating neuronal cells transfected with small

inhibitory RNA (siRNA) directed toward AHR (siAHR)

are presented in Fig. 10a-b. In our experimental conditions,

over 60 % transfection could be achieved. Following the

transfection and exposure of cells to MC, the expression of

AHR was found to be down regulated significantly at all

the time points studied during the neuronal differentiation.

MC-induced repression of neuronal markers, viz. nestin,

SYP, PSA-NCAM, CREB, AMPA, NR2A receptors, was

found to be regained in the cells ablated of AHR and

receiving of MC (4 lM) exposure. Western blots analysis

of AHR protein showed a significantly reduced expression

of AHR protein after transfection. This demonstrates the

specificity of the siRNA to AHR. Thus, AHR plays a key

role in the MC-induced repression of neuronal markers.

The unchanged expression of neuronal markers following

MC exposure is due to the loss of AHR and other possible

transcriptional modifiers associated with its presence.

Discussion

We were able to optimize the conditions in our study to

allow for long-term proliferation, growth and neuronal

differentiation of human umbilical cord blood stem cells

with the purity of more than 90 %. The phenotypic and

physiological maturity of the cells seen as a significantly

high magnitude of neuronal proteins’ expression could be

achieved in 8 days’ time (Singh et al. 2012). These dif-

ferentiating neuronal cells expressed various neuronal

markers of early, mid and late stages of development. The

initial increase in the levels of nestin at day 2 of differ-

entiation followed by gradual decrease at day 4 and day 8

supports the theory of the triggering of neuronal differen-

tiation by day 2 (Haque et al. 2012; McGuckin et al. 2004).

We observed a gradual increase in the expression of neu-

ronal markers (PSA-NCAM, SYP and CREB) from day 2

to 8 of differentiation, while the other neuronal markers

(AMPA and NR2A receptors) have shown the expression

by day 4 of differentiation and peaked by day 8. The trend

of expression of neuronal markers was consistent with the

Fig. 9 Developmental neurotoxic response of MC in differentiating

neuronal cells derived from human cord blood hematopoietic stem

cells. a MC induced altered expression in transcripts of selected

neuronal marker genes in human HSC-derived differentiating neuro-

nal cells at days 2, 4 and 8 (2DT, 4DT and 8DT, respectively). b-actin

was used as internal control to normalize the data. MC-induced

alterations in mRNA expression are expressed in relative quantity

compared with respective unexposed control groups (RQ values of

controls = 1). Reliability of specific products was checked by

melting curve analysis as well as by running the product on 2 %

agarose gel. * p \ 0.05, ** p \ 0. 001. b MC induced alterations in

the protein expression of selected neuronal markers in differentiating

neuronal cells at days 0, 2 and 4. b-actin was used as loading control

to normalize the data. MC-induced alterations in protein expression

are expressed in relative quantity compared with respective unex-

posed control groups. C2: unexposed control cells at day 2 of

differentiation; T2: differentiating cells at day 2 exposed to MC

(4 lM) for 24 h; C4: unexposed control cells at day 4 of differen-

tiation; T4: differentiating cells at day 4 exposed to MC (4 lM) for

24 h; C8: unexposed control cells at day 8 of differentiation; T8:differentiating cells at day 8 exposed to MC (4 lM) for 24 h

Neuromol Med

123

Author's personal copy

earlier findings observed in different in vitro systems

(Sanchez-Ramos et al. 2001; Zigova et al. 2002). The

physiological differentiation of cells into neuronal subtypes

was also confirmed by the down regulation in the expres-

sion of stemness markers, viz. CD34, Thy1, CD133 and

Nanog all through the neuronal differentiation. The

increased expression of Nanog at day 2 might be due to the

intracellular competition between the proliferation and

differentiation processes.

Though there was a significant expression and activity

of CYP1A1, AHR and phase II xenobiotic metabolizing

GSTP1-1, all through the differentiation of neuronal cells,

the early differentiating (day 2) neuronal cells had the

highest expression and activity of these markers of

metabolism. This may be suggested as that the early dif-

ferentiating neuronal cells are metabolically more active.

Such significant expression and activity of CYP1A1 and

AHR in differentiating neuronal cells has been suggested

due to the possible association of xenobiotic metabolizing

enzymes in neuronal development and physiological role in

brain (Gervasini et al. 2004; Miksys and Tyndale 2002;

Singh et al. 2012). The role of adrenoreceptors has also

been reported in the regulation of CYP1A1 gene that

causes a reduction in the levels of noradrenaline and a

depletion of catecholamine in central nervous system,

which subsequently down regulates the expression of

CYP1A1 (Konstandi et al. 2004; Konstandi et al. 2005).

MC is known to activate AHR that induces the expres-

sion of various genes including CYP1A1 for the metabo-

lism of MC (Kumagai et al. 2012). Thus, the 3D structure

of human-specific AHR was developed for the first time

using homology molecular modeling and was used in

molecular docking studies to understand the binding

properties. We observed a strong binding of MC to mod-

eled AHR target protein with a high docking score. Such

MC-AHR binding was found to induce significant expres-

sion of CYP1A1. At the same time, MC and AHR inde-

pendently as well as in combinations were found to show

the strong binding with neurogenesis inducing transcription

factor CREB. The key mediator of MC toxicity is the AHR

which upon activation translocates from cytoplasm to

nucleus, dimerizes with ARNT and interacts with other

transcription factors to modulate the expression of various

genes via binding to xenobiotic-responsive elements

(XREs) of promoter sequence of target genes (Omiecinski

et al. 2011). Our in silico data also supported the induction

of CYP1A1 by MC through binding at XREs with AHR

and ARNT. The data further revealed the highest stable

binding of MC to neurogenesis-related transcription factor

CREB with maximum docking energy via hydrophobic

interactions at Arg301, Lys-304/305 and Ser-300 residues.

The docking studies showed that MC modulates the

activity of CREB, NMDA and AMPA receptors. We

observed that the MC has interacted with target proteins

with conserved hydrophobic amino acid residues in binding

pockets, thus leading to more stability and potency. To

better understand the binding nature of MC to AMPA, the

MC-AMPA docked structure was compared with the

Fig. 10 Role of AHR in MC-induced developmental neurotoxicity in

differentiating neuronal cells derived from hUCBSCs. a Real-time

PCR analysis for altered expression in transcripts of selected neuronal

marker genes in differentiating neuronal cells at days 2, 4 and 8 (2DT,

4DT and 8DT) following the transfection of siAHR and exposure of

cells to MC. MC-induced repression of neuronal markers, viz. nestin,

SYP, NCAM, CREB, AMPA, NR2A receptors, was found to be

regained in the cells ablated of AHR and receiving of MC (4 lM)

exposure. b-actin was used as internal control to normalize the data.

MC-induced alterations in mRNA expression are expressed in relative

quantity compared with respective unexposed control groups (RQ

values of controls = 1). * p \ 0.05, ** p \ 0.001. b MC-induced

alterations (regain) in the protein expression of selected neuronal

markers in differentiating neuronal cells at days 0, 2 and 4 following

the transfection of siAHR. b-actin was used as loading control to

normalize the data. MC-induced alterations in protein expression are

expressed in relative quantity compared with respective unexposed

control groups. C2: unexposed control transfected cells at day 2 of

differentiation; T2: differentiating transfected cells at day 2 exposed

to MC (4 lM) for 24 h; C4: unexposed control transfected cells at day

4 of differentiation; T4: differentiating transfected cells at day 4

exposed to MC (4 lM) for 24 h; C8: unexposed control transfected

cells at day 8 of differentiation; T8: differentiating transfected cells at

day 8 exposed to MC (4 lM) for 24 h

Neuromol Med

123

Author's personal copy

AMPA receptor complexed with {[7-morpholin-4-yl-2,

3-dioxo-6- (trifluoromethyl)–3, 4-dihydroquinoxalin-1

(2H)-yl] methyl} phosphonic acid inhibitor. The AHR

protein was found to dock with CREB protein with highest

docking score of 25.48, suggesting very strong hydropho-

bic interaction. Thus, the in silico data confirm the inter-

action of MC to AHR, ARNT, CREB, NMDA (NR2A) and

AMPA. We also observed strong MC binding with CREB

at the active pocket with common amino acid residues,

where ATP would initially bind and phosphorylate the

optimum functionality of CREB. Thus, it seems that MC

influences the phosphorylation and subsequent activity of

CREB by competitive inhibition, as the binding energy of

MC is significantly greater than ATP. The data clearly

indicate that MC exerts potent neurotoxic effects by

altering the receptor-mediated neurotransmission as it has

strong and stable binding with ion channel neurotransmitter

receptors, viz. AMPA and NMDA, and modulating capa-

bility of the activity of neurogenesis transcription factor

CREB. Strong and highly stable protein–protein interaction

between AHR and CREB further confirmed the MC/AHR-

mediated modulation of physiological functionality of

CREB and associated mechanism of neurogenesis. Hence,

MC and AHR are considered to be the key players in

neurotoxic response. To confirm the binding specificity of

MC to these target proteins, mutational simulation study

was performed. The decreased binding of MC to mutant

proteins confirmed the strong binding and stability of MC

to these target proteins.

Taking the leads from in silico data where MC exposure

exerted neurotoxic responses by triggering the activation of

AHR and subsequent cascade of events via affecting CREB

(neurogenesis) and receptors AMPA, NMDA (neurotrans-

mission) and metabolism (CYP1A1 and GSTP1-1),etc., the

in vitro experiments were carried out in differentiating

human neuronal cells derived from cord blood stem cells.

In the linearity with in silico data, exposure of MC induced

the expression of AHR significantly in neuronal cells all

through the differentiation. MC-induced expression of

AHR was highest at day 2 of differentiation, and thereafter,

a reduction in magnitude of induction was seen. Such

induction of AHR in early differentiating cells could be

well correlated with the MC-induced altered expression of

neuronal and metabolism markers studied in the investi-

gation. For instance, our data indicated that neurons are

more vulnerable to MC at early stage of differentiation at

days 2 and 4, while mature neurons at day 8 were less

affected. The gene expression of the neuronal markers, viz.

nestin, PSA-NCAM and CREB (early markers), was more

affected at day 2, and AMPA and NR2A (midterm mark-

ers) at day 4 of differentiation. SYP was more affected at

day 8 of differentiation, as SYP is a mature neuronal

marker required for synapse formation. Thus, by affecting

neural cell adhesion molecule (NCAM) expression in

neurons, MC/AHR may affect the cell-to-cell adhesion/

interaction, cell migration, neurite extension, synaptic

plasticity and various intracellular signaling pathways

(Barouki et al. 2007; Gassmann et al. 2010). NCAM

induces the neurite outgrowth via fibroblast growth factor

receptor (Kleene and Schachner 2004). MC significantly

decreased the expression of PSA-NCAM at early stage of

differentiation that indicates a delay in cell–cell interaction

that might affect the neuronal cell–cell communication and

neurite growth in further differentiation. There are two

types of ligand gated ion channels, that is, ionotropic glu-

tamate receptors, viz. AMPA receptor (GluA1, A2, A3,

A4) and NMDA (GluN1, N2A, N2B, N2C, N2D), which

are involved in glutamate transmission during brain

development (Thomas et al. 2011). Their activities are

reported to be altered by intracellular and extracellular

signaling. AMPA receptor is reported to control synaptic

transmission at post-synaptic membrane and neural plas-

ticity in the central nervous system (CNS) (Huls et al.

2011). In the present investigation, MC inhibited the

expression of CREB, AMPA and NMDA, thus interfering

in the process of neurogenesis. Transfection study to

repress the expression of AHR and its effect on down-

stream targets (neuronal markers) resulted in regain of

neuronal markers and further confirmed the involvement of

AHR and MC in developmental neurotoxicity of MC.

AHR, a ligand-activated transcription factor and mem-

ber of basic helix-loop-helix/Per-Arnt-Sim transcription

factor super family, is reported to be expressed in various

regions of the developing brain. Upon binding with MC,

the activated AHR translocates from cytoplasm to the

nucleus where it dimerizes with aryl hydrocarbon nuclear

translocator (ARNT) and binds to xenobiotic response

element (XRE) to induce the expression of several xeno-

biotic metabolizing enzymes, viz. CYP1A1, 1B1 and GSTs

(Hankinson 1995; Shimada et al. 2002). AHR has also been

reported as key player in various cellular processes, viz.

cell fate determination, cell adhesion, proliferation,

migration and differentiation (Barouki et al. 2007; Fox

et al. 2010). AHR interacts with p65/RelA to transactivate

the cMYC which subsequently promotes cell proliferation

(Kim et al. 2000). The regulation of AHR involves phos-

phorylation by various protein kinases. The activator of

AHR regulates the signaling of c-Jun N-terminal kinase

(JNK), mitogen-activated protein kinases (MAPK) and

extracellular signal-regulated kinases (ERK) (Tan et al.

2002). The MAPKs are the key player in various cellular

process, viz. cell signaling, cell differentiation, carcino-

genesis and cell death (Henklova et al. 2008). Thus, AHR

activation in association with estrogen receptor signaling

can lead to apoptotic cell death in neocortical and hippo-

campal region of the brain (Kajta et al. 2009). MC (ligands

Neuromol Med

123

Author's personal copy

of AHR) has been reported to affect the proliferation and

differentiation of osteoblast under both in vivo and in vitro

conditions (Naruse et al. 2002). Petroff et al. (2011) have

shown that AHR pathway altered sperm penetration and

early embryonic development in the rat following the

exposure of AHR agonist 2, 3, 7, 8-tetrachloro-dibenzo-p-

dioxin (TCDD). In another study, AHR restored the pro-

liferative activity in human primary cerebellar brain tumor

and advocated for its therapeutic targeting for the treatment

of brain tumors(Dever and Opanashuk 2012).

Conclusion

In summary, our wet laboratory data confirm the in silico

finding that MC significantly induces the expression and

activity of AHR, CYP1A1, GSTP1-1 and reduces the

expression of CREB, AMPA and NMDA receptors.

Together, the results of molecular docking, real-time PCR,

western blot analysis and substrate-specific catalytic

activity suggest that MC possibly interferes with neuronal

transmission and significantly inhibits the expression and

activity of various developmental stage-specific neuronal

markers (Fig. 11). The data also identify the suitability of

differentiating neuronal cells derived from human cords

blood stem cells as an in vitro tool to screen and understand

the mechanisms involved in drug/chemical-induced

developmental neurotoxicity. Our data also confirm that

lactational or gestational exposure of MC may impair brain

function during development of fetal brain and the brain of

newly born children. It has also been concluded that the

early stage of neuronal development is metabolically more

active than mature cells, hence more vulnerable to the

polycyclic aromatic hydrocarbons.

Acknowledgments The Director, Indian Institute of Toxicology

Research, Lucknow, India, is acknowledged for providing the Labo-

ratory and infrastructure support. Council of Scientific & Industrial

Research, New Delhi, India, is acknowledged for providing fellow-

ship to Mr. AK Singh.

Fundings Financial support from Department of Biotechnology,

Ministry of Science & Technology, Government of India, New Delhi,

India [Grant No. 102/IFD/SAN/PR1524/2010–2011]; Department of

Science and Technology, Ministry of Science & Technology, Gov-

ernment of India, New Delhi, India [Grant No. SR/SO/Z 36/2007/91/

10]; and Council of Scientific & Industrial Research, Government of

India, New Delhi, India [Grant No. BSC0111/INDEPTH/CSIR Net-

work Project] is acknowledged. The funders had no role in study

design, data collection and analysis, decision to publish, or prepara-

tion of the manuscript.

References

Bal-Price, A. K., Hogberg, H. T., Buzanska, L., Lenas, P., van Vliet,

E., & Hartung, T. (2010). In vitro developmental neurotoxicity

(DNT) testing: Relevant models and endpoints. Neurotoxicology,

31, 545–554.

Barouki, R., Coumoul, X., & Fernandez-Salguero, P. M. (2007). The

aryl hydrocarbon receptor, more than a xenobiotic-interacting

protein. FEBS Letters, 581, 3608–3615.

Batty, G. D., Der, G., & Deary, I. J. (2006). Effect of maternal

smoking during pregnancy on offspring’s cognitive ability:

Empirical evidence for complete confounding in the US national

longitudinal survey of youth. Pediatrics, 118, 943–950.

Breier, J. M., Radio, N. M., Mundy, W. R., & Shafer, T. J. (2008).

Development of a high-throughput screening assay for chemical

effects on proliferation and viability of immortalized human

neural progenitor cells. Toxicological Sciences, 105, 119–133.

Dever, D. P., & Opanashuk, L. A. (2012). The aryl hydrocarbon

receptor contributes to the proliferation of human medulloblas-

toma cells. Molecular Pharmacology, 81, 669–678.

Ferguson, C. S., & Tyndale, R. F. (2011). Cytochrome P450 enzymes

in the brain: Emerging evidence of biological significance.

Trends in Pharmacological Sciences, 32, 708–714.

Fox, D. A., Opanashuk, L., Zharkovsky, A., & Weiss, B. (2010).

Gene-chemical interactions in the developing mammalian ner-

vous system: Effects on proliferation, neurogenesis and differ-

entiation. Neurotoxicology, 31, 589–597.

Gabb, H. A., Jackson, R. M., & Sternberg, M. J. (1997). Modelling

protein docking using shape complementarity, electrostatics and

biochemical information. Journal of Molecular Biology, 272,

106–120.

Gassmann, K., Abel, J., Bothe, H., Haarmann-Stemmann, T., Merk,

H. F., Quasthoff, K. N., et al. (2010). Species-specific differen-

tial AhR expression protects human neural progenitor cells

against developmental neurotoxicity of PAHs. Environmental

Health Perspectives, 118, 1571–1577.

Gervasini, G., Carrillo, J. A., & Benitez, J. (2004). Potential role of

cerebral cytochrome P450 in clinical pharmacokinetics: Modu-

lation by endogenous compounds. Clinical Pharmacokinetics,

43, 693–706.

Grandjean, P., & Landrigan, P. J. (2006). Developmental neurotox-

icity of industrial chemicals. Lancet, 368, 2167–2178.

Hankinson, O. (1995). The aryl hydrocarbon receptor complex.

Annual Review of Pharmacology and Toxicology, 35, 307–340.

Haque, A., Yue, X. S., Motazedian, A., Tagawa, Y., & Akaike, T.

(2012). Characterization and neural differentiation of mouse

Fig. 11 Schematic flowchart depicting the mechanisms involved in

MC-induced AHR-mediated neurotoxicity of differentiating neuronal

cells derived from human umbilical cord blood stem cells

Neuromol Med

123

Author's personal copy

embryonic and induced pluripotent stem cells on cadherin-based

substrata. Biomaterials, 33, 5094–5106.

Henklova, P., Vrzal, R., Ulrichova, J., & Dvorak, Z. (2008). Role of

mitogen-activated protein kinases in aryl hydrocarbon receptor

signalling. Chemico-Biological Interactions, 172, 93–104.

Huls, S., Hogen, T., Vassallo, N., Danzer, K. M., Hengerer, B., Giese,

A., et al. (2011). AMPA-receptor-mediated excitatory synaptic

transmission is enhanced by iron-induced alpha-synuclein olig-

omers. Journal of Neurochemistry, 117, 868–878.

Kajta, M., Wojtowicz, A. K., Mackowiak, M., & Lason, W. (2009).

Aryl hydrocarbon receptor-mediated apoptosis of neuronal cells:

A possible interaction with estrogen receptor signalling. Neuro-

science, 158, 811–822.

Kalani, K., Yadav, D. K., Khan, F., Srivastava, S. K., & Suri, N.

(2011). Pharmacophore, QSAR, and ADME based semisynthesis

and in vitro evaluation of ursolic acid analogs for anticancer

activity. Journal of Molecular Modeling, 18, 3389–3413.

Kapoor, N., Pant, A. B., Dhawan, A., Dwievedi, U. N., Seth, P. K., &

Parmar, D. (2006). Cytochrome P450 1A isoenzymes in brain

cells: Expression and inducibility in cultured rat brain neuronal

and glial cells. Life Sciences, 79, 2387–2394.

Kashyap, M. P., Singh, A. K., Siddiqui, M. A., Kumar, V., Tripathi,

V. K., Khanna, V. K., et al. (2010). Caspase cascade regulated

mitochondria mediated apoptosis in monocrotophos exposed

PC12 cells. Chemical Research in Toxicology, 23, 1663–1672.

Katchalski-Katzir, E., Shariv, I., Eisenstein, M., Friesem, A. A.,

Aflalo, C., & Vakser, I. A. (1992). Molecular surface recogni-

tion: Determination of geometric fit between proteins and their

ligands by correlation techniques. Proceedings of the National

Academy of Sciences of the United States of America, 89,

2195–2199.

Kim, W., Gazourian, L., Quadri, S. A., Romieu-Mourez, R., Sherr, D.

H., & Sonenshein, G. E. (2000). The RelA NF-kappaB subunit

and the aryl hydrocarbon receptor (AhR) cooperate to transac-

tivate the c-myc promoter in mammary cells. Oncogene, 19,

5498–5506.

Kleene, R., & Schachner, M. (2004). Glycans and neural cell

interactions. Nature Reviews Neuroscience, 5, 195–208.

Konstandi, M., Johnson, E. O., Marselos, M., Kostakis, D., Fotopo-

ulos, A., & Lang, M. A. (2004). Stress-mediated modulation of

B(alpha)P-induced hepatic CYP1A1: Role of catecholamines.

Chemico-Biological Interactions, 147, 65–77.

Konstandi, M., Kostakis, D., Harkitis, P., Marselos, M., Johnson, E.

O., Adamidis, K., et al. (2005). Role of adrenoceptor-linked

signaling pathways in the regulation of CYP1A1 gene expres-

sion. Biochemical Pharmacology, 69, 277–287.

Kumagai, T., Suzuki, H., Sasaki, T., Sakaguchi, S., Miyairi, S.,

Yamazoe, Y., et al. (2012). Polycyclic aromatic hydrocarbons

activate CYP3A4 gene transcription through human pregnane X

receptor. Drug Metabolism and Pharmacokinetics, 27, 200–206.

Liu, M., Hurn, P. D., & Alkayed, N. J. (2004). Cytochrome P450 in

neurological disease. Current Drug Metabolism, 5, 225–234.

Liu, J., Li, J., Liu, Y., Chan, H. M., Zhao, Y., Cai, Z., et al. (2011).

Comparison on gestation and lactation exposure of perfluori-

nated compounds for newborns. Environment International, 37,

1206–1212.

McGuckin, C. P., Forraz, N., Allouard, Q., & Pettengell, R. (2004).

Umbilical cord blood stem cells can expand hematopoietic and

neuroglial progenitors in vitro. Experimental Cell Research, 295,

350–359.

Miksys, S. L., & Tyndale, R. F. (2002). Drug-metabolizing

cytochrome P450 s in the brain. Journal of Psychiatry and

Neuroscience, 27, 406–415.

Naruse, M., Ishihara, Y., Miyagawa-Tomita, S., Koyama, A., &

Hagiwara, H. (2002). 3-Methylcholanthrene, which binds to the

arylhydrocarbon receptor, inhibits proliferation and differentiation

of osteoblasts in vitro and ossification in vivo. Endocrinology, 143,

3575–3581.

Nebert, D. W., & Russell, D. W. (2002). Clinical importance of the

cytochromes P450. Lancet, 360, 1155–1162.

Nielsen, J. K., Vikstrom, A. C., Turner, P., & Knudsen, L. E. (2011).

Deoxynivalenol transport across the human placental barrier.

Food and Chemical Toxicology, 49, 2046–2052.

Omiecinski, C. J., Vanden Heuvel, J. P., Perdew, G. H., & Peters, J.

M. (2011). Xenobiotic metabolism, disposition, and regulation

by receptors: From biochemical phenomenon to predictors of

major toxicities. Toxicological Sciences, 120(Suppl 1), S49–S75.

Petroff, B. K., Valdez, K. E., Brown, S. B., Piasecka, J., & Albertini,

D. F. (2011). The aryl hydrocarbon receptor agonist 2,3,7,8-

tetrachloro-dibenzo-p-dioxin (TCDD) alters early embryonic

development in a rat IVF exposure model. Reproductive

Toxicology, 32, 286–292.

Polanska, K., Hanke, W., Sobala, W., Brzeznicki, S., & Ligocka, D.

(2009). Exposure of smoking pregnant women to polycyclic

aromatic hydrocarbons. Medycyna Pracy, 60, 103–108.

Radio, N. M., Breier, J. M., Shafer, T. J., & Mundy, W. R. (2008).

Assessment of chemical effects on neurite outgrowth in PC12

cells using high content screening. Toxicological Sciences, 105,

106–118.

Rodgman, A., Smith, C. J., & Perfetti, T. A. (2000). The composition

of cigarette smoke: A retrospective, with emphasis on polycyclic

components. Human and Experimental Toxicology, 19, 573–595.

Sanchez-Ramos, J. R., Song, S., Kamath, S. G., Zigova, T., Willing,

A., Cardozo-Pelaez, F., et al. (2001). Expression of neural

markers in human umbilical cord blood. Experimental Neurol-

ogy, 171, 109–115.

Schreiber, T., Gassmann, K., Gotz, C., Hubenthal, U., Moors, M.,

Krause, G., et al. (2010). Polybrominated diphenyl ethers induce

developmental neurotoxicity in a human in vitro model:

Evidence for endocrine disruption. Environmental Health Per-

spectives, 118, 572–578.

Scobioala, S., Klocke, R., Kuhlmann, M., Tian, W., Hasib, L.,

Milting, H., et al. (2008). Up-regulation of nestin in the infarcted

myocardium potentially indicates differentiation of resident

cardiac stem cells into various lineages including cardiomyo-

cytes. FASEB J, 22, 1021–1231.

Shao, J., Stapleton, P. L., Lin, Y. S., & Gallagher, E. P. (2007).

Cytochrome p450 and glutathione s-transferase mRNA expres-

sion in human fetal liver hematopoietic stem cells. Drug

Metabolism and Disposition, 35, 168–175.

Shimada, T., Inoue, K., Suzuki, Y., Kawai, T., Azuma, E., Nakajima,

T., et al. (2002). Aryl hydrocarbon receptor-dependent induction

of liver and lung cytochromes P450 1A1, 1A2, and 1B1 by

polycyclic aromatic hydrocarbons and polychlorinated biphenyls

in genetically engineered C57BL/6J mice. Carcinogenesis, 23,

1199–1207.

Siddiqui, M. A., Singh, G., Kashyap, M. P., Khanna, V. K., Yadav, S.,

Chandra, D., et al. (2008). Influence of cytotoxic doses of

4-hydroxynonenal on selected neurotransmitter receptors in PC-

12 cells. Toxicology in Vitro, 22, 1681–1688.

Singh, A. K., Kashyap, M. P., Jahan, S., Kumar, V., Tripathi, V. K.,

Siddiqui, M. A., et al. (2012). Expression and inducibility of

cytochrome P450s (CYP1A1, 2B6, 2E1, 3A4) in human cord

blood CD34(?) stem cell-derived differentiating neuronal cells.

Toxicological Sciences, 129, 392–410.

Slotkin, T. A., & Seidler, F. J. (2010). Diverse neurotoxicants

converge on gene expression for neuropeptides and their

receptors in an in vitro model of neurodifferentiation: Effects

of chlorpyrifos, diazinon, dieldrin and divalent nickel in PC12

cells. Brain Research, 1353, 36–52.

Talge, N. M., Neal, C., & Glover, V. (2007). Antenatal maternal

stress and long-term effects on child neurodevelopment: How

Neuromol Med

123

Author's personal copy

and why? Journal of Child Psychology and Psychiatry, 48,

245–261.

Tan, Z., Chang, X., Puga, A., & Xia, Y. (2002). Activation of

mitogen-activated protein kinases (MAPKs) by aromatic hydro-

carbons: Role in the regulation of aryl hydrocarbon receptor

(AHR) function. Biochemical Pharmacology, 64, 771–780.

Tayebati, S. K., Di Tullio, M. A., Ricci, A., & Amenta, F. (2009).

Influence of dermal exposure to the pyrethroid insecticide

deltamethrin on rat brain microanatomy and cholinergic/dopa-

minergic neurochemistry. Brain Research, 1301, 180–188.

Thapar, A., Fowler, T., Rice, F., Scourfield, J., van den Bree, M.,

Thomas, H., et al. (2003). Maternal smoking during pregnancy

and attention deficit hyperactivity disorder symptoms in off-

spring. American Journal of Psychiatry, 160, 1985–1989.

Thomas, C. G., Tian, H., & Diamond, J. S. (2011). The relative roles

of diffusion and uptake in clearing synaptically released

glutamate change during early postnatal development. Journal

of Neuroscience, 31, 4743–4754.

Tilson, H. A. (2000). Neurotoxicology risk assessment guidelines:

Developmental neurotoxicology. Neurotoxicology, 21, 189–194.

True, L. D., Zhang, H., Ye, M., Huang, C. Y., Nelson, P. S., von

Haller, P. D., et al. (2010). CD90/THY1 is overexpressed in

prostate cancer-associated fibroblasts and could serve as a cancer

biomarker. Modern Pathology, 23, 1346–1356.

Valentiner, U., Muhlenhoff, M., Lehmann, U., Hildebrandt, H., &

Schumacher, U. (2011). Expression of the neural cell adhesion

molecule and polysialic acid in human neuroblastoma cell lines.

International Journal of Oncology, 39, 417–424.

Wells, P. G., Lee, C. J., McCallum, G. P., Perstin, J., & Harper, P. A.

(2010). Receptor- and reactive intermediate-mediated mecha-

nisms of teratogenesis. Handb Exp Pharmacol, pp 131–162.

Zigova, T., Song, S., Willing, A. E., Hudson, J. E., Newman, M. B.,

Saporta, S., et al. (2002). Human umbilical cord blood cells

express neural antigens after transplantation into the developing

rat brain. Cell Transplantation, 11, 265–274.

Neuromol Med

123

Author's personal copy