Tris-(2,3-dibromopropyl) Isocyanurate Induced Oxidative Stress in the Human Neuronal Cell Lines and in Rat Brains

Wei Feng, Yu Li, Jing Zhang, Xuefei Lyu and Yulin Deng

(School of Life Science, Beijing Institute of Technology, Beijing 100081, China)

Abstract: Tris-(2, 3-dibromopropyl) isocyanurate (TBC) is a heterocyclic hexabrominated additive flame retardant, which is bio-accumulative, and can cause reproductive, endocrine disrupting, and neurotoxic effects. The present study was aimed at further evaluating the oxidative stress induced by TBC in the human neuronal cells and in rat brains. The results demonstrated that TBC caused apoptosis of U251 and SH-SY5Y cells in a dose- and time-dependent manner and that U251 cells were more sensitive to TBC than that of SH-SY5Y cells. Meanwhile, 1 μg/mL of TBC can significantly induce the production of MDA in U251 cells, indicating that oxidative stress occurred after TBC short-term exposure (24 h). Similarly, in vivo administration of 0.5 mg/kg of TBC in rats for 7 days led to low growth rates and a significant increase of reactive oxygen species (ROS) and nitrogen species (RNS) in the brains. However, enzymes related to antioxidation, total superoxide dismutase (T-SOD) and reduced glutathione (GSH), were not affected obviously. This might indicate that 7-day exposure was not long enough to weaken antioxidant defence in the brains. Altogether, the results indicated that oxidative stress was induced by short-time TBC exposure.

Key words: tris-(2, 3-dibromopropyl) isocyanurate(TBC); oxidative stress; human neuronal cell lines; rat brain

Brominated flame retardants (BFRs) have been widely used in electronics, furniture, and clothes as useful additives to enhance flame resistance. However, some BFRs persist in the environment, bio-accumulate[1-3], and cause harmful effects on the environment and human health[4]. Moreover, previous studies revealed that BFRs can induce neurotoxicity[5-8]and immunotoxicity[6,8-10]. Given the adverse effect on ecosystems and humans, their use was banned by the Stockholm Convention on Persistent Organic Pollutants. Since 2009, hexabromobiphenyl, hexabromodiphenyl ether, heptabromodiphenyl ether, tetrabromodiphenyl ether, pentabromodiphenyl ether, and hexabromocyclododecane were successively added to the forbidden list[11]. As a result, there has been a stable growth in the demand for BFRs substitutes[12]. As a heterocyclic hexabrominated additive flame retardant, tris-(2, 3-dibromopropyl) isocyanurate (TBC) is thermally stable, durable, and resistant to photodegradation. It has been widely used in polyolefins, polyphenyl alkenes, unsaturated polyesters, synthetic rubbers, and fibers[13]. With the forbidden production of some BFRs worldwide, increased production and use of TBC can be expected[14]. TBC was first identified in water, sediment, and biota samples near a manufacturing factory in southern China in 2009[15]. Since then, studies focusing on quantitative detection[12,15-17], spatial distribution[18-19], time trends[18], environmental behaviour[14], and toxicity[3,20-25]of TBC in the environment have been carried out. Until now, only a few studies on TBC toxicity have been conducted. The results obtained showed that TBC exposure causes reproductive and endocrine disrupting effects in zebrafish[22], damages the inflation of the gas bladder of zebrafish[20], and also induces significant liver and lung toxicity in mice[25]. TBC possesses a high octanol-water partition coefficient (log Kow=7.37) and a high bioconcentration factor (log BCF=4.30), indicating that TBC was bio-accumulative[15]. TBC was also verified to be one of the causative compounds inducing neuronal toxicity in environmental samples using neuronal toxicity-directed analysis[21]. Furthermore, TBC induces significant hippocampal neurotoxicity in adult rats and mice[3,24]. The published literature on oxidative stress induced by TBC exposure was limited. In the present study, in vitro and in vivo experiments were designed to investigate the oxidative stress induced by TBC exposure. In the in vitro experiments, U251 cells and SH-SY5Y cells were exposed to gradient concentrations of TBC for 24 h or 48 h, respectively, and then cytotoxicity, apoptosis, and cell oxidative stress were measured. In the in vivo experiments, rats were exposed to TBC at concentrations of 0.5 mg/kg and 2 mg/kg via intraperitoneal injection for 7 days, then the brains were collected and several related oxidative stress indicators were measured.

1 Experimental

1.1 Materials

Tris-(2, 3-dibromopropyl) isocyanurate (TBC, purity >97%) and chloral hydrate were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO), Dulbecco’s modified Eagle’s medium (DMEM), and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum (FBS) for SH-SY5Y was purchased from Zhejiang Tianhang Biotechnology Co., Ltd. (Zhejiang, China). Antibiotics (penicillin 10 000 U/mL streptomycin 10 000 mg/mL) and trypsin were ordered from Beijing Xinjinke Biotechnology Co., Ltd.(Beijing, China). Corning Culture Dishes (Corning, NY, USA) were used for cell culture. The kits of malondialdehyde (MDA), hydrogen peroxide, reduced glutathione (GSH), and total superoxide dismutase (T-SOD) were all ordered from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Corn oil was obtained from Yihai Kerry Investments Co., Ltd. (Shanghai, China). Quick Start Bradford protein assay kit was purchased from Bio-Rad Laboratories, Inc. (CA, USA). All other reagents with analytical grade were bought from Beijing Chemical Plant (Beijing, China).

1.2 Cell cultures

Two in vitro models, U251 cells and SH-SY5Y cells, were chosen herein to investigate oxidative stress of TBC. Both of them are human cell lines and have been widely used as in vitro models for neurotoxicity studies. U251 cells were derived from a malignant glioblastoma tumour and SH-SY5Y cells were from a neuroblastoma cell line.

Cells were cultured in at 37 ℃ in 10 cm dishes in a humidified incubator with 5% CO2. They were propagated in 10 mL of DMEM medium supplemented with penicillin-streptomycin and 10% FBS.

1.3 Assessment of TBC effects on cell viability by the MTT assay

Tetrazolium-based colorimetric (MTT) assay was used to measure cell viability. U251 and SH-SY5Y cells were seeded into 96-well plates (4×104cells/well), respectively, and cultured for 24 h. The cells were then exposed to gradient concentrations of TBC (0, 0.1, 1 or 10 μg/mL) for 24 h and 48 h, respectively. After that, the culture medium was removed, and new culture medium with 2 mg/mL of MTT was added for another 4 h culture. The medium was then aspirated again and 150 μL of DMSO was added as a solvent of formazan. After incubation for 5 min, the absorbance value of each well was measured under the wavelength of 570 nm by a microplate reader (Bio-Rad, CA, USA).

1.4 Assessment of TBC-induced effects on apoptosis by flow cytometry

Cells were separated into 4 groups (1 dishper group), and cultured in dishes for 24 h. After that, cells were exposed to TBC at concentrations of 0, 0.1, 1 or 10 μg/mL. Then, cells were digested from dishes by trypsin followed by washing with PBS. Cells (～5×105) were collected and stained separately by Annexin V-FITC and propidium iodide (PI). Flow cytometry(Beckman, CA, USA)was used to analyze the apoptotic effect in each sample.

1.5 Assessment of TBC-induced effects on oxidative stress in cultured cells

To evaluate the effect of TBC on the intracellular oxidative stress level, cells were separated into four groups (3 dishes each), and cultured for 24 h. They were then exposed to TBC at concentrations of 0, 0.1, 1 and 10 μg/mL for 24 h. Cells (～3×106/dish) were collected in PBS and cells in the same group were mixed. Finally, the malondialdehyde (MDA) content was determined by the kit according to the manufacturer’s protocol.

1.6 Animals and treatment

Male Sprague-Dawley rats aged 3-5 weeks (93.6-135.3 g) were purchased from the Institute of Laboratory Animal Sciences. They were housed in plastic cages at a temperature of 22 ℃ and humidity higher than 40%. They were supplied with water and standard food, and a regular light cycle of 12/12 hours was used. After 7 days of acclimation, the rats were randomly divided into the following three groups with 10 animals per group: one control group and two treated groups. The two treated groups received an intraperitoneal injection of TBC at a dose of 0.5 mg/kg and 2 mg/kg body weight per day for 7 days, respectively. The control group received the same volume of corn oil in the same way. Their body weights were measured before and after exposure.

The rats were sacrificed 24 h after the last injection. Rats were anesthetized by intraperitoneal injection of 50 mg/kg of sodium pentobarbital before cardiacperfusion. The brain tissue was taken from the sacrificed rats and was preserved at -80 ℃ for further experiments.

1.7 Assessment of TBC-induced effects on oxidative stress indicators in rat brains

10% whole rat brain homogenate was obtained by mechanically disrupting the brains on ice. Samples were centrifuged independently according to the manufacturer’s protocols. The concentration of protein in the brain homogenate was determined by Bradford protein assays. The contents of malondialdehyde (MDA), hydrogen peroxide (H2O2), nitrogen monoxide (NO), reduced glutathione (GSH), and superoxide dismutase (SOD) in rat brains after TBC treatment were measured according to the manufacturer’s protocols.

2 Results and Discussion

2.1 Effect of TBC exposure on viability of U251 and SH-SY5Y cells

Cell viability assays are usually used for testing the cytotoxicity of chemicals. MTT assay was used to measure cells viability after exposure to gradient concentrations of TBC. Results of TBC-induced effects on cells viability are shown in Fig.1.

Fig.1 Viability of U251 and SH-SY5Y cells exposed to TBC at the concentrations of 0, 0.1, 1, and 10 μg/mL for 24 h and 48 h by colorimetric MTT assays. Values are represented as means ± S.D. (n=5). Asterisks indicate a statistically significant difference with that of the control group without TBC exposure (t test,* p<0.05, ** p<0.01, *** p<0.001)

Results showed that TBC exerted an inhibiting effect on viability in U251 and SH-SY5Y cells in a time- and dose-dependent manner. As indicated by Fig.1a, 10 mg/mL of TBC significantly inhibited the viability of U251 cells after exposure for 24 h (p<0.05). Moreover, after exposure of U251 cells to gradient concentrations of TBC for 48 h, the inhibiting effect on cell viability was dose-dependent, and even 0.1 μg/mL of TBC significantly inhibited the viability of U251 cells (p<0.05). However, compared to the results obtained in U251 cells, SH-SY5Y cells were less sensitive to TBC and showed higher cell viability. The results presented in Fig.1b indicate that TBC, at any of the applied concentrations, did not inhibit cell viability after exposure for 24 h, and even 1 μg/mL of TBC significantly enhanced viability of SH-SY5Y cells. The reasons remain to be found in the further studies. After exposure of SH-SY5Y cells to TBC for 48 h, a significant decrease in cell viability was observed only at the highest concentration (p<0.001). The inhibiting effect of TBC exposure for 48 h on the viability of SH-SY5Y cells observed in this study was in agreement with those in the study of Dong et al.[24], and the toxicity of TBC on cell viability was further validated.

2.2 TBC-induced effects on apoptosis of U251 and SH-SY5Y cells

Apoptosis is one of the mechanisms underlying cytotoxicity. Previous studies have demonstrated that BFRs could decrease the viability of cells by inducing apoptosis[7,26- 27]. An apoptotic effect induced by TBC exposure was detected by flow cytometry in the present study.

The results of apoptosis in U251 cells exposed to TBC are shown in Fig.2. We found that TBC-induced apoptosis in U251 cells was dose-dependent. With the increase of TBC concentration, apoptosis of U251 cells was enhanced from 26.1% to 46.1% (10 μg/mL). On the contrary, after exposure to gradient concentrations of TBC for 24 h, almost no apoptosis appeared in SH-SY5Y cells (Fig.3). These results indicate that U251 cells were more vulnerable to TBC compared to SH-SY5Y cells, which were consistent with the results on cell viability (Fig.1).

2.3 TBC-induced oxidative stress in U251 and SH-SY5Y cells

Indicators of oxidative stress are often used as markers for the evaluation of toxic effects.Lipid peroxidation (LPO) is one of the major mechanisms underlying oxidative stress, and MDA content has been widely used as an index reflecting this condition. Here, changes in MDA contents of U251 and SH-SY5Y cells after short-term (24 h) exposure to TBC were determined and the results are shown in Fig.4.

Fig.2 Apoptosis in U251 cells induced by TBC exposure at concentrations of 0, 0.1, 1, and 10 μg/mL for 24 h by flow cytometry. Cells were separated into four different subpopulations, dead necrotic (B1), dead apoptotic (B2), live non-apoptotic (B3), and live apoptotic cells (B4)

Fig.3 Apoptosis in SH-SY5Y cells induced by TBC exposure at concentrations of 0, 0.1, 1, and 10 μg/mL for 24 h by flow cytometry. Cells were separated into four different subpopulations, dead necrotic (B1), dead apoptotic (B2), live non-apoptotic (B3), and live apoptotic cells (B4)

Fig.4 MDA contents of U251 and SH-SY5Y cells exposed to different concentrations of TBC (0, 0.1, 1, and 10 μg/mL) for 24 h (t test, * p<0.05, ** p<0.01)

For U251 cells (Fig.4a), the MDA content without TBC exposure was 0.55 nmol/mg prot. After exposure to TBC for 24 h, the MDA content significantly increased. This result reflects that lipid peroxidation really occurred in U251 cells after short-term exposure to TBC (p<0.05). Comparing with the obtained data of cell viability (Fig.1) that shows exposure to 1 μg/mL of TBC for 24 h did not decrease cell viability, it might be concluded that measured changes in the level of oxidative stress is a more sensitive indicator to evaluate the toxic effects of TBC exposure. However, the obtained oxidative stress levels changed by TBC exposure were not dose-dependent. The amounts of MDA in U251 cells exposed to 10 μg/mL of TBC was 0.76 nmol/mg prot and thus, lower than that of 1 μg/mL of TBC. This might reflect the self-repairing ability of organisms facing exposure to pollutants.

For SH-SY5Y cells (Fig.4b), the MDA content without TBC exposure was 0.96 nmol/mg prot. After exposure to different concentrations of TBC, there was no noticeable change in oxidative stress levels.

Together with the results of cell viability and apoptosis,we found that U251 cells may be more vulnerable than SH-SY5Y when exposed to TBC. However, the underlying mechanisms for the discrepancy of the two cell lines remain to be studied.

2.4 Effects of TBC on the levels of oxidative stress indicators in rat brains

To further validate the results of oxidative stress levels in the TBC-exposed cells, the levels of oxidative stress indicators in rat brains treated with TBC (0.5 mg/kg and 2 mg/kg) were measured. The determined oxidative stress indicators included MDA, nitrogen monoxide (NO), hydrogen peroxide (H2O2), total superoxide dismutase (T-SOD), and reduced glutathione (GSH). All the results are shown in Fig.5.

The level of H2O2induced by 0.5 mg/kg of TBC was significantly enhanced (p<0.001) compared with that of the control group. However, at the higher TBC concentration (2 mg/kg), the H2O2content was as low as that of the control group (Fig.5a). As for NO, both 0.5 mg/kg and 2 mg/kg of TBC significantly induced its production (p<0.05). However, the NO amount in the 0.5 mg/kg TBC-exposed group was higher than that in the 2 mg/kg TBC-exposed group (Fig.5b). Resembling the trends observed with H2O2and NO, the MDA level was significantly enhanced under the exposure to 0.5 mg/kg TBC, and it decreased to the baseline level at the TBC concentration of 2 mg/kg (Fig.5e). The obtained results of MDA, H2O2, and NO indicate that TBC might induce oxidative stress only by increasing the level of ROS and NOS in short-term exposure and that the higher concentration of TBC was less effective in increasing reactive oxygen formation.

Fig.5 H2O2, NO, T-SOD, GSH, and MDA contents in whole rat brains treated with TBC at the concentration of 0.5 mg/kg and 2 mg/kg. Values are represented as means ±S.D. (n=6). Asterisks indicate a statistically significant difference with that of the control group (t test,* p<0.05, *** p<0.001)

It must be noted that increases in the levels of MDA, H2O2, and NO were also associated with a growth decrease. The weight gains in 3 groups (0 mg/kg, 0.5 mg/kg or 2 mg/kg) were 52.17±2.09 g, 39.65±2.11 g, and 46.81±2.66 g, respectively. The weight gain in the group treated with 0.5 mg/kg of TBC, in contrast to the group treated with 2 mg/kg of TBC, was significantly lower than that in the control group (p<0.01). The results of weight gain might reflect the toxicity of different concentrations of TBC to rats from another aspect.

Different from the changes of H2O2, NO and MDA levels, almost no change has occurred in the level of the antioxidant enzymes, T-SOD (Fig.5c), and GSH (Fig.5d) in rat brains exposed to different concentrations of TBC. Previous studies have reported that 2, 2′, 4, 4′-tetrabromodiphenyl ether (PBDE-47), in a concentration of 2.06 μM, caused oxidative damage by decreasing SOD and GSH levels[28]. Cells fromGclmknockout mice were more sensitive to DE-71, which means that GSH could modulate the neurotoxicity of DE-71[29-30]. However, the antioxidant parameters here showed no significant changes. Compared with the results of a 6-months exposure[3], the 1-week exposure in our study was not long enough for the brains to weaken, or to significantly weaken the antioxidant defence. However, their antioxidant defence was not strong enough to handle the excessive free radical formation.

Interestingly, all these changes did not happen in the group treated with 2 mg/kg of TBC. The mechanism behind this observation remains unclear and needs to be further studied. Considering that the antioxidant ability of other organs was not covered here, the application of 2 mg/kg of TBC might change the activity of some enzymes in other organs and by that, eliminate excessive free radicals. For example, unpublished data in our team showed that 2 mg/kg of TBC could significantly increase the level of catalase (CAT) in rat livers.

3 Conclusion

We conclude from the obtained results that short-term exposure to TBC can cause measurable changes in oxidative stress both in vitro and in vivo. We suppose that both oxidation effects and physiological changes may be reversed by adding exogenous ROS scavengers, and thus oxidative stress may be the mechanism of toxicity caused by TBC exposure. The relationship between oxidation effects and physiological changes remains to be further investigated in the future study.