Abstract

The status of xenobiotic metabolism in developing human brain cells is not known. The reason is nonavailability of developing human fetal brain. We investigate the applicability of the plasticity potential of human umbilical cord blood stem cells for the purpose. Characterized hematopoietic stem cells are converted into neuronal subtypes in eight days. The expression and substrate-specific catalytic activity of the cytochrome P450s (CYPs) CYP1A1 and 3A4 increased gradually till day 8 of differentiation, whereas CYP2B6 and CYP2E1 showed highest expression and activity at day 4. There was no significant increase in the expression of CYP regulators, namely, aryl hydrocarbon receptor (AHR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), and glutathione-S-transferase (GSTP1-1) during differentiation. Differentiating cells showed significant induction in the expression of CYP1A1, 2B6, 2E1, 3A4, AHR, CAR, PXR, and GSTP1-1 when exposed to rifampin, a known universal inducer of CYPs. The xenobiotic-metabolizing capabilities of these differentiating cells were confirmed by exposing them to the organophosphate pesticide monocrotophos (MCP), a known developmental neurotoxicant, in the presence and absence of a universal inhibitor of CYPs—cimetidine. Early-differentiating cells (day 2) were found to be more vulnerable to xenobiotics than mature well-differentiated cells. For the first time, we report significant expression and catalytic activity of selected CYPs in human cord blood hematopoietic stem cell–derived neuronal cells at various stages of maturity. We also confirm significant induction in the expression and catalytic activity of selected CYPs in human cord blood stem cell–derived differentiating neuronal cells exposed to known CYP inducers and MCP.

Cytochrome P450s (CYPs) have been identified as the functional brain enzymes that catalyze the metabolic activation and detoxification of a variety of xenobiotics reaching the brain (Ferguson and Tyndale, 2011; Nebert and Russell, 2002). Along with xenobiotic metabolism, CYPs are involved in metabolism of various endogenous chemicals, namely, fatty acids, hormones, neurotransmitters, steroids, cholesterol, and vitamins (Liu et al., 2004). Thus, brain CYPs have been reported to play an important role in controlling brain activity, behavior, susceptibility, and neuronal disorders (Ferguson and Tyndale, 2011). Studies have shown that CYPs are not distributed uniformly in the brain, with some brain regions exhibiting higher expressions of CYPs (Dutheil et al., 2008; Dutheil et al., 2009). In vitro studies using primary cultures of rat brain neuronal and glial cells (Kapoor et al., 2006b; Kapoor et al., 2007) and immortal cell lines of neuronal and glial origin (Howard et al., 2003; Stacey and Viviani, 2001) have also shown unequal magnitude of expression and inducibility of CYPs in these cells. Besides the brain cells, the expression and inducibility of various CYPs have also been reported in cultured human bone marrow (Bernauer et al., 2000), macrophages (Quinn et al., 2005), and mononuclear cells (Asghar et al., 2002). Thus, these cultured cells have proven to be a powerful tool to study and elucidate the expression, activity, and regulation of CYPs in the brain. But most previous studies were restricted to the adult brain and/or adult terminally differentiated neuronal and glial cells/cell lines. It has been documented that the developing brain in the fetus is more vulnerable to drugs/environmental chemicals even at doses usually safe in adults (Bal-Price et al., 2010; Grandjean and Landrigan, 2006). In part, this might be due to the metabolic incapability of the developing brain cells during neurogenesis and due to a looser placental barrier, when compared with the blood-brain barrier, against chemical exposures to the developing brain (Bal-Price et al., 2010). However, there is a complete lack of literature on the expression and inducibility of xenobiotic-metabolizing CYPs in the developing brain, because of nonavailability of developing fetal brain tissue during gestation period. Cultured pluripotent stem cells are worthy models to gain mechanistic insights of metabolism and toxicity of xenobiotics. Human leukemia hematopoietic stem cell (HSC) lines KG-1 and U937 have shown significant induction in the mRNA expression of CYPs on exposure to different metabolites of benzene (Henschler and Glatt, 1995). Kousalová et al. (Kousalová et al., 2004) have reported the expression and activity of CYP2E1 in HSCs. Soucek et al. (2005) have reported the constitutive expression (mRNA and protein) of CYP1B1 and CYP2E1in undifferentiated HSCs and progenitor cells. The expression of CYPs at transcriptional level has been reported in fetal liver HSCs (Shao et al., 2007).

To the best of our knowledge, no information is available on the constitutive expression and inducibility of xenobiotic-metabolizing CYPs in developing human brain cells. Thus, the present investigations were conducted to study constitutive expression of selected xenobiotic-metabolizing CYPs (CYP1A1, 2B6, 2E1, and 3A4), regulator receptors (aryl hydrocarbon receptor [AHR], constitutive androstane receptor [CAR], and pregnane X receptor [PXR]), and the phase II metabolizing enzyme glutathione-S-transferase (GSTP1-1) in human umbilical cord blood stem cell–derived neuronal cells all through the development. HSC-derived developing neuronal cells were also used to study the induction/inhibition in the expression of CYPs, regulator receptors, and GSTP1-1 following exposure of cells to CYP inducer/inhibitor, respectively. Responsiveness of differentiating neuronal cells to CYPs, receptors, and GSTP1-1 expression was studied by exposing the cells to a known developmental neurotoxic organophosphate pesticide, monocrotophos (MCP).

MATERIALS AND METHODS

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, MO, 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 PeproTech (Rocky Hill, NJ). All the antibodies were procured from Chemicon International, CA, and Abcam, CA. Culture wares and plastic wares were procured from Nunc, Denmark, and Corning Inc., NY. Autoclaved Milli-Q water was used in all the experiments.

Ethical Clearance for Collection and Transportation of Human Tissues

The entire study was performed following the protocols and procedures approved by the Institutional Human Ethical Committees of Indian Institute of Toxicology Research (IITR), Lucknow, India, and CSM Medical University, Lucknow, India. The informed consent of parents was obtained prior to collecting blood from umbilical cord.

Isolation and Purification of Human Umbilical Cord Blood HSCs

A total of 103 blood samples (approximately 40ml/cord) were collected from the cord vein in sterile containers having anticoagulant citrate dextrose buffer and immediately transported to IITR, Lucknow, India, for further processing. Blood was diluted 1:1 with Dulbecco’s phosphate-buffered saline (PBS) without Ca2+ and Mg2+, pH 7.5 (Stem Cell Technologies). Then, cord blood mononuclear cells were segregated by negative immunodepletion of CD3+, CD14+, CD19+, CD38+, and CD66b+ cells using RossetteSep cord blood CD34 Preenrichment cocktail (Stem Cell Technologies, catalog no. 15631C) as per manufacturer’s instructions, followed by Ficollpaque (1.077g/cm3, Stem Cell Technologies) density-gradient centrifugation (700 × g for 30min). CD34+ HSCs were isolated from mononuclear (1 × 108 cells/ml) cells using automated robotic and magnetic cell separator RoboSep (Stem Cell Technologies, catalog no. 20000) and EasySep human cord blood CD34 positive selection kit (Stem Cell Technologies, catalog no. 18056) having monoclonal bispecific antibodies against human antigen CD34 and iron-core dextran-coated nanoparticles as per manufacturer’s instructions.

Undifferentiated Proliferation and Bulk Production of HSCs

Freshly isolated HSCs were cultured in plastic 25-cm2 ultralow attachment culture flask (Corning Inc.) at a density of 1 × 105 cells/ml in 5ml of serum containing myeloCult medium (Stem Cell Technologies, catalog no. H5150) supplemented with hydrocortisone (10–6M) and other growth factors, viz., rhbFGF (50ng/ml), rhSCF (25ng/ml), rhTPO (25ng/ml), and rhFLT-3 ligand (10ng/ml; PeproTech). Cells were maintained in suspension in humidified atmosphere at 37°C and 5% CO2. Half of the medium was changed biweekly. At each passage, cells were checked for CD34+ marker to ascertain the purity of stem cells. Cells were subcultured at the confluence of 85–90% in Myelocult medium supplemented with hydrocortisone and growth factor cocktails, as mentioned earlier.

Differentiation of HSCs into Neuronal Subtypes

Confluent growing cells were subcultured as per the experimental demand and allowed to adhere in Corning Synthemax six-well culture plate and 75-cm2 culture flask (Corning Inc.). The cells were then induced to differentiate into neuronal subtypes in serum-free neurobasal medium (Gibco-BRL) supplemented with N2, B27 supplements, rhNGF (50ng/ml), rhFGF-basic (50ng/ml), rhTPO (25ng/ml), rhSCF (25ng/ml), and retinoic acid (10–5M) for a period of up to 12 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 and stemness at various points of maturity.

Experimental Design

Identification of Noncytotoxic Doses of Rifampin, Cimetidine, and Monocrotophos

Rifampin and cimetidine, universal inducer and inhibitor of CYPs, respectively, and an organophosphate pesticide—MCP, a known developmental neurotoxicant, were used in the study. Prior to use in the experiments, noncytotoxic doses of these chemicals were ascertained using standard endpoint of cytotoxicity, i.e., MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-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 × 104 cells/well) at various maturity levels (days 0, 2, 4, and 8) were incubated with medium containing different concentrations of cimetidine (50–1000µM), rifampin (50–5000µM), or MCP (10–2 to 10–8M) for 6–72h and the MTT assay was done. Tetrazolium bromide salt (10 µl/well; 5mg/ml of stock in PBS) was added 4h prior to the completion of respective incubation periods. Then, the reaction mixture was carefully taken out and 200 μl of culture-grade dimethylsulfoxide was added to each well by pipetting up and down several times until the content was homogenized. After 10min, the color was read at 550nm using a multiwell microplate reader (Synergy HT; Bio-Tek). The untreated controls were also run simultaneously under identical conditions.

Exposure of Differentiating Cells to Rifampin, Cimetidine, and mcp

Cells at various maturity levels, i.e., at days 0, 2, 4, and 8, were either exposed to 250μM cimetidine (group I), 250μM rifampin (group II), 10–5M of MCP (group III), 250μM cimetidine for 1h followed by 10–5M of MCP (group IV), or 250μM rifampin plus 10–5M MCP concurrently (group V), or left unexposed (group VI). The exposure periods were 3, 6, and 12h for mRNA, protein expression, and enzymatic activity studies, respectively. Cells were harvested following the respective exposures and processed for transcriptional, translational expression, and enzymatic activity studies.

Transcriptional Changes

Transcriptional changes in marker genes associated with neuronal development, stemness, and xenobiotic metabolism were studied in the experimental and control groups. Total RNA was isolated using Gene Elute mammalian total RNA Miniprep Kit (Sigma; catalog no. RTN-70). Total RNA (1 μg) was reverse transcribed into cDNA by SuperScript III first-strand cDNA synthesis kit (Invitrogen Life Science, catalog no. 18080-051). Quantitative real-time PCR (qRT-PCR) for neuronal marker genes was done using TaqMan Low Density Array (TLDA microfluidic card; Applied Biosystems). The expression of marker genes of stemness and xenobiotic metabolism were studied using SYBR Green dye (Applied Biosystems). 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 performed using the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems). Real-time reactions were performed in triplicate wells for each sample and β-actin was used as an internal control to normalize the data. Exposure-induced alterations are expressed in relative quantification.

TABLE 1

Enzyme Activity of CYP1A1 (EROD), CYP2B6 (PROD), CYP2E1 (NDMA-d), and CYP3A4 (EMD) in Differentiating Neuronal Cells at Days 0, 2, 4, and 8

CYPs activity Neuronal maturity
 in days Unexposed
 control Cimetidine
 (12h) Rifampin
 (12h) MCP (12h) Cimetidine (1h),
 then MCP (12h) Coexposure to rifampin
 and MCP (12h) 
EROD activitya 0D 14.7±1.3 6.17±0.52* 22.45±3.2* 24.63±3.7** 19.35±1.77 35.89±4.3** 
2D 18.2±2.0 8.33±0.73* 35.43±4.3** 39.27±4.8** 38.6±3.2** 44.77±4.4** 
4D 20.3±1.8 11.09±0.8** 29.3±3.2** 31.42±3.6** 29.93±3.0** 39.88±4.1** 
8D 24.9±2.7 13.2±1.5** 31.56±2.4 38.13±4.3** 37.79±4.0** 40.77±4.8** 
PROD activityb 0D 18.3±2.1 10.08±0.9** 20.23±2.2 27.3±3.1** 21.8±1.9 35.54±3.6** 
2D 22.2±1.8 13.73±1.7** 28.18±3.0 32.8±3.8** 25.8±2.3 31.72±2.6** 
4D 27.4±2.8 20.18±1.2 35.23±3.3 38.68±4.0* 37.44±4.1* 43.59±4.7** 
8D 24.6±2.5 20.33±1.8 30.47±2.5 31.37±2.6* 32.92±2.9** 34.75±3.0** 
NDMA-d activityc 0D  8.8±0.7 6.32±0.5 9.67±0.8 11.3±1.6 13.17±1.8** 14.12±2.1** 
2D 12.3±1.4 10.8±1.2 15.57±1.0 18.22±1.9** 24.6±2.3** 23.48±2.5** 
4D 15.4±1.7 13.53±1.5 16.34±1.8 24.83±2.3** 23.5±2.0** 31.77±2.8** 
8D 10.2±0.9 9.64±0.8 11.67±1.1 13.39±1.5 18.33±2.2** 15.28±1.7** 
EMD activityd 0D 24.2±2.6 18.73±1.9 36.71±3.8** 39.33±4.4** 38.17±4.1** 41.8±4.4** 
2D 27.6±3.0 21.56±2.6 54.08±6.2** 50.17±4.8** 51.42±5.7** 58.32±6.3** 
4D 30.3±2.4 23.82±1.8 40.48±4.4* 43.7±5.1** 38.77±4.2 45.5±4.8** 
8D 35.8±3.8 27.39±2.8 47.32±5.0* 50.6±5.8* 51.19±5.4** 56.58±5.3** 
CYPs activity Neuronal maturity
 in days Unexposed
 control Cimetidine
 (12h) Rifampin
 (12h) MCP (12h) Cimetidine (1h),
 then MCP (12h) Coexposure to rifampin
 and MCP (12h) 
EROD activitya 0D 14.7±1.3 6.17±0.52* 22.45±3.2* 24.63±3.7** 19.35±1.77 35.89±4.3** 
2D 18.2±2.0 8.33±0.73* 35.43±4.3** 39.27±4.8** 38.6±3.2** 44.77±4.4** 
4D 20.3±1.8 11.09±0.8** 29.3±3.2** 31.42±3.6** 29.93±3.0** 39.88±4.1** 
8D 24.9±2.7 13.2±1.5** 31.56±2.4 38.13±4.3** 37.79±4.0** 40.77±4.8** 
PROD activityb 0D 18.3±2.1 10.08±0.9** 20.23±2.2 27.3±3.1** 21.8±1.9 35.54±3.6** 
2D 22.2±1.8 13.73±1.7** 28.18±3.0 32.8±3.8** 25.8±2.3 31.72±2.6** 
4D 27.4±2.8 20.18±1.2 35.23±3.3 38.68±4.0* 37.44±4.1* 43.59±4.7** 
8D 24.6±2.5 20.33±1.8 30.47±2.5 31.37±2.6* 32.92±2.9** 34.75±3.0** 
NDMA-d activityc 0D  8.8±0.7 6.32±0.5 9.67±0.8 11.3±1.6 13.17±1.8** 14.12±2.1** 
2D 12.3±1.4 10.8±1.2 15.57±1.0 18.22±1.9** 24.6±2.3** 23.48±2.5** 
4D 15.4±1.7 13.53±1.5 16.34±1.8 24.83±2.3** 23.5±2.0** 31.77±2.8** 
8D 10.2±0.9 9.64±0.8 11.67±1.1 13.39±1.5 18.33±2.2** 15.28±1.7** 
EMD activityd 0D 24.2±2.6 18.73±1.9 36.71±3.8** 39.33±4.4** 38.17±4.1** 41.8±4.4** 
2D 27.6±3.0 21.56±2.6 54.08±6.2** 50.17±4.8** 51.42±5.7** 58.32±6.3** 
4D 30.3±2.4 23.82±1.8 40.48±4.4* 43.7±5.1** 38.77±4.2 45.5±4.8** 
8D 35.8±3.8 27.39±2.8 47.32±5.0* 50.6±5.8* 51.19±5.4** 56.58±5.3** 

a(CYP1A1 Activity): Specific activity in pmoles resorufin/min/mg protein.

b(CYP2B6 Activity): Specific activity in pmoles resorufin/min/mg protein.

c(CYP2E1 Activity): Specific activity in nmol HCHO/min /mg protein.

d(CYP3A4 Activity): Specific activity in nmol HCHO/min/mg protein.

*p < 0.05; **p < 0.01. All values are given as mean ± SE of three experiments.

Translational Changes

Immunocytochemical localization for marker proteins of neurons and CYPs. Immunocytochemical localization of marker proteins of neurons and CYPs in differentiating neuronal cells at days 0, 2, 4, and 8 was conducted following the protocol described earlier (Kashyap et al., 2011). Briefly, HSCs (1 × 104 cells/well) were seeded in a laminin (25 µg/ml)-coated eight-well chamber slide (Labtek, Campbell) and incubated for 24h to adhere onto the surface. The adhered HSCs were induced to differentiate into neuronal subtypes up to day 8. At various maturities (days 0, 2, 4, and 8 ), the cells were fixed in 4% paraformaldehyde for 20min. The cells were then washed with PBS twice and incubated for 15min in PBS containing 0.02% Triton X 100 and 0.1% BSA to block the nonspecific binding site. Cells were then washed with PBS and incubated at room temperature for 2h with primary antibodies against specific proteins, viz., rabbit antihuman acetylcholinesterase (1:500), neurofilament-M (1:500), nestin (1:200), synaptophysin (1:500), CYP1A1 (1:750), 2B6 (1:750), 2E1 (1:750), and 3A4 (1:750); and mouse antihuman TUJ-1 (1:500), PSD95 (1:500), NCAM (1:500), NGF (1:500), AHR (1:750), CAR (1:750), PXR (1:750), and GSTP1-1 (1:500; all antibodies were from Chemicon International, except those for TUJ-1, NF-M, and GSTP1-1, which were procured from Stem Cell Technologies, Sigma, and Calbiochem, respectively). All the antibodies were diluted in PBS containing 0.02% Triton X 100 and 0.1% BSA. Following incubation with primary antibodies, cells were washed three times with PBS for 5min each to remove the unbound antibodies. Then, fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse (1:500) and rhodamine-conjugated goat anti-rabbit (1:1000) antibodies were added to each well and kept on a rocker shaker in dark for 2h at room temperature. Cells were then washed with PBS three times for 5min each. Thereafter, the cells were visualized under an upright fluorescence microscope (Nikon Eclipse 80i equipped with Nikon DS-Ri1 12.7-megapixel camera, Japan) using specific filters for FITC and rhodamine. For each marker, 20 randomly selected microscopic fields were captured and analyzed for fluorescence intensity with the help of Leica Qwin 500 Image Analysis Software (Leica, Germany). The values were expressed in mean ± SE of percent area for fluorescence intensity covered. The values of undifferentiated cells at day 0 were used as control to calculate the differentiation-induced alterations in the expression of each marker protein.

Western blot analysis for markers of neuronal cells, stemness, and xenobiotic metabolism. The altered expression of marker proteins of neuronal cells (NES, NeuN, NF-M, NF-H, TUJ-1, PSD95, SYP, GAP43, NGF, BDNF, acetylcholinesterase [ACHE], and NCAM), stemness (CD34, CD133, and NANOG), and xenobiotic metabolism (CYP 1A1, 2B6, 2E1, 3A4, AHR, CAR, PXR, and GSTP1-1) were studied in differentiating cells at days 0, 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 respective exposures, cells were scraped, pelleted, and lysed using CelLytic M Cell Lysis Reagent (Sigma) in the presence of protein inhibitor cocktail (Sigma). After protein estimation by Bradford’s Reagent (Fermentas Inc., Glen Burnie, MD), equal amounts (30 µg/well) of denatured proteins were loaded onto 10% Tricine–SDS gel (Schägger, 2006) and blotted onto polyvinylidene fluoride membrane (Millipore) by wet transfer method using transfer buffer (25mM Tris [pH 8.3], 190mM glycine, and 20% methanol) at 250 mA current for 2h. Nonspecific binding was blocked with 2% BSA and 3% nonfat dry milk powder in TBST (20mM Tris-HCl [pH 7.4], 137mM NaCl, and 0.1% Tween 20) for 2h at 37°C. After blocking, the membranes were incubated overnight at 4°C with primary antibodies specific for CD34, CD133, NANOG, AHR, CAR, PXR, 3A4 (1:1000, Abcam), 1A1, 2B6, 2E1, Nestin, NeuN, NCAM, PSD95, SYP, GAP43, NGF, BDNF, ACHE (1:1000, Chemicon), GSTP1-1 (1:1000, Calbiochem), TUJ-1 (1:1000, Stem Cell Technologies), NF-M, NF-H, and β-actin (1:2000, Sigma) in blocking buffer (pH 7.5). The membrane was then incubated for 2h at room temperature with secondary anti-primary immunoglobulin G (IgG)–conjugated horseradish peroxidase (Chemicon). Then the blots were developed using SuperSignal West Femto Chemiluminescent Substrate (ThermoFisher Scientific) and Bio-Rad Versa Doc Imaging System 4000 (Bio-Rad, Philadelphia, PA). The densitometry for protein-specific bands was conducted in Gel Documentation System (Alpha Innotech) with the help of Alpha Ease FC Stand Alone V.4.0 software.

Enzyme Activity

Following the completion of exposure period (12h), cells were harvested from treatment and control groups and processed for microsome preparation following the protocol described earlier by us (Kapoor et al., 2006a). In brief, cells were scraped and collected in PBS at 4°C and pelleted by centrifuging at 500 × g for 10 min. The cell pellet was resuspended in microsomal dilution buffer containing 0.1% (vol/vol) glycerol, 0.25mM protease inhibitor cocktail, 0.01M EDTA, and 0.1mM dithiothreitol. The cells were then sonicated thrice at 15 Hz for 10 s each. Following sonication, the cells were again centrifuged at 9000 × g for 20min. The supernatant was then further centrifuged at 105,000 × g for 60min, to isolate the microsomal fraction. The microsomal pellet thus obtained was then resuspended in microsomal dilution buffer and protein estimation was done by Bradford’s Reagent (Fermentas Inc.). The activities of 7-ethoxyresorufin-O-deethylase (EROD) for CYP1A1, 7-pentoxyresourfin-O-dealkylase (PROD) for CYP2B6, N-nitrosodimethylamine demethylase (NDMA-d) for CYP2E1, and erythromycin demethylase (EMD) for CYP3A4 were determined following the methods described earlier by us (Kapoor et al., 2006a; Kapoor et al., 2006b; Kapoor et al., 2007; Yadav et al., 2006) using a Perkin Elmer LS 55 Luminescence spectrophotometer.

Statistical Analysis

The results are expressed as mean and SE of means (mean ± SE) for at least three experiments. One-way ANOVA followed by post hoc Dunnett’s test was employed to detect differences between the treated and control groups. A value of p < 0.05 was taken to indicate significant differences.

RESULTS

Isolation, Purification, and Culture of HSCs

Following purification, 80–85% pure population of CD34+ stem cells with more than 95% viability could be achieved. Purified stem cells were cultured in selective myelocult culture medium supplemented with cytokines and growth factor cocktail for more than 4 months up to passages 12–15. At each passage, cells were checked for the expression of stem cell markers. In the cultures without growth factors and cytokines, a rapid decrease in cell number was observed during the first 3 weeks and by week 4, no cells were alive, whereas cells receiving the support of growth factor and cytokine cocktails were found to have progressive and steady increase of cell production. Cells reaching confluence of more than 80% were passaged and transferred to fresh culture flasks for further proliferation up to 12–15 passages. These purified, smaller, and rounded cells increased in size and formed large colonies within 1 week. Some single cells and colonies adhered to the substratum of culture flask. Numerous cells within adhered clusters started to send out clearly distinct smaller cells on their surface or in close vicinity to the cell (Figs. 1A and 1B).

FIG. 1.

Microphotographs of cultured human cord blood stem cells, colony, and neuronal differentiation. Images were captured at ×200 magnification using Leica phase contrast microscope equipped with Leica IM50 software (Leica Microsystems). (A) Isolated purified population of human HSCs in culture at day 0. (B) A full-grown colony of cells with loosely adhered cells in the periphery and dissociating cells. (C) Early-differentiating neuronal cells from HSCs at day 2. (D) Neuronal differentiation in HSCs at day 4. (E) Well-differentiated HSC-derived neuronal cells at day 8. (F) Morphological maturity of neuronal cells at day 12.

FIG. 1.

Microphotographs of cultured human cord blood stem cells, colony, and neuronal differentiation. Images were captured at ×200 magnification using Leica phase contrast microscope equipped with Leica IM50 software (Leica Microsystems). (A) Isolated purified population of human HSCs in culture at day 0. (B) A full-grown colony of cells with loosely adhered cells in the periphery and dissociating cells. (C) Early-differentiating neuronal cells from HSCs at day 2. (D) Neuronal differentiation in HSCs at day 4. (E) Well-differentiated HSC-derived neuronal cells at day 8. (F) Morphological maturity of neuronal cells at day 12.

Differentiation of HSCs Into Neuronal Subtypes

Morphological studies: At day 2 of differentiation, the cytoplasm started retracting to form contracted cell bodies and cytoplasmic extensions. By day 4, the cell bodies increased in size and became spherical with multiple cell processes, exhibiting neuronal appearance. By the end of day 8, more than 95% of cells acquired neuronal morphology, exhibiting a refractive cell body with extended dendrite-like cytoplasmic projections, and in the areas of higher cell density, differentiated cells were arranged in a network-like structure. Beyond 8 days’ differentiation, an insignificant increase in the neurite structure and cell body size was observed. Approximately 5% of cells remained undifferentiated and round in shape (Fig. 1C–F).

Transcriptional Changes in Stem Cell and Neuronal Marker Genes

qRT-PCR analysis revealed that the mRNA expression of markers associated with stemness, viz., CD34, CD133, MYC, NANOG, and SHH were affected significantly (p < 0.001) with progress of differentiation. The quantitative reduction in expression was most severe for CD34, i.e., 1.85±0.08-fold and 0.85±0.01-fold on days 2 and 4, respectively. Expression of CD34 was not detectable by day 8 of differentiation. Expression of CD133 decreased significantly from day 0 (1.02±0.02-fold) to day 8 (0.03±0.001-fold). The expression of SHH could be detected only at day 2 of differentiation (0.31±0.001-fold), when compared with undifferentiated cells at day 0. In case of NANOG, a biphasic response was observed. At days 2 and 4, expression was increased (4.82±0.57-fold and 3.04±0.03-fold, respectively), and, thereafter, at day 8, was reduced to 0.9±0.05-fold of control. However, all the values were significantly higher than those of undifferentiated cells at day 0 (Fig. 2A). The expression of mRNA of neuronal markers increased with progression of differentiation in general, except in the cases of TUJ-1 and NGF. The expression reached the highest levels, i.e., 18.45±1.5-, 34.1±2.36-, 60.88±4.92-, 20.6±1.38-, 4.38±0.21-, and 21.66±1.61-fold of undifferentiated control cells, at day 8 for NCAM, SYP, NF-M, NF-H, BDNF, and PSD95, respectively. Marker associated with early differentiation, viz., nestin (NES), recorded significantly higher expression at day 2 (20.22±1.35-fold), and thereafter, a reduction in subsequent differentiation, i.e., day 4: 16.43±1.22-fold, and day 8: 9.83±1.07-fold. However, the overall expression of nestin was higher than that of undifferentiated cells. Moreover, the expression of NGF increased significantly (56.07±3.29 and 62.15±4.23) by days 2 and 4, respectively, but was reduced by day 8 (25.37±2.45-fold) when compared with the expression in undifferentiated control cells (Fig. 2B). The expression of neurogenesis-related transcription factors, such as CREB (2.25±0.16-fold) and artemin (ARTN, 2.37±0.13), increased all through differentiation and peaked by day 8 (Fig. 2B).

FIG. 2.

Transcriptional changes in differentiating neuronal cells derived from human HSCs. (A) Relative quantification of altered mRNA expression of stemness genes in differentiating neuronal cells at days 0, 2, 4, and 8 (0D, 2D, 4D, and 8D, respectively). β-actin was used as internal 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 (denoted by parallel line). *= p < 0.05, **= p < 0.001. (B) Relative quantification of altered mRNA expression of selected genes involved in neuronal development in differentiating neuronal cells at days 0, 2, 4, and 8. β-actin was used as internal control to normalize the data. The data are expressed in relative quantity (mean ± SE) compared with the expression in cells at day 0 (denoted by parallel line). *= p < 0.05, **= p < 0.001.

FIG. 2.

Transcriptional changes in differentiating neuronal cells derived from human HSCs. (A) Relative quantification of altered mRNA expression of stemness genes in differentiating neuronal cells at days 0, 2, 4, and 8 (0D, 2D, 4D, and 8D, respectively). β-actin was used as internal 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 (denoted by parallel line). *= p < 0.05, **= p < 0.001. (B) Relative quantification of altered mRNA expression of selected genes involved in neuronal development in differentiating neuronal cells at days 0, 2, 4, and 8. β-actin was used as internal control to normalize the data. The data are expressed in relative quantity (mean ± SE) compared with the expression in cells at day 0 (denoted by parallel line). *= p < 0.05, **= p < 0.001.

Translational Changes in Neuronal and Stem Cell Markers

Data of Western blot analyses are presented in Figure 3A. Data show the significant alterations in stemness markers, viz., CD34 (0.83-, 0.32-, and 0.05-fold of undifferentiated control), CD133 (0.27-, 0.06-, and 0.03-fold of undifferentiated control), NANOG (1.18-, 1.05-, and 0.69-fold of undifferentiated control) at days 2, 4, and 8 of differentiation, respectively. Early-differentiation marker nestin shows an initial increase, i.e., 6.93-fold, at day 2 of differentiation but this was down to 3.88- and 3.02-fold of control at days 4 and 8 of differentiation, respectively. A gradual increase in the expression of mature neuronal marker proteins was observed all through differentiation, which peaked by day 8, i.e., NeuN (14.17-fold), NF-M (4.23-fold), NF-H (15.5-fold), PSD95 (12.23-fold), and GAP43 (21.97-fold) of control, respectively. Markers such as TUJ-1, SYP, NGF, BDNF, and NCAM have shown maximum expression by day 4 of differentiation, i.e., 5.83-, 11.31-, 5.22-, 4.07-, and 9.77-fold of control, respectively. The continuous increase in the expression of ACHE, i.e., 9.99-, 15.34-, and 16.42-fold of control at days 2, 4, and 8, respectively, indicate the formation of acetyl cholinesterase–expressing neuronal cells. Similar trends in the expression of these neuronal marker proteins are also observed in immunocytochemical localization (Figs. 3B and 3C).

FIG. 3.

Translational changes in differentiating neuronal cells derived from human HSCs. (A) Western blot analysis of expression of selected marker proteins of stemness and neuronal development in stem cell–derived neuronal cells all through differentiation. The values obtained at day 0 were considered basal, i.e., relative quantification in expression at days 2, 4, and 8 (0D, 2D, 4D, and 8D, respectively) was done comparing the values at day 0. β-actin was used as internal control to normalize the data. (B) Immunocytochemical localization for the quantification of alterations in the expression of proteins of selected neuronal markers in stem cell–derived neuronal cells at various stages of maturity. Data are expressed as mean ± SE of percent area of expression in the snapped microscopic fields. A minimum of 20 microscopic fields were snapped for each group. *= p < 0.05, **= p < 0.001. (C) Representative microphotographs of immunocytochemical localization of neuronal marker proteins in stem cell–derived neuronal cells at various maturity, i.e., days 0, 2, 4, and 8 (0D, 2D, 4D, and 8D, respectively). Images were captured at ×400 magnification using Nikon Eclipse 80i upright fluorescence microscope equipped with Nikon DS-Ri1 12.7 megapixel camera, Japan. The specific filters used were for FITC and rhodamine. The captured photographs were analyzed for fluorescence intensity with the help of Leica Qwin 500 Image Analysis Software (Leica, Germany).

FIG. 3.

Translational changes in differentiating neuronal cells derived from human HSCs. (A) Western blot analysis of expression of selected marker proteins of stemness and neuronal development in stem cell–derived neuronal cells all through differentiation. The values obtained at day 0 were considered basal, i.e., relative quantification in expression at days 2, 4, and 8 (0D, 2D, 4D, and 8D, respectively) was done comparing the values at day 0. β-actin was used as internal control to normalize the data. (B) Immunocytochemical localization for the quantification of alterations in the expression of proteins of selected neuronal markers in stem cell–derived neuronal cells at various stages of maturity. Data are expressed as mean ± SE of percent area of expression in the snapped microscopic fields. A minimum of 20 microscopic fields were snapped for each group. *= p < 0.05, **= p < 0.001. (C) Representative microphotographs of immunocytochemical localization of neuronal marker proteins in stem cell–derived neuronal cells at various maturity, i.e., days 0, 2, 4, and 8 (0D, 2D, 4D, and 8D, respectively). Images were captured at ×400 magnification using Nikon Eclipse 80i upright fluorescence microscope equipped with Nikon DS-Ri1 12.7 megapixel camera, Japan. The specific filters used were for FITC and rhodamine. The captured photographs were analyzed for fluorescence intensity with the help of Leica Qwin 500 Image Analysis Software (Leica, Germany).

Constitutive Expressions (mRNA and Protein) of CYPs in Differentiating Neuronal Cells

Real-time data reveal the significant upregulation in mRNA expression levels of CYP1A1, 2B6, 2E1, and 3A4 mRNA during neuronal differentiation (Fig. 4A). The expression of mRNA for CYP1A1 and 3A4 recorded a gradual increase from day 2 (1.38±0.5 and 1.83±0.1-fold) of neurogenesis to day 4 (9.12±0.5 and 8.72±1.64-fold), peaking at day 8 (58.8±3.52 and 29.51±3.58-fold), when compared with day 0 control. The expression of CYP2B6 and 2E1 peaked at day 4 (37.3±3.0 and 11±0.83-fold), was significantly induced by day 2 (16.0±1.85 and 7.0±0.64-fold), and reached a minimum at day 8 (7.2±1.02 and 4.0±0.46-fold). Among the CYP regulator receptors, only CAR was found to have significant induction in mRNA expression.

FIG. 4.

Constitutive expression of cytochrome P450s (CYPs), their regulator receptors, and GSTP1-1 in differentiating neuronal cells. (A) Relative quantification of altered mRNA expression of CYPs, GSTP1-1, and regulator receptors in differentiating neuronal cells at days 2, 4, and 8 (2D, 4D, and 8D, respectively). β-actin was used as internal control to normalize the data. The data are expressed in relative quantity (mean ± SE) compared with the expression at day 0 (denoted by parallel line). *= p < 0.05, **= p < 0.001. (B) Western blot analysis for relative quantification of altered protein expression of CYPs, GSTP1-1, and regulator receptors in differentiating neuronal cells at days 2, 4, and 8 (2D, 4D, and 8D, respectively). β-actin was used as internal control to normalize the data. The data are expressed in relative quantity compared with the expression at day 0. (C) Immunocytochemical localization for relative quantification of altered protein expression of CYPs, GSTP1-1, and regulator receptors in differentiating neuronal cells at days 2, 4, and 8 (2D, 4D, and 8D, respectively). Data are expressed in mean ± SE of percent area of expression in the snapped microscopic fields. A minimum of 20 microscopic fields were snapped for each group. *= p < 0.05, **= p < 0.001. (D) Representative microphotographs of immunocytochemical localization of proteins of CYPs, GSTP1-1, and regulator receptors in stem cell–derived neuronal cells at various maturity, i.e., days 0, 2, 4, and 8 (0D, 2D, 4D, and 8D, respectively). Images were captured at ×400 magnification using Nikon Eclipse 80i upright fluorescence microscope equipped with Nikon DS-Ri1 12.7 megapixel camera, Japan. The specific filters used were for FITC and rhodamine. The captured photographs were analyzed for fluorescence intensity with the help of Leica Qwin 500 Image Analysis Software (Leica, Germany).

FIG. 4.

Constitutive expression of cytochrome P450s (CYPs), their regulator receptors, and GSTP1-1 in differentiating neuronal cells. (A) Relative quantification of altered mRNA expression of CYPs, GSTP1-1, and regulator receptors in differentiating neuronal cells at days 2, 4, and 8 (2D, 4D, and 8D, respectively). β-actin was used as internal control to normalize the data. The data are expressed in relative quantity (mean ± SE) compared with the expression at day 0 (denoted by parallel line). *= p < 0.05, **= p < 0.001. (B) Western blot analysis for relative quantification of altered protein expression of CYPs, GSTP1-1, and regulator receptors in differentiating neuronal cells at days 2, 4, and 8 (2D, 4D, and 8D, respectively). β-actin was used as internal control to normalize the data. The data are expressed in relative quantity compared with the expression at day 0. (C) Immunocytochemical localization for relative quantification of altered protein expression of CYPs, GSTP1-1, and regulator receptors in differentiating neuronal cells at days 2, 4, and 8 (2D, 4D, and 8D, respectively). Data are expressed in mean ± SE of percent area of expression in the snapped microscopic fields. A minimum of 20 microscopic fields were snapped for each group. *= p < 0.05, **= p < 0.001. (D) Representative microphotographs of immunocytochemical localization of proteins of CYPs, GSTP1-1, and regulator receptors in stem cell–derived neuronal cells at various maturity, i.e., days 0, 2, 4, and 8 (0D, 2D, 4D, and 8D, respectively). Images were captured at ×400 magnification using Nikon Eclipse 80i upright fluorescence microscope equipped with Nikon DS-Ri1 12.7 megapixel camera, Japan. The specific filters used were for FITC and rhodamine. The captured photographs were analyzed for fluorescence intensity with the help of Leica Qwin 500 Image Analysis Software (Leica, Germany).

Western blot analyses for translational changes have shown similar trends as the transcriptional level (Fig. 4B). The expression of protein for CYP1A1 and 3A4 gradually increased from day 2 (1.85- and 3.03-fold of control) of neurogenesis to day 4 (2.25- and 4.69-fold of control) and peaked at day 8 (3.00- and 6.16-fold of control). The protein expression of CYP2B6 and 2E1 peaked at day 4 (3.73- and 2.12-fold of control), remained significantly elevated at day 8 (2.54- and 1.4-fold of control), followed by day 2 (1.72- and 1.63-fold of control). Unlike the transcriptional changes, the CYP regulator receptors (CAR and PXR) were significantly upregulated all through differentiation.

The findings of immunocytochemical localization (Figs. 4C and 4D) further confirm the trend of Western blot analyses, as a gradual increase in the expression of CYP1A1 and 3A4 proteins was recorded; this reached a maximum at day 8 (37.2% ± 1.1 and 31.45% ± 2.58 of control). The expression pattern of immunocytochemical localization for most of the proteins studied was similar for Western blot analyses and transcriptional expression levels.

Constitutive Catalytic Activity of CYPs in Differentiating Neuronal Cells

The substrate-specific CYP catalytic activity reveals the increase in metabolic capability of the cells upon differentiation (Table 2). The EROD and EMD activity for CYP1A1 and 3A4 recorded a gradual increase from day 2 (18.2±2.0 pmoles resorufin/min/mg protein and 27.6±3.0nM HCHO/min/mg protein) of neurogenesis to day 4 (20.3±1.8 pmoles resorufin/min/mg protein and 30.3±2.4nM HCHO/min/mg protein), peaking at day 8 (24.9±2.7 pmoles resorufin/min/mg protein and 35.8±3.8nM HCHO/min/mg protein) when compared with day 0 control (14.7±1.3 pmoles resorufin/min/mg protein and 24.2±2.6nM HCHO/min/mg protein). The PROD and NDMA-d activity peaked at day 4 (27.4±2.8 pmoles resorufin/min/mg protein and 15.4±1.7nM HCHO/min/mg protein), was significantly induced by day 2 (22.2±1.8 pmoles resorufin/min/mg protein and 12.3±1.4nM HCHO/min/mg protein), and reached a minimum at day 8 (21.6±1.8 pmoles resorufin/min/mg protein and 10.2±0.9nM HCHO/min/mg protein).

TABLE 2

Real-Time PCR Primer Sequences for Genes of Human Stem Cell Markers, Cytochrome P450s, Their Receptor Regulators, and GSTP1-1

Genes Forward primer Reverse primer Reference 
CD34 5′-gccctgctggctgtcttg-3′ 5′-gctgcggcgattcatca-3′ Primer express 3.0 
CD133 5′-cgtgattttttactacctgg-3′ 5′-gggtggcatgcctgtcatag-3′ Primer express 3.0 
NANOG 5′-aaatctaagaggtggcagaa-3′ 5′-cttctgcgtcacaccattgc-3′ Primer express 3.0 
CYP1A1 5′-accttccgacactcttccttcg-3′ 5′-aagcggatttgtcttggtgaa-3′ Shao et al. (2007
CYP2B6 5′-tggaggatggtggtgaagaag-3′ 5′-tgccatcaaggataggcaag-3′ Primer express 3.0 
CYP2E1 5′-tgccatcaaggataggcaag-3′ 5′-caacaaaagaaacaactccatga-3′ Primer express 3.0 
CYP3A4 5′-cacagatccccctgaaattaagctta-3′ 5′-aaaattcaggctccacttacggtg-3′ Shao et al. (2007) 
AHR 5′-cggctgggcaccatga-3′ 5′-ctgggattggctttactgttttct-3′ Primer express 3.0 
CAR 5′-cacatgggcaccatgtttga-3′ 5′-aagggctggtgatggatgaa-3′ Primer express 3.0 
GSTP1-1 5′-gtagtttgcccaaggtcaag-3′ 5′-agccacctgaggggtaag-3′ Primer express 3.0 
β-Actin 5′-aaccccaaggccaaccg-3′ 5′-agggatagcacagcctgga-3′ Shao et al. (2007) 
Genes Forward primer Reverse primer Reference 
CD34 5′-gccctgctggctgtcttg-3′ 5′-gctgcggcgattcatca-3′ Primer express 3.0 
CD133 5′-cgtgattttttactacctgg-3′ 5′-gggtggcatgcctgtcatag-3′ Primer express 3.0 
NANOG 5′-aaatctaagaggtggcagaa-3′ 5′-cttctgcgtcacaccattgc-3′ Primer express 3.0 
CYP1A1 5′-accttccgacactcttccttcg-3′ 5′-aagcggatttgtcttggtgaa-3′ Shao et al. (2007
CYP2B6 5′-tggaggatggtggtgaagaag-3′ 5′-tgccatcaaggataggcaag-3′ Primer express 3.0 
CYP2E1 5′-tgccatcaaggataggcaag-3′ 5′-caacaaaagaaacaactccatga-3′ Primer express 3.0 
CYP3A4 5′-cacagatccccctgaaattaagctta-3′ 5′-aaaattcaggctccacttacggtg-3′ Shao et al. (2007) 
AHR 5′-cggctgggcaccatga-3′ 5′-ctgggattggctttactgttttct-3′ Primer express 3.0 
CAR 5′-cacatgggcaccatgtttga-3′ 5′-aagggctggtgatggatgaa-3′ Primer express 3.0 
GSTP1-1 5′-gtagtttgcccaaggtcaag-3′ 5′-agccacctgaggggtaag-3′ Primer express 3.0 
β-Actin 5′-aaccccaaggccaaccg-3′ 5′-agggatagcacagcctgga-3′ Shao et al. (2007) 

Cytotoxicity Assessment

In general, there was no significant variation in the sensitivity of cells against exposure to cimetidine, rifampin, and MCP at days 0, 2, 4, and 8 of differentiation. All the concentrations (50–250μM) of cimetidine were found to be noncytotoxic, whereas dose-dependent decrease in percent cell viability was recorded in cells exposed to 500 and 1000μM of cimetidine (Fig. 5). Rifampin was found safe upto the concentration of 250μM at all time points of exposure (Fig. 6). MCP (10–8–10–5M) exposure up to 12h was not cytotoxic. MCP (10–6–10–2M) exposure for 48 and 72h showed a dose-dependent cytotoxic response (Fig. 7).

FIG. 5.

Identification of noncytotoxic doses of cimetidine in stem cell–derived neuronal cells at various stages of differentiation, i.e., at days 0, 2, 4, and 8 (0D, 2D, 4D, and 8D, respectively). Cells were exposed to cimetidine (50–1000μM) for 6–72h. 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.

*= p < 0.05, **= p < 0. 001.

FIG. 5.

Identification of noncytotoxic doses of cimetidine in stem cell–derived neuronal cells at various stages of differentiation, i.e., at days 0, 2, 4, and 8 (0D, 2D, 4D, and 8D, respectively). Cells were exposed to cimetidine (50–1000μM) for 6–72h. 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.

*= p < 0.05, **= p < 0. 001.

FIG. 6.

Identification of noncytotoxic doses of rifampin in stem cell–derived neuronal cells at various stages of differentiation, i.e., at days 0, 2, 4, and 8 (0D, 2D, 4D, and 8D, respectively). Cells were exposed to rifampin (50–5000μM) for 6–72h. 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.

*= p < 0.05, **= p < 0. 001.

FIG. 6.

Identification of noncytotoxic doses of rifampin in stem cell–derived neuronal cells at various stages of differentiation, i.e., at days 0, 2, 4, and 8 (0D, 2D, 4D, and 8D, respectively). Cells were exposed to rifampin (50–5000μM) for 6–72h. 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.

*= p < 0.05, **= p < 0. 001.

FIG. 7.

Identification of noncytotoxic doses of MCP in stem cell–derived neuronal cells at various stages of differentiation i.e., at days 0, 2, 4, and 8 (0D, 2D, 4D, and 8D, respectively). Cells were exposed to MCP (10-2 to 10-8M) for 6-72h. 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. *= p < 0.05, **= p < 0. 001.

FIG. 7.

Identification of noncytotoxic doses of MCP in stem cell–derived neuronal cells at various stages of differentiation i.e., at days 0, 2, 4, and 8 (0D, 2D, 4D, and 8D, respectively). Cells were exposed to MCP (10-2 to 10-8M) for 6-72h. 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. *= p < 0.05, **= p < 0. 001.

Xenobiotic-Induced Alterations in the Expression (mRNA and Protein) of CYPs and GSTP1-1 in Differentiating Neuronal Cells

In real-time PCR, expression of mRNA in control cells was compared with that in cells exposed to rifampin (250µM), MCP (10–5M), or coexposure to rifampin and MCP for 3h. For Western blot analyses, cells were exposed to either cimetidine (250µM), rifampin (250µM), MCP (10–5M), cimetidine exposure followed by MCP exposure, and coexposure to rifampin and MCP for 6h.

Day 0: The expression of mRNAs of CYP1A1, CYP2B6, CYP2E1, and CYP3A4 was induced significantly (1.89±0.12-, 2.88±0.17-, 3.54±0.49-, and 4.7±0.56-fold of respective controls) following the exposure to rifampin, a universal inducer of CYPs. However, rifampin could not induce significant alterations in the expression of mRNAs of AHR, CAR, and GSTP1-1 (Fig. 8A). Stem cells showed significant responsiveness to MCP exposure as expression levels of mRNA for CYP1A1 (1.77±0.1-fold), CYP2B6 (5.54±0.63-fold), CYP2E1 (2.53±0.16-fold), CYP3A4 (5.23±0.6-fold), and GSTP1-1 (2.07±0.11-fold) were elevated significantly. Coexposure to rifampin and MCP was found to be additive significantly, leading to the induction of mRNA expression of CYP1A1 (3.4±0.24-fold), CYP3A4 (5.44±0.25-fold), CAR (2.24±0.18-fold), and GSTP1-1 (2.65±0.22-fold).

FIG. 8.

Altered expression of CYPs, receptors, and GSTP1-1 in differentiating neurons (day 0) exposed to xenobiotics. (A) MCP and/or rifampin–induced alterations in the mRNA expression of CYPs, their regulator receptors, and GSTP1-1 genes in human HSCs (day 0). β-actin was used as internal control to normalize the data. MCP and/or rifampin–induced alterations in mRNA expression are expressed in relative quantity compared with respective unexposed control groups (denoted by parallel line). 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) MCP/rifampin/cimetidine exposure–associated alterations in the protein expression of CYPs, their regulator receptors, and differentiation into neuronal cells (day 0). β-actin was used as internal control to normalize the data. Xenobiotic-induced alterations in protein expression are expressed in relative quantity compared with respective unexposed control groups. Lane 1: unexposed control; Lane 2: cells exposed to cimetidine (250μM) for 6h; Lane 3: cells exposed to rifampin (250μM) for 6h; Lane 4: cells exposed to MCP (10–5M) for 6h; Lane 5: cells exposed to cimetidine for 1h, then removal of cimetidine and reexposure to MCP (10–5M) for 6h; Lane 6: cells receiving coexposure to rifampin and MCP for 6h.

FIG. 8.

Altered expression of CYPs, receptors, and GSTP1-1 in differentiating neurons (day 0) exposed to xenobiotics. (A) MCP and/or rifampin–induced alterations in the mRNA expression of CYPs, their regulator receptors, and GSTP1-1 genes in human HSCs (day 0). β-actin was used as internal control to normalize the data. MCP and/or rifampin–induced alterations in mRNA expression are expressed in relative quantity compared with respective unexposed control groups (denoted by parallel line). 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) MCP/rifampin/cimetidine exposure–associated alterations in the protein expression of CYPs, their regulator receptors, and differentiation into neuronal cells (day 0). β-actin was used as internal control to normalize the data. Xenobiotic-induced alterations in protein expression are expressed in relative quantity compared with respective unexposed control groups. Lane 1: unexposed control; Lane 2: cells exposed to cimetidine (250μM) for 6h; Lane 3: cells exposed to rifampin (250μM) for 6h; Lane 4: cells exposed to MCP (10–5M) for 6h; Lane 5: cells exposed to cimetidine for 1h, then removal of cimetidine and reexposure to MCP (10–5M) for 6h; Lane 6: cells receiving coexposure to rifampin and MCP for 6h.

Western blot analyses showed that cimetidine could not significantly block the expression of the CYP proteins, except in the case of CYP1A1 (0.39-fold of control respectively); rather, the cells showed upregulation of GSTP1-1 (1.22-fold of control) and AHR (2.65-fold of control). Rifampin exposure was capable of inducing significant protein expression of CYP1A1 (1.91-fold of control) and AHR (8.63-fold of control). MCP exposure induced significant protein expression of CYP1A1 (1.86-fold of control), CYP2B6 (1.41-fold of control), GSTP1-1 (1.9-fold of control), and AHR (7.14-fold of control). Preexposure to cimetidine could not directly influence the CYP induction in MCP-exposed cells, except in the case of CYP1A1, CYP2B6, and AHR. Rather, the induction in the protein expression of CYP3A4 and GSTP1-1 was found to be more in cimetidine-preexposed cells receiving MCP exposure than in cells receiving MCP alone. Additive effect of coexposure to rifampin and MCP was clearly seen in the protein expression of CYP2B6 (4.1-fold of control), CYP2E1 (1.69-fold of control), and CAR (1.68-fold of control; Fig. 8B).

Day 2: Cells receiving rifampin exposure showed statistically significant upregulation in the expression of mRNAs of all the genes studied. Cells were also responsive to MCP, as mRNA expression of all the genes were upregulated, i.e., CYP1A1 (11.87±0.48-fold), CYP2B6 (6.24±0.51-fold), CYP2E1 (3.43±0.25-fold), CYP3A4 (9.73±0.77-fold), AHR (2.31±0.22-fold), CAR (2.48±0.08-fold), and GSTP1-1 (6.56±0.2-fold) of control. Though the overall magnitude of induction was higher than the levels induced by rifampin, in general, coexposure to rifampin and MCP showed statistically highly significant additive effect in the induction of mRNA expression of all the genes studied except GSTP1-1 (Fig. 9A).

FIG. 9.

Altered expression of CYPs, receptors, and GSTP1-1 in differentiating neurons (day 2) exposed to xenobiotics. (A) MCP and/or rifampin–induced alterations in the mRNA expression of CYPs, their regulator receptors, and GSTP1-1 genes in stem cell–derived neuronal cells at day 2 of differentiation. β-actin was used as internal control to normalize the data. MCP and/or rifampin–induced alterations in mRNA expression are expressed in relative quantity compared with respective unexposed control groups (denoted by parallel line). 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) MCP/rifampin/cimetidine exposure–associated alterations in the protein expression of CYPs, their regulator receptors, and GSTP1-1 in stem cell–derived neuronal cells at day 2 of differentiation. β-actin was used as internal control to normalize the data. Xenobiotic-induced alterations in protein expression are expressed in relative quantity compared with respective unexposed control groups. Lane 1: unexposed control; Lane 2: cells exposed to cimetidine (250μM) for 6h; Lane 3: cells exposed to rifampin (250μM) for 6h; Lane 4: cells exposed to MCP (10–5M) for 6h; Lane 5: cells exposed to cimetidine for 1h, then removal of cimetidine and reexposure to MCP (10–5M) for 6h; Lane 6: cells receiving coexposure to rifampin and MCP for 6h.

FIG. 9.

Altered expression of CYPs, receptors, and GSTP1-1 in differentiating neurons (day 2) exposed to xenobiotics. (A) MCP and/or rifampin–induced alterations in the mRNA expression of CYPs, their regulator receptors, and GSTP1-1 genes in stem cell–derived neuronal cells at day 2 of differentiation. β-actin was used as internal control to normalize the data. MCP and/or rifampin–induced alterations in mRNA expression are expressed in relative quantity compared with respective unexposed control groups (denoted by parallel line). 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) MCP/rifampin/cimetidine exposure–associated alterations in the protein expression of CYPs, their regulator receptors, and GSTP1-1 in stem cell–derived neuronal cells at day 2 of differentiation. β-actin was used as internal control to normalize the data. Xenobiotic-induced alterations in protein expression are expressed in relative quantity compared with respective unexposed control groups. Lane 1: unexposed control; Lane 2: cells exposed to cimetidine (250μM) for 6h; Lane 3: cells exposed to rifampin (250μM) for 6h; Lane 4: cells exposed to MCP (10–5M) for 6h; Lane 5: cells exposed to cimetidine for 1h, then removal of cimetidine and reexposure to MCP (10–5M) for 6h; Lane 6: cells receiving coexposure to rifampin and MCP for 6h.

At the translational level, cimetidine inhibited the expression of all the xenobiotic-metabolizing proteins and their regulators, except CYP2E1 (1.22-fold of control). Except CYP2B6 and AHR, rifampin exposure induced upregulation in the expression of protein of all the CYPs and regulators. Similar to rifampin, MCP also induced significant protein expression of all the CYPs and GSTP1-1. However, there was a reduction in the protein expression of all the CYP regulators, i.e., AHR, CAR, and PXR. Cimetidine pretreatment did not cause significant effect on CYP responsiveness against MCP exposure except in the case of CYP1A1. Coexposure to rifampin and MCP showed additive effect in the induction of most of the CYPs and regulators studied (Fig. 9B).

Day 4: More or less similar levels of induction in the expression of mRNAs of all the CYP genes and regulators were observed against exposure to rifampin/MCP. The upregulation of these genes was below threefold of control, except in the case of CYP2E1 and CAR (7.8±0.88- and 4.68±0.51-fold of control, respectively) following exposure to MCP. Following coexposure to rifampin and MCP, a clear-cut and statistically highly significant (p < 0.001) additive effect in the upregulation of mRNA expression was observed for all the genes studied, viz., CYP1A1 (8.88±0.62-fold), 2B6 (7.74±0.82-fold), 2E1 (5.02±0.63-fold), 3A4 (4.32±0.46-fold), and GSTP1-1 (2.66±0.16-fold; Fig. 10A).

FIG. 10.

Altered expression of CYPs, receptors, and GSTP1-1 in differentiating neurons (day 4) exposed to xenobiotics. (A) MCP and/or rifampin–induced alterations in the mRNA expression of CYPs, their regulator receptors, and GSTP1-1 genes in stem cell–derived neuronal cells at day 4 of differentiation. β-actin was used as internal control to normalize the data. MCP and/or rifampin–induced alterations in mRNA expression are expressed in relative quantity compared with respective unexposed control groups (denoted by parallel line). 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) MCP/rifampin/cimetidine exposure–associated alterations in the protein expression of CYPs, their regulator receptors, and GSTP1-1 in stem cell–derived neuronal cells at day 4 of differentiation. β-actin was used as internal control to normalize the data. Xenobiotic–induced alterations in protein expression are expressed in relative quantity compared with respective unexposed control groups. Lane 1: unexposed control; Lane 2: cells exposed to cimetidine (250μM) for 6h; Lane 3: cells exposed to rifampin (250μM) for 6h; Lane 4: cells exposed to MCP (10–5M) for 6h; Lane 5: cells exposed to cimetidine for 1h, then removal of cimetidine and reexposure to MCP (10–5M) for 6h; Lane 6: cells receiving coexposure to rifampin and MCP for 6h.

FIG. 10.

Altered expression of CYPs, receptors, and GSTP1-1 in differentiating neurons (day 4) exposed to xenobiotics. (A) MCP and/or rifampin–induced alterations in the mRNA expression of CYPs, their regulator receptors, and GSTP1-1 genes in stem cell–derived neuronal cells at day 4 of differentiation. β-actin was used as internal control to normalize the data. MCP and/or rifampin–induced alterations in mRNA expression are expressed in relative quantity compared with respective unexposed control groups (denoted by parallel line). 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) MCP/rifampin/cimetidine exposure–associated alterations in the protein expression of CYPs, their regulator receptors, and GSTP1-1 in stem cell–derived neuronal cells at day 4 of differentiation. β-actin was used as internal control to normalize the data. Xenobiotic–induced alterations in protein expression are expressed in relative quantity compared with respective unexposed control groups. Lane 1: unexposed control; Lane 2: cells exposed to cimetidine (250μM) for 6h; Lane 3: cells exposed to rifampin (250μM) for 6h; Lane 4: cells exposed to MCP (10–5M) for 6h; Lane 5: cells exposed to cimetidine for 1h, then removal of cimetidine and reexposure to MCP (10–5M) for 6h; Lane 6: cells receiving coexposure to rifampin and MCP for 6h.

Cimetidine exposure caused a range of downregulations in the protein expression of all the CYPs and their regulators studied. MCP exposure was found to induce the expression of all the proteins studied. Interestingly, pretreatment with cimetidine was found to induce the responsiveness of cells receiving MCP exposure. Additive response of coexposure to rifampin and MCP was clearly seen in the upregulation of protein expression of all CYPs, GSTP1-1, and CAR (Fig. 10B).

Day 8: Similar to day 4, the expression levels of mRNAs were more pronounced for all the CYPs and their regulators only in cells coexposed to rifampin and MCP (Fig. 11A). At the translational level, cimetidine inhibited the protein expression of all the CYPs except CYP3A4 and AHR. Rifampin/MCP also showed a range of induction in protein expression of different CYPs, with the maximum induction for CYP3A4, i.e., 2.35- and 3.00-fold of control, respectively. As at other time points, at day 8 of differentiation, MCP-rifampin coexposure showed additive effects in the upregulated protein expression of all the CYPs and regulators except CYP2E1 and PXR (Fig. 11B).

FIG. 11.

Altered expression of CYPs, receptors, and GSTP1-1 in differentiating neurons (day 8) exposed to xenobiotics. (A) MCP and/or rifampin–induced alterations in the mRNA expression of CYPs, their regulator receptors, and GSTP1-1 genes in stem cell–derived neuronal cells at day 8 of differentiation. β-actin was used as internal control to normalize the data. MCP and/or rifampin–induced alterations in mRNA expression are expressed in relative quantity compared with respective unexposed control groups (denoted by parallel line). 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) MCP/rifampin/cimetidine exposure–associated alterations in the protein expression of CYPs, their regulator receptors, and GSTP1-1 in stem cell–derived neuronal cells at day 8 of differentiation. β-actin was used as internal control to normalize the data. Xenobiotic-induced alterations in protein expression are expressed in relative quantity compared with respective unexposed control groups. Lane 1: unexposed control; Lane 2: cells exposed to cimetidine (250μM) for 6h; Lane 3: cells exposed to rifampin (250μM) for 6h; Lane 4: cells exposed to MCP (10–5M) for 6h; Lane 5: cells exposed to cimetidine for 1h, then removal of cimetidine and reexposure to MCP (10–5M) for 6h; Lane 6: cells receiving coexposure to rifampin and MCP for 6h.

FIG. 11.

Altered expression of CYPs, receptors, and GSTP1-1 in differentiating neurons (day 8) exposed to xenobiotics. (A) MCP and/or rifampin–induced alterations in the mRNA expression of CYPs, their regulator receptors, and GSTP1-1 genes in stem cell–derived neuronal cells at day 8 of differentiation. β-actin was used as internal control to normalize the data. MCP and/or rifampin–induced alterations in mRNA expression are expressed in relative quantity compared with respective unexposed control groups (denoted by parallel line). 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) MCP/rifampin/cimetidine exposure–associated alterations in the protein expression of CYPs, their regulator receptors, and GSTP1-1 in stem cell–derived neuronal cells at day 8 of differentiation. β-actin was used as internal control to normalize the data. Xenobiotic-induced alterations in protein expression are expressed in relative quantity compared with respective unexposed control groups. Lane 1: unexposed control; Lane 2: cells exposed to cimetidine (250μM) for 6h; Lane 3: cells exposed to rifampin (250μM) for 6h; Lane 4: cells exposed to MCP (10–5M) for 6h; Lane 5: cells exposed to cimetidine for 1h, then removal of cimetidine and reexposure to MCP (10–5M) for 6h; Lane 6: cells receiving coexposure to rifampin and MCP for 6h.

Xenobiotic-Induced Catalytic Activity of CYPs

The microsomal fraction from both undifferentiated stem cells and differentiating neuronal cells showed significant induction in CYP-dependent EROD (CYP1A1), PROD (CYP2B6), NDMA-d (CYP2E1), and EMD (CYP3A4) activities (Table 2). The trend in the induction of CYP activities was fairly correlative with the data of Western blot analyses. As anticipated, cimetidine, a known inhibitor of CYPs, causes significant (p < 0.001) inhibition in the catalytic activity of CYP1A1 (6.17±0.52, 8.33±0.73, 11.09±0.8, and 13.2±1.5 pmoles resorufin/min/mg protein) and CYP2B6 (10.08±0.9, 13.73±1.7, 20.18±1.2, and 20.33±1.8 pmoles resorufin/min/mg protein) at all the points of differentiation, i.e., days 0, 2, 4, and 8, respectively. However, the inhibitory effects could not reach significant levels in the cases of CYP2E1 and CYP3A4 (Table 2).

A significant induction in the activity of CYP1A1, CYP2B6, and CYP3A4 was observed in differentiating neuronal cells exposed to rifampin. Differentiating neuronal cells showed significant responsiveness to MCP, as the catalytic activity of all the studied CYPs was increased significantly (p < 0.001). The magnitude of MCP-induced catalytic activity was greater than the induction levels following exposure to rifampin. Cells coexposed to rifampin and MCP for 12h showed an additive effect in the induction of catalytic activity of all the CYPs. The cells showed maximum expression and induction in the activity of CYP3A4 all through differentiation, i.e., days 0, 2, 4, and 8. In all the exposure groups, induction in the activity of CYP1A1, 2E1, and 3A4 was found to be the maximum at day 2 of differentiation, whereas CYP2B6 showed maximum induction in activity at day 4 of differentiation (Table 2).

DISCUSSION

The frequency of CD34+ HSCs ranged from 0.6 to 1.37% (N = 103) among mononuclear cells, with percent cell viability between 85 and 95%. Factors such as storage time, magnetic effects during processing of HSCs, etc., may account for 5–15% loss of cell viability. It has been documented that the variation in number of HSCs in cord blood largely depends on the gestational age, mode of delivery, and positioning of neonate after delivery; and the variation is significantly higher in cord blood than in bone marrow (Wahid et al., 2012; da Silva et al., 2009; Wagner-Souza et al., 2008). In the present investigations, the prolonged maintenance and massive expansion of primitive HSCs suggest the extensive self-renewal and proliferation capacity of cells in undifferentiated state. Our findings also demonstrate that the cocktail of selected known regulators of early hematopoiesis and antiapoptotic agents was capable of maintaining the HSC cultures for longer periods (Bernstein and Delaney, 2012; Liu et al., 2012; Zheng et al., 2005).

The cord blood–derived CD34+ HSCs/non-HSCs have a high commitment toward neuronal lineage, similar to fetus-derived neural stem cells (Buzanska et al., 2006; Chen et al., 2005; Zangiacomi et al., 2008). Using a cocktail of various growth factors and rhNGF in serum-free neurobasal medium, we found that CD34+ HSCs can be induced to undergo morphologic and phenotypic changes consistent with developing neuronal cells. NGF is a potent neurotrophic factor that regulates the development, maintenance, and function of nervous system (Dutta et al., 2011; Huang and Reichardt, 2001). NGF is structurally related to BDNF, NT-3, and NT-4, which signal through its receptor TrkA (tropomyosin-related kinase), and plays a crucial role in the development of the sympathetic nervous system. These differentiated cells showed a typical neuron-like morphology and expressed various cell type–specific markers for neurons. The real-time PCR, immunocytochemical localization, and Western blot data revealed that significant mRNA transcripts and antigens of various neuronal markers, viz., NCAM, SYP, PSD 95, ACHE, NF-M, NF-H, and TUJ-1, were elevated after the onset of differentiation at day 2 and these markers became more significantly elevated at the end of day 8. Neuron-associated growth factors, NGF and BDNF, also increased from day 2 to day 8 at transcriptional as well as translational levels. The expression of CREB was upregulated from day 2 to day 8. CREB is a neuron-associated transcription factor that promotes neuronal differentiation by activating upstream regulators like MAP kinases (Herold et al., 2011). The mRNA and protein expression of ACHE increased after the onset of differentiation from day 2 and continued to be upregulated till day 8, which confirms that the neurons differentiated from stem cells might be of cholinergic type. We also observed elevated expressions of ARTN during differentiation, which is known to support survival, neurite outgrowth, cell migration, and differentiation of peripheral neurons (Airaksinen and Saarma, 2002). From this study, it might be inferred that the differentiated neurons are peripheral cholinergic neurons. We observed maximum protein expression of nestin, a well-known marker of neural precursor cell, at day 2, which proves the initiation of neuronal differentiation at this time point. The findings are also in accordance with the observations of Sanchez-Ramos et al. (2001) and Zigova et al. (Zigova et al., 2002). In general, morphological and physiological maturation in neuronal cells was achieved by day 8 of differentiation. With the progress of neuronal differentiation, the expression level of hematopoietic stemness markers, viz., CD34, CD133, Nanog, SHH, and MYC was reduced gradually and diminished by day 8 of differentiation. The gradual suppression of these transcription factors and stemness markers confirm the commitment of stem cells toward neuronal cell differentiation (Laurenti et al., 2008). The initial increase in stemness markers at day 2 may be suggested due to the remodeling of cell physiology, i.e., competition between proliferation and differentiation induction in the cells.

Limited reports show the expression and activity of selected CYPs in undifferentiated HSCs derived from bone marrow or peripheral blood (Czekaj et al., 2005; Kousalová et al., 2004; Soucek et al., 2005). For instance, bone marrow CD34+ stem cells of humans, rats, and rabbits show the expression of CYP2E1 (Bernauer et al., 2000). The catalytic activity of CYP2E1 has been reported only in human peripheral blood–derived CD34+ stem cells exposed to chlorzoxazone (Kousalová et al., 2004). Induced mRNA expression of CYP1A1 has also been reported in primitive HSCs against the challenge of hydroxylated metabolites of benzene (Henschler and Glatt, 1995). The expression of CYP1A1, 1A2, and 2E1 proteins in fetuses, newborns, pregnant rats, and human placenta has been reported (Czekaj et al., 2005). CD34+ HSCs show expression and inducibility of AHR and CYP1A1/1B1 following exposure to benzo[a]pyrene, a known carcinogen, thus suggesting the possible targets for such carcinogens (van Grevenynghe et al., 2005). Human fetal liver HSCs express mRNAs of CYP 1A1, 2E1, 3A4, and 3A5 at very low levels and various isoforms of the phase II metabolizing enzyme, glutathione S- transferase, at significant higher levels (Shao et al., 2007).

Stem cell (embryonic and mesenchymal)–derived hepatocytes are the only examples reported for expression and inducibility of CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 (Sa-ngiamsuntorn et al., 2011; Takayama et al., 2012). For the first time, we report the expression and inducibility of CYPs (1A1, 2B6, 2E1, and 3A4), their regulators (AHR, CAR, and PXR), and the phase II enzyme GSTP1-1 in developing neuronal cells derived from human HSCs. We found an increase in the expression and activity of CYPs with the progress of neuronal cell differentiation, which suggests the possible association of CYP activity in the neuronal development of developing brain. These differentiating cells responded significantly to rifampin (a known CYP inducer), cimetidine (a known CYP inhibitor), and MCP, a widely used organophosphate pesticide and known developmental neurotoxicant. Thus, our data identifies the CYP-mediated xenobiotic metabolic capabilities of human umbilical cord blood stem cell–derived neuronal cells at various stages of maturity. Buzanska et al. (Buzanska et al., 2009) have also shown the developmental stage–dependent susceptibility of cord blood–derived neural stem cells to a panel of neurotoxicants and reported that undifferentiated or early committed cells were more fragile than neuronally differentiated cells. Earlier, we have demonstrated the association of CYP (CYP1A1, 1A2, 2B1, 2B2, and 2E1) induction with oxidative stress and mitochondria-mediated apoptosis in PC12 cells exposed to MCP (Kashyap et al., 2011). We observed inconsistency between the mRNA and protein expressions of CYPs at a few study time points. This might be due to their regulation via transcriptional, posttranscriptional, translational, and posttranslational mechanisms under the influence of various endogenous factors, including insulin, glucagon, growth hormones, leptin, epidermal growth factor, etc. (Novak and Woodcroft, 2000). Xenobiotic inducers were also found to alter CYP expression levels through increased/decreased transcription and/or translational efficiency and stabilization of mRNA and protein from degradation (half-life for turnover; Hargrove and Schmidt, 1989; Novak and Woodcroft, 2000). Micro RNA(s)–mediated posttranscriptional regulation of human PXR receptor, which directly affects the expression of cytochrome P450 3A4/2B6, has also been demonstrated (George et al., 1995; Nakajima and Yokoi, 2011; Takagi et al., 2008). Studies have also shown that regulation of CYP induction is a complex process, involves tissue-specific factors, and is dependent on inducer pharmacokinetic or pharmacodynamic properties (Matheny et al., 2004). Souidi et al. (Souidi et al., 2005) have identified the family of orphan nuclear receptors AHR, PXR, CAR, and retinoid X receptor (RXR) in brain. AHR regulates and induces the expression of CYP1A1 in response to xenobiotics. PXR and CAR mediate the effects of xenobiotics and therapeutic drugs on regulation of the CYP3A4, 2B6, and 2E1 genes (Xie and Evans, 2001).

Cimetidine, which is known as an antagonist of the H2 receptor, is used as a medicine to reduce gastric acid secretion in the treatment of various gastrointestinal disorders. The CYP inhibitory mechanism of cimetidine is primarily based on its binding with the heme group of CYPs, thus inhibiting expression at the protein level (Stadel et al., 2008). Cimetidine does not interfere with transcription machinery of CYP for expression and induction of mRNAs. In the present investigations, cimetidine significantly inhibits the expression of CYPs at the protein level. However, a preexposure to cimetidine for 1h could not inhibit the MCP (12h)-induced expression and activity of CYPs. It is anticipated that even upon the withdrawal of cimetidine exposure after 1h, the heme group–bound cimetidine remained in the cells but could not interfere with MCP-induced alterations in the levels of CYPs. In general, chemical-induced differential inhibition of CYPs has been reported by both reversible and irreversible phenomena (Fowler and Zhang, 2008), In addition, the binding of cimetidine to CYPs is reported to be weak and reversible (Sorkin and Darvey, 1983). Upregulation in the expression of CYPs, their receptors, and the phase II metabolizing enzyme GSTP1-1 under the influence of rifampin confirms the responsiveness of differentiating neuronal cells to xenobiotics. In general, the magnitude of expression of CYPs studied was highest at day 2 of differentiation, followed by those at days 0, 4, and 8, respectively, in differentiated cells . Such stage-specific vulnerability of the cells may be due to the complexity in the tightly controlled mechanism of cell differentiation/maturation and higher intracellular metabolic activities in early stages of differentiation (Bal-Price et al., 2010). Upregulation in the expression (mRNA and protein) and substrate-specific catalytic activity of CYPs (1A1, 2B6, 2E1, and 3A4), related receptor regulators (AHR, CAR, and PXR), and phase II enzyme GSTP1-1 against MCP exposure also confirms the metabolizing capabilities of human cord blood stem cell–derived neuronal cells for a wide range of xenobiotics, including pesticides, organic solvents, polycyclic aromatic hydrocarbons, drugs, etc. Similar patterns of induction of selected xenobiotic metabolizing CYPs and associated toxicological consequences has also been reported in PC12 cells, a rat pheochromocytoma cell line (Kashyap et al., 2011). Though there is no direct evidence for the involvement of CYPs in brain development, evidences have been shown for the role of CYP systems in early embryonic development of mice (Otto et al., 2003).

SUMMARY

Human HSC–derived developing neuronal cells significantly express xenobiotic-metabolizing cytochrome P450s (CYP1A1, 2B6, 2E1, and 3A4), related receptor regulators (AHR, CAR, and PXR), and phase II metabolizing enzyme GSTP1-1. Rifampin, a known universal inducer of CYP, induces significant upregulation in the expression of CYP1A1, 2B6, 2E1, 3A4, AHR, CAR, PXR, and GSTP1-1 in these cells. Xenobiotic-metabolizing capability of human HSC–derived neuronal cells could be demonstrated against MCP, a known developmental neurotoxicant organophosphate pesticide. In general, undifferentiated (day 0) and early-differentiated (day 2) cells were found to be more vulnerable to xenobiotics than mature, fully differentiated (day 8) cells. Our study provides the evidence for stage-specific xenobiotic metabolizing capability of developing neurons, which could be a homogenous tool to predict human-specific developmental neurotoxicity and to identify stage-specific biomarkers of exposure and effect. The human stem cell–based in vitro system could be utilized as a prescreening tool to assess the neurotoxicity/developmental neurotoxicity potential of environmental chemicals/drugs.

FUNDING

Council of Scientific & Industrial Research (CSIR), New Delhi, India, (Supra Institutional Project SIP-08); Department of Biotechnology, New Delhi, India (102/IFD/SAN/PR-1524/ 2010-201).

Acknowledgments

The authors are grateful to the Director, Indian Institute of Toxicology Research, Lucknow, India, for his keen interest in the study. Council of Scientific & Industrial Research (CSIR), New Delhi, India, is acknowledged for providing fellowship to A.K.S. We have no conflict of interest with anybody working in the area and among the authors in the manuscript.

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Author notes

Disclaimer: The authors certify that all research involving human subjects was done under full compliance with all government policies and the Helsinki Declaration.