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Developmental neurotoxic effects of a low dose of TCE on

3D neurosphere system

Journal: Biochemistry and Cell Biology

Manuscript ID bcb-2017-0089.R2

Manuscript Type: Article

Date Submitted by the Author: 06-Sep-2017

Complete List of Authors: Abdraboh, Mohamed; Mansoura University Faculty of Science, Zoology Abdeen, Sherif; Mansoura University Faculty of Science, Zoology Salama, Mohamed; Mansoura University, Faculty of Medicine, Toxicology Department Elhussiny, Mahmoud; Mansoura University, Faculty of Medicine, MERC research center

El-Sherbini, Yasser M.; Oxford Brookes University, Faculty of health and life Science Eldeen, Nada; Mansoura University Faculty of Science, Zoology

Is the invited manuscript for consideration in a Special

Issue? : N/A

Keyword: Trichloroethylene, Neurotoxicity, neurospheres, astrocytes, cell proliferation, GFAP

https://mc06.manuscriptcentral.com/bcb-pubs

Biochemistry and Cell Biology

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Developmental neurotoxic effects of a low dose of TCE on 1

3D neurosphere system 2

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Abdraboh M.E.1*, Abdeen S.H.

1, Salama M.

2, El-Husseiny M.

2, Yasser El- 4

Sherbini Y. M.

3, Eldeen N.M.

1 5

1Department of Zoology, Faculty of Science, Mansoura University, Egypt. 6

2 Department of Toxicology, Faculty of Medicine, Mansoura University, Egypt. 7

3 Faculty of health and life science, Oxford Brookes University, Oxford, UK. 8

9

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* Corresponding author contacts 11

Department of Zoology, Faculty of Science, Mansoura University, Egypt 12

Email: [email protected] 13

Cell: +201002996239 14

15

Authors declares no conflict of interest 16

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Running title: TCE toxicity on NSCs 18

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Abstract 21

Trichloroethylene (TCE) is one of the industrial toxic byproducts which consequently exist in 22

air, soil and water. Several studies illustrated the toxicity effect of high doses of TCE on 23

biological functions of several organs. This study aims to highlight the toxic impact of a low 24

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dose of TCE (1µM.) on the development of rat neural stem cells (NSCs). The sub ventricular 25

zone (SVZ) of Rat pup's brains was collected, minced and harvested cells were cultured in the 26

presence of neural growth factors B27/N2 to develop neurospheres. cells were then exposed 27

to a dose of 1µM TCE for 1 and 2 weeks. The outcomes indicated a remarkable inhibitory 28

effect of TCE on NSCs differentiation capacity which was confirmed by downregulation of 29

astrocytes marker Gfap. TCE inhibitory effect on NSCs proliferation was identified by the 30

reduction of neurospheres diameter, Ki67 expression and cell cycle arrest at G1/S phase. 31

Immunolabelling with annexin V indicated the proapoptotic effect of TCE exposure. PCR 32

results revealed a TCE mediated suppression of the antioxidant enzyme Sod1 expression. The 33

current paper illustrated for the first a detailed examination of the toxic effect of an 34

environmental low dose of TCE on NCSs at the transcriptional, translational and functional 35

levels. 36

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Key words: Trichloroethylene, Neurotoxicity, neurospheres, astrocytes, cell 38

proliferation, Gfap 39

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Background 51

TCE is a volatile organic solvent which is predominantly involved in many industrial 52

activities such as detergents and metal degreasing processes (Otsuki et al. 2016; 53

Varshney et al. 2015). Recently, carcinogenic effect of TCE was identified in organs 54

where it is metabolized such as liver and kidney (Lash et al. 2014; Vlaanderen et al. 55

2013). Furthermore, TCE has been announced as a cardiac teratogen as the long term 56

exposure to TCE altered not only the calcium ion concentration at cardiac myocytes 57

but also the genes that are involved in regulation of calcium homeostasis (Caldwell et 58

al. 2008; Selmin et al. 2008). In the same context, the cytotoxic effect of TCE on 59

human epidermal keratinocyte was confirmed to be associated with accumulation of 60

intracellular calcium (Ali et al. 2015). In addition, exposure to TCE oxidative 61

metabolites, specially trichloroacetic acid, trichloroethanol and/or dichloroacetic acid, 62

was associated with DNA damage and other serious genotoxic effects by altering the 63

expression of cell cycle, tumor suppression and apoptosis regulatory proteins 64

(Varshney et al. 2015). 65

The cytotoxicity of TCE on central nervous system was addressed by several 66

literatures that highlighted the effect of TCE on human and mammalian brain 67

development (Bal et al. 2015; Chiu et al. 2013; Hogberg et al. 2013; Mundy et al. 68

2015). TCE was illustrated to decrease myelination of rat brain hippocampus which 69

in turn led to cognitive dysfunction of memory and learning abilities (Isaacson et al. 70

1990; Tiwari et al. 2015). 71

In an epidemiological study, the consumption of well water contaminated with TCE 72

led to high scores for low intelligence, depression, memory recall, and various mood 73

disorders (Reif et al. 2003). Similarly, children of mothers with an occupational 74

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exposure to TCE suffered from developmental disorders reflected by low 75

neurobehavioral scores and poor visual sharpness (Laslo-Baker et al. 2004). There are 76

some evidences correlated the exposure to TCE and progression of Parkinson's 77

disease and autism (Gash et al. 2008; Windham et al. 2006). 78

Several literatures elucidated that prenatal exposure to TCE inhibited the cardiogenic 79

differentiation process of human embryonic stem cells (Gilbert et al. 2014; Jiang et al. 80

2015; Wang et al. 2015). 81

Neurosphere is a three-dimensional cell culture technique that has been developed for 82

in vitro studies of neural stem cells (NSCs) proliferation, self-renewal and 83

differentiation potential into neurons, astrocytes and oligodendrocytes. In this 84

approach cells are grown in a form of non-adhesive heterogenous clusters of NSCs 85

with GFAP and β-tubulin III-positive cells in the core and nestin positive progenitor 86

cells in the periphery (Campos 2004; Gil‐Perotín et al. 2013; Moore et al. 2010). In 87

neurosphere system, the ability of NSCs to proliferate is indicated by the change at 88

the neurosphere diameter. Meanwhile, the ability for self-renewal is determined by 89

the ability of single cell subclone to form a fresh neurosphere (Ladiwala et al. 2012). 90

This study aims to evaluate for the first time, the possible neurotoxic effect of low 91

dose of TCE which is described to be human safe, on the proliferation, viability and 92

differentiation of NSCs using neurosphere approach. 93

Materials and methods 94

NSCs collection and culture 95

Brains of Sprague dawley rat pups (3-5 days) were collected; minced and then digested 96

using 0.1mg/ml collagenase enzyme for 10 min. To stop collagenase activity, minced 97

tissues were transferred into falcon tubes supplied with complete NSCs growth medium 98

of high-glucose DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, 1% 99

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L-glutamine (Gibco, CA, USA) and enriched with neural growth factors N2/B27 100

(invitrogen, CA, USA). After that, Cells were collected by centrifuging the suspension 101

at 800 rpm for 5 min. Supernatant was discarded and cell pellet were cultured in 102

complete NSCs medium for two weeks till neurospheres were formed then it separated 103

control and TCE (1µM) treated groups (Sigma, CA, USA) (Pastrana et al. 2011). The 104

dose of TCE (1µM) was chosen as a representative dose of the environmental 105

concentration of TCE on surface water (1µg/L) according to U.S. Environmental 106

Protection Agency report (https://www.epa.gov/sites/production/files/2016- 107

09/documents/trichloroethylene.pdf). 108

Cell differentiation 109

The capability of NSCs to differentiate into astrocytes in the presence and absence of 110

TCE was determined by immunolabeling of astrocytes using anti-mouse Gfap (1:60 111

dilution) and goat anti-mouse (1:500 dilution) (Sigma, CA, USA) after two weeks of 112

NSCs culturing. Cell suspension (~106 cells) from each group was transferred to a 113

microcentrifuge tube and spin at 800 rcf for 3 min. Cell pellet were then processed for 114

immunocytochemistry using the Vector lab detection kit system (Vector Lab, CA, USA) 115

(Wang et al. 2014). 116

Cell proliferation 117

Neurosphere were sub-cultured into two groups of control and TCE treated cells 118

(receiving fresh dose of TCE every two days) for two weeks. The time dependent 119

change at neurospheres diameter in TCE treated and untreated groups was estimated 120

using cell profiler software (Cell Profiler, version 2.1; Broad Institute, 121

http://www.cellprofiler.org). 122

Flow cytometry studies 123

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Cell cycle analyses 124

Cell suspensions of NSCs (1x 105 cells) were collected, washed in phosphate buffered 125

saline (PBS) and fixed in 70% ethanol at 4°C overnight. Then, the cells were 126

centrifuged at 380xg for 5 min at room temperature. The supernatant was discarded 127

and the cell pellet was re-suspended in 100 µL of PBS. Cells were incubated with 128

propidium iodide (PI) for 30 min at room temperature, and then filtered through a 30 129

mm pore diameter nylon mesh filter of flow cytometry tube to eliminate nuclear 130

clumps. The cell cycle distribution and percentage of apoptosis was estimated from 131

the resulting figure (FACS can, Becton Dickinson, Germany). 132

Cell apoptosis (Annexin V) 133

Alcohol fixed cells were placed in the presence of 2.5% of FITC- conjugated annexin 134

V for 30 min, then PI were added for extra 30 min. The incidence of cell apoptosis 135

and necrosis was indicated using FACScan software (Becton Dickinson, Germany). 136

Ki67 expression 137

Proliferative activity of NSCs within one and two weeks of TCE treatment was 138

determined by fixing and permeabilizing 1 × 105 cells using 70% ethanol. Cells were 139

labeled with anti Ki67-FITC conjugated monoclonal antibody (Santa Cruz, CA) 140

according to manufacturer's protocol. Afterwards, the cells were washed in PBS, re- 141

suspended and immediately acquired on flow cytometer chambers (Becton Dickinson, 142

Germany). Data were analyzed using FACScan software (Becton Dickinson, 143

Germany); proliferative activity was expressed as percentage of Ki67 positive cells. 144

Reverse transcriptase polymerase chain reaction (RT-PCR) 145

Total RNA was isolated from control and TCE treated NSCs using the RNeasy Mini 146

kit (Qiagen, CA, USA), according to the manufacturer’s protocol. 1.0 µg of extracted 147

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total RNA was reverse-transcribed using Superscript cDNA synthesis kit (invitrogen, 148

CA, USA) by incubating samples in the PCR machine at 50°C for 30 minutes, initial 149

PCR activation step (95°C for 5 minutes), followed by 27 PCR cycles. The semi 150

quantitative RT-PCR was conducted using oligonucleotide primers of superoxide 151

dismutase (Sod1) (sense) 5'-CGAGCATGGGTTCCATGTC-3', (anti-sense)5'- 152

CTGGACCGCCATGTTTCTT-3', Myleoperoxidase (Mpo) (sense)5'- 153

GCTGAATGTGTTGTTCCAAGTC-3', (anti-sense) 5'- 154

TGATGGTGCGGTATTTGTCC-3' and glyceraldehyde-3-phosphate dehydrogenase 155

(Gapdh) (sense)5'-CCA GGG CTG CCT TCT CTT GT-3', (anti-sense) 5'-CTG TGC 156

CGT TGA ACT TGC CG -3'. For detection of expression of antioxidant enzymes 25 157

pg of cDNA were applied in each PCR cycle consisted of 95°C for 30 seconds, 60°C 158

for SOD1 (35 cycle) and 61°C for MPO (27 cycle) and (GAPDH) (35 cycle) for 30 159

seconds each, followed by 72°C for 1 minute and the final extension was at 72°C for 160

10 minutes. The RT-PCR products were examined by electrophoresis in 1% agarose 161

gel containing 0.2 µg/mL ethidium bromides. 162

Statistical Analysis 163

Differences between mean values were assessed for statistical significance using a 164

two-tailed Student's t-test (GraphPad Prism 5.0 software, La Jolla, CA). P values < 165

0.05 were considered statistically significant. 166

Results 167

I- Effect of TCE on production of neural rosettes and neurogenesis 168

In order to isolate and develop neuronal progenitor cells (NPCs), Collagenase 169

digested brains were cultured in medium supplied with neural growth factors 170

(N2/B27) for 12 days for production of neurospheres (data not shown). The 171

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neurogenesis and differentiation of subcultured neural stem cells in presence and 172

absence of TCE was monitored for extra 12 days after primary spheres formation (3-5 173

days). The change at cells shape, aggregation and adherence to flasks surface was 174

determined at different time points. By day twelve the clustered cells started to form 175

neural rosettes of differentiated NPCs that can adhere to the culture flasks surface. On 176

the other hand, the cultured neurospheres in presence of TCE failed to form 177

differentiated NPCs and to adhere to culture flasks surface (Fig. 1a). The failure of 178

NPCs to differentiate into astrocytes was further confirmed by immunolabelling of 179

NPCs of both groups with glial fibrillary acidic protein (Gfap) using 180

immunocytochemistry approach, whereas untreated NPCs showed positive Gfab 181

astrocytes unlike the TCE treated neurospheres which showed no signs of astrocytes 182

(Fig. 1b). 183

II- Effect of TCE on NSCs proliferation 184

NPCs were cultured in presence and absence of TCE to evaluate the impact of TCE 185

on their ability to proliferate forming neurospheres. The diameter of neurospheres in 186

both groups was measured throughout the experiment for 12 days as an indication of 187

cell proliferation capability. The results indicated a significant effect of TCE in 188

lessening the cell ability to proliferate throughout the time points (Fig. 2a). In order to 189

confirm these results, the expression of cell proliferation marker Ki67 was detected in 190

cells of both groups after one week and two weeks of TCE treatment. The 191

flowcytometric data indicated a significant effect of TCE on downregulating Ki67 192

expression in a time dependent manner (Fig 2b). Cell cycle analyses of NPCs at week 193

one and week two post TCE treatment indicated a significant effect of TCE in 194

inducing cell cycle arrest at G1/S phase by week two. Meanwhile, the data revealed a 195

significant induction of cell mortality in TCE treated cells at sub G1 phase (Fig. 2c) 196

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III- TCE induces NSCs apoptosis and necrosis 198

To assess the effect of TCE on the viability of treated NPCs, Annexin V labeling of 199

treated cells at week one and two was conducted using flowcytomtry approach. A 200

significant reduction at cell viability at both time points was indicated in TCE treated 201

cells. Meanwhile, week one was associated with more tendency towards cell necrosis 202

while week two showed remarkable up-regulation at number of cells in late apoptosis 203

phase showed after two weeks of TCE treatment (Fig. 3) 204

IV- Effect of TCE on oxidative stress 205

In order to elucidate the mechanism of action by which TCE exerts its impact on 206

NSCs to proliferation and survival. The effect of TCE on the expression of two main 207

anti-oxidant enzymes Sod1 and Mpo was observed at week one and two post TCE 208

treatment. The expression levels of Sod1 and Mpo mRNA was estimated using PCR. 209

Bands density was quantified and the data were normalized against the expression of 210

the house keeping gene Gapdh. The results revealed a significant time dependent 211

effect of TCE on diminishing Sod1 expression. This time dependent effect was clearly 212

demonstrated at week two post treatment. Interestingly, contradictorily to Sod1 data 213

the neurospheres treatment with TCE leads to a significant up-regulation of Mpo 214

expression at week two of treatment (Fig. 4). 215

Discussion 216

TCE is one of the toxic compounds that are merely present in air, soil and water 217

systems due to environmental pollution. The occupational exposure, of people living 218

in such areas, for chronic low doses of this chemical has been reported to cause severe 219

damage to kidney, intestine, liver and brain (Khan et al. 2009; Lock et al. 2006; 220

Vlaanderen et al. 2013). 221

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In this study, we were determined to assess the possible neurotoxic effect of chronic 222

exposure to a low dose of TCE on NSCs differentiation, proliferation and cell 223

survival. The effect of TCE on NPCs production was assessed by the treatment of 224

neurospheres with 1µM of TCE for two weeks. The ability of NPCs to differentiate 225

was first indicated via the ability of neurosphere differentiated peripheral cells to 226

adhere to culture flask substrate which was not the case in TCE treated neurospheres 227

(Fig. 1a). The differentiation of NPCs in neurospheres periphery into astrocytes was 228

illustrated by Chojnacki and Weiss whom demonstrated a protocol for generation of 229

astrocytes, neurons, and oligodendrocytes from mammalian CNS stem cells 230

(Chojnacki and Weiss 2008). 231

These phenotypical outcomes were further confirmed by immunolabeling of NPCs of 232

both groups with the astrocytes marker GFAB. The absence of GFAB immunolabeled 233

cells in the presence of TCE pointed out and confirmed the inability of neurosphere 234

NPCs to differentiate into astrocytes compared to untreated cells which showed 235

positive immunolabeled astrocytes (Fig. 1b). 236

On other hand, the effect of TCE on NPCs proliferation was first assessed by 237

observing the alteration at cultured neurospheres diameters for 12 days in response to 238

TCE. The data indicated a TCE significant time dependent reduction in the 239

proliferative capacity ability of NPCs which was indicated by the decrease at the 240

mean diameter of neurospheres in comparison with untreated group (Fig. 2a). The 241

change at neurosphere diameter was considered as a parameter that reflects the ability 242

of the neurosphere NPCs to proliferate (Mori et al. 2006; Sachewsky et al. 2014). 243

These results were further confirmed by detecting the expression level of the cell 244

proliferation marker Ki67 in both groups. A significant down-regulation of Ki67 245

expression levels in NPCs was detected after one and two weeks of TCE treatment 246

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(Fig. 2b). Furthermore, the cell cycle analyses indicated a significant effect of TCE in 247

inducing G1/S cell cycle arrest after two weeks of cell exposure to TCE (Fig. 2c). 248

A recent study elucidated a dose dependent biphasic effect of TCE treated L-02 249

hepatocye cell line, in which short term (12, 24 and 48 hrs) exposure to low doses of 250

TCE showed a significant increase at cell proliferation rate while high doses (2, 4 and 251

8 mmol) significantly inhibited cell proliferation (Xu et al. 2013). These results still 252

support our hypothesis of the cumulative effect of chronic TCE exposure (1 and 2 253

weeks) to such a low dose of 1µM on inhibiting NPCs proliferation. 254

Moreover, long term inhalation of TCE vapors for 3 months caused neuropathological 255

alterations at hippocampus demonstrated by the induction of astroglial cells 256

hypertrophy and inhibition of cell proliferation (Haglid et al. 1981). 257

On the other hand, the significant increase at cell population of sub G1 phase during 258

cell cycle analyses indicated a significant effect of TCE on induction of cell death 259

(Fig. 2c). To elucidate whether this induction at cell death in presence of TCE was 260

due to cell apoptosis and/or necrosis, the NPCs double labeling with the early 261

apoptotic marker annexin V and PI revealed significant induction of cell necrosis by 262

week one of TCE treatment which was downregulated in week two. Meanwhile, the 263

TCE pro-apoptotic effect started by week one and progressed to the second week 264

(Fig.3) The first week necrotic effect was most likely a prompt response of NPCs for 265

the sudden exposure to TCE, the survived cells suffered later from programmed cell 266

death due to the cumulative TCE exposure effect. 267

In a recent occupational study on the exposure of lock industries workers to TCE and 268

its metabolites, the collected blood samples showed significant signs of apoptosis. A 269

significant up-regulation of pro-apoptotic P53 and its downstream target Bax was 270

indicated in the workers blood samples (Varshney et al. 2015). Moreover, TCE 271

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exposure significantly intervened in mitochondrial signaling by activation of caspase 272

9 and 3 dependent cell apoptosis (McDermott and Heffron 2013; Shen et al. 2009). 273

In order to elucidate the mechanism of action by which TCE exerts its previously 274

stated effects, the effect of TCE on anti-oxidant enzymes cellular levels was 275

investigated as a possible target of TCE signaling. The expression of Sod1 and Mpo as 276

two major anti-oxidant enzymes was assessed in NPCs after one and two weeks of 277

TCE exposure. Densitometrical quantification of PCR products showed a significant 278

effect of TCE in inhibiting Sod1 expression only at week two of treatment. 279

Meanwhile, the transcription of Mpo was significantly upregulated at both time points 280

in comparison to their controls of untreated cells (Fig 4). 281

This imbalance in antioxidant enzymes expression indicated a development of an 282

oxidative stress in TCE treated group. TCE application to skin was found to cause 283

skin irritation by up-regulating NO generation with an initial up-regulation of SOD 284

activity which thereafter down-regulated with further concentration increment (Shen 285

et al. 2008). In a study of oxidative stress status in patients diagnosed with 286

arthrofibrosis, the up-regulation at MPO expression was found to have a significant 287

negative feedback on expression of SOD 1 of arthrofibrotic fibroblast cultured cells 288

(Freeman et al. 2009). Recently, the downregulation of SOD 1 expression was 289

illustrated as the main target of TCE dependent oxidative stress affecting motor 290

neuron in both in vitro and in vivo studies (Otsuki et al. 2016). 291

Hence, these data highlighted for the first time the possible developmental 292

neurotoxic effect of low doses of TCE even within the acceptable health and safety 293

range on NSCs. This neurotoxic effect of chronic exposure to such low dose of TCE 294

on neural rosettes development and NPCs proliferation and survival was conducted 295

via TCE dependent cellular oxidative stress 296

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Figures legends 429

Figure 1. Effect of TCE on NPCs differentiation (A.) Effect of TCE on formation 430

of neurospheres and generation of neural rosettes. Subjected neurospheres to TCE 431

formed small neurospheres and failed to differentiate which was indicated by the 432

failure of neurospheres to attach to the flask substrate. Meanwhile, Control group 433

showed a fully formed neurospheres of differentiated NPCs. (B.) Characterization of 434

NPCs differentiation into astrocytes. Immunocytochemical localization of astrocytes 435

with Gfap monoclonal antibody. Positive Gfap labeling in control group and its 436

absence in TCE group revealed the success of NSCs to developed into early 437

astrocytes only in absence of TCE. The data were a repeat of 3 different experiments. 438

439

Figure 2. TCE treatment demolishing NSCs proliferation. (A.) Measurement of 440

neurospheres mean diameters indicated a significant effect of TCE on diminishing 441

neurosphere growth rate. (B.) The expression of cell proliferation marker Ki67 was 442

significantly downregulated in week one and of treatment. (C.) Cell cycle analysis 443

indicated a significant effect of TCE in ceasing cell cycle progression in week 1 and 2 444

of treatment. The data were a repeat of 3 different experiments. All data were 445

expressed as the mean of positive cells ± SD. Significance was denoted as * P<0.05, ** 446

P<0.001 and *** P<0.0001. 447

448

Figure 3. Effect of TCE on the viability of neurospheres. Flowcytometric analyses 449

of the propidium iodide and annexin V showed (A.) a significant decrease at cell 450

viability by week one and two of treatment, (B.) which is correlated with significant 451

upregulation in necrotic cells mainly in week one. (C.) Early apoptosis, the significant 452

increase of early apoptosis was much more noticed by week one than week two of 453

treatment. (D.) the significant increase in population of cells at late apoptosis was the 454

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prominent effect by second week of treatment. The data were a repeat of 3 different 455

experiments. All data were expressed as the mean of positive cells ± SD. Significance 456

was denoted as * P<0.05, ** P<0.001 and *** P<0.0001. 457

458

459

Figure 4. Effect of TCE on the cellular oxidative stress. The level of antioxidant 460

enzymes RNA expression in control and TCE treated cells was detected using semi- 461

quantitative RT-PCR. (A.) A marked decrease at Sod1 transcripts were noticed after 2 462

weeks of TCE treatment. Meanwhile, a marked increase at Mpo transcripts level were 463

noticed by weeks one and two of treatment. (B.) Densitometrical analysis of both 464

Sod1 and Mpo band densities, after normalization against Gapdh levels, revealed a 465

significant time dependent effect of TCE in lessen expression of Sod1 and 466

upregulating Mpo expression. All data were expressed as the mean of positive cells ± 467

SD. The data were a repeat of 3 different experiments. Significance was denoted as * 468

P<0.05, ** P<0.001 and *** P<0.0001 as compared to MPO controls, and denoted as $ 469

P<0.05, $$

P<0.001 and $$$

P<0.0001 as compared to SOD1 controls. 470

471

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Figure 3. Effect of TCE on the viability of neurospheres. Flowcytometric analyses of the propidium iodide and annexin V showed (A.) a significant decrease at cell viability by week one and two of treatment, (B.) which is correlated with significant upregulation in necrotic cells mainly in week one.(C.) Early apoptosis, the significant increase of early apoptosis was much more noticed by week one than week two of treatment. (D.) the significant increase in population of cells at late apoptosis was the prominent effect by second week of treatment. The data were a repeat of 3 different experiments. All data were expressed as the mean of positive cells ± SD. Significance was denoted as * P<0.05, ** P<0.001 and *** P<0.0001.

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