环境中有机磷酸酯阻燃剂(TDCP、TECP和TCPP)对人胚肾细胞(HEK 293)的影响

环境中有机磷酸酯阻燃剂(TDCP、TECP和TCPP)对人胚肾
细胞(HEK 293)的影响
任相浩;陈雅岚;寇莹莹
【摘要】水环境中的微污染物有机磷酸酯如三(2-氯乙基)磷酸酯(TCEP)、三(2-氯-异丙基)磷酸酯( TCPP)和三(1,3-二氯丙基)磷酸酯(TDCP)主要用作聚氨酯泡沫塑料的阻燃剂.物理、化学和生物处理很难完全消除这些污染物.研究阻燃剂在环境水平和较高浓度下对人细胞系的细胞毒性和细胞周期效应.结果表明,在浓度为0. 001 mg/L和0. 01 mg/L时,只有TDCP具有轻微的细胞毒性,而当这三种化学物质浓度100 mg/L以上时对细胞毒性有显著的诱导作用. TCEP和TCPP的EC50分别为276. 8 mg/L和58. 4 mg/L.三种药物均能抑制CDK 4的表达,TDCP能增加CDK 2和cyclin E的表达,而TCEP和TCPP在CDK 2和cyclin E的表达上表现出自差现象. TDCP和TCEP使细胞数量减少,细胞形态发生改变.可见三种化学物质对HEK 293细胞的杀伤作用可能是通过抑制CDK 4调节蛋白而产生的.
【期刊名称】《科学技术与工程》
【年(卷),期】2019(019)010
【总页数】7页(P254-260)
【关键词】有机磷阻燃剂;微量污染物;环境水平;细胞毒性;细胞周期调节蛋白;人体细胞
【作者】任相浩;陈雅岚;寇莹莹
【作者单位】北京建筑大学环境与能源工程学院城市雨水系统与水环境教育重点实验室,北京100044;北京建筑大学环境与能源工程学院城市雨水系统与水环境教育重点实验室,北京100044;北京建筑大学环境与能源工程学院城市雨水系统与水环境教育重点实验室,北京100044
【正文语种】中文
【中图分类】X171.5
Being a major component of chlorinated organophosphate flame retardants, tris-(1,3-dichloro-isopropyl)-phosphate (TDCP), tris-(2-chloroethyl)-phosphate (TCEP) and tris-(2-chloro-isopropyl)-phosphate (TCPP) are mostly used in polyurethane foams. These retardants as typical micropollutants present in surface water, wastewater treatment plant effluent, ocean and drinking water from ng/L to μg/L level normally called as environmental level [1—3]. The elimination of these three organophosphate retardants is 0～30% by conventional wastewater treatment plant and membrane bioreactor (MBR), about 50% by ozonization, and 83%～96% by biologically active slow sand filters and GAC filtration[3—7]. Even in reverse osmosis (RO) and nanofiltration (NF), maximum removal efficiency is 95% [6,8]. Therefore, the importance of low concentration of these fire retardants remained in aquatic environment has been increasing on human health[9].
In cytotoxic study of these organophosphate retardants, only few people have investigated toxic effects using in vitro test, especially on human cells,
whereas major studies were progressed by in vivo test[10]. Föllmann and Wober[11] studied cytotoxic, genotoxic, mutagenic and estrogenic effects of TCEP and TCPP using hamster fibroblasts (V79) cells with various concentrations from low ng/L to high mg/L, and detected slight cytotoxic effect at mg/L level. Dishaw et al[12] investigated neurotoxicity of TDCP, TCEP and TCPP using PC12 cells, and the results showed that those chemicals might affect neurodevelopment. Ren et al[13] studied cytotoxic and molecular toxic effects of TCEP on primary cultured rabbit renal proximal tubule cells, and TCEP induced cytotoxic and molecular toxic effects at environmental level. However, in spite of sensitive detection, the culture of primary animal cells was not easy to be handled in the experiments.
In this research, a typical human cell line, HEK293 cells, was used to investigate toxic effects of target chemicals conducted from waster sources, where the culture of cell lines is much easier than primary cells [14,15]. Due to investigation of environmentally typical micropollutants, both environmental concentration (0.001 mg/L and 0.01 mg/L) and higher concentrations were considered with 48 h exposure time on the
cells[13,16]. Expression of cell cycle regulatory proteins was also investigated to study mechanism of cytotoxic effect, where cell cycle regulatory proteins control eukaryotic cell growth that is composed of the gap 1 phase (G1), DNA synthesis phase (S), gap 2 phase (G2), and mitosis phase (M)[17]. Thus, the purpose in this study was to investigate cytotoxic and molecular toxic effect of TDCP, TCEP and TCPP at environmental and
higher level on the typical human cell line.
1 Materials and methods
1.1 Chemical samples and antibodies
Tris-(2-chloroethyl)-phosphate (TCEP) (CAT-No.119660-25G) was from Sigma-Aldrich (USA). Tris-(1,3-dichloro-isopropyl)-phosphate (TDCP) (CAT-No. 203-06892) and tris-(2-chloro-isopropyl)-phosphate (TCPP) (CAT-No. 200-13811) were purchased from Wako (Japan). Primary antibodies of cyclin dependent kinase 4 (CDK4) (CAT-No.sc-23896), cyclin dependent kinase 2 (Cdk2) (CAT-No.sc-6248), cyclin E (CAT-No.sc-247) and β-actin (CAT-No.sc-47778) were acquired from Santa Cruz Biotechnology (USA). Goat anti-mouse (CAT-No.sc-2005) as a secondary antibody was also obtained from Santa Cruz Biotechnology (USA).
1.2 Cell culture
Human embryonic kidney 293 cells were purchased from American Type Culture Collection (ATCC CRL-1573). The cells were cultured in Dulbecco’s modified eagle medium (DMEM) (GIBCO 11995-065, Invitrogen, USA) with addition of 10% fetal bovine serum (FBS) (GIBCO 16000-044, Invitrogen, USA). The cells were maintained in CO2 incubator with 37 ℃ and 5% CO2 concentration. In the sub-culture, HEK293 cells were washed by Dulbecco’s phosphate-buffered saline (PBS) (GIBCO 142000-075, Invitrogen, USA) solution and then harvested by trypsin (GIBCO 12604-013, Invitrogen, USA).
1.3 Sulforhodamine B (SRB) assay and cell morphological analysis
Ten thousands cells of HEK293 cells were cultivated to each well in 96-well
plate and maintained in CO2 incubator for 12 h, where DMEM culture media was used to culture the cells. These cells were then treated by the three chemicals with twelve different concentrations for 48 h. Fifty micro-liter of 50% cold TCA was added to each well slowly and kept in the refrigerator for 1 h with 4 ℃. The plate was washed by tap water, and then dried for 1 h in room temperature. One hundred micro-liter of 0.4% SRB was added to each well and dried more than 30 min. Again, the plate was washed five times by 1% acetic acid, and then dried for 2 h in room temperature. One hundred fifty micro-liter of 10 mmol/L unbuffered Tris was added to each well and detected by microplate reader with 550 nm. Also, with 48 h exposure of three chemicals to HEK293 cells, the cell morphology was detected by an inverted microscopy (Olympus IX-51) with 20×10 magnification.
1.4 Western blotting assay
HEK293 cells cultivated in 60Ø dish were washed twice with ice-cold PBS, and lysed in SDS buffer. The lysed cells were collected and moved to E-tube. The Etube was boiled at 90 ℃ for 5 min. The boiled cells were triturated 20 times by 1 mL syringe and then centrifuged at 12 000 rpm for 10 min. The supernatants were collected as a total cell fraction. The protein was quantified by Quant-iTTM protein assay (Q33211, Invigtrogen, USA). The cell homogenates (30 μg of protein) were separated on Nupage 10% Bis-Tris Gel (NP0315BOX, Invitrogen, USA) with 20× NuPA GE MOPS SDS Running Buffer (NP0001, Invitrogen, USA). The separated proteins were transferred from the gel to nitrocellulose membrane (IB3010-01, Invitrogen,
USA) by iBlot Transfer (692404, Invitrogen, USA). The membranes were then washed with water and then blocked with 5% skimmed milk powder in TBS-T (10 mmol/L Tris-HCl, pH 7.6, 150 mmol/L NaCl, 0.05% Tween-20) for 1 h. The blocked membrane were washed three times with TBS-T, and treated with primary antibody at 4 ℃ for overnight. After treatment of secondary antibody, the bands were visualized with enhanced chemiluminescence (Amersham Pharmacia Biotech, England, UK).
1.5 Statistical analysis
The results were expressed as the mean ± standard error (SE), and were also analyzed by t-test and ANOVA, where P<0.05 and P<0.01 were considered significant.
2 Results and analyese
2.1 Cytotoxic effects of three environmental chemicals on HEK293 cells Cytotoxicity of TDCP, TCEP and TCPP was observed from μg/L to mg/L level by SRB assay. TDCP slightly decreased cell viability at environmental concentrations (0.001 mg/L and 0.01 mg/L), showing 95.0% and 96.6% of the control, respectively [Fig.1(a)]. From 50 mg/L, TDCP significantly decreased cell viability, ranging in 82.2%～87.4% of the control. TCEP did change cell viability from 0.001 mg/L to 75 mg/L, compared with the control, but significantly decreased from 100 mg/L, 27.7%～94.1% of the control [Fig.1(b)]. TCPP inFig.1(c) did not affect cell viability until 1 mg/L, but significantly decreased from 50 mg/L and upper concentrations, showing 2
3.5%～71.4% of the control.
*represents for P<0.05 vs control; ** represents for P<0.01 vs controlFig.1
Cytotoxic effects of TDCP, TCEP and TCPP on HEK293 cells for 48 h exposure with different concentrations
The half effective concentration (EC50) as one expressional way of cytotoxic effects was also calculated using Origen 6.1 software based on the cell viability data shown above (Table 1). Because there was no dose dependent change in TDCP treated samples, no EC50 value was possibly evaluated. The EC50 of TCEP was 276.8 mg/L and TCPP was four times lower than TCEP, showing 58.4 mg/L.
Table 1 EC50 values of three target chemical samples for 48 h exposureEnvironmental chemicalsEC50 value in HEK293 cells/(mg·L-
1)TDCPNDTCEP276.8TCPP58.4
ND: not detected.
2.2 Morphological and numerical effect of three chemicals
Table 2 showed morphological and numerical effects of HEK293 cells on TDCP, TCEP and TCPP at three different concentrations through microscopic study with 48 h chemical exposure. In the TDCP treated cells, morphological and numerical change was not clearly found at 0.01 mg/L, but cell number was slightly decreased at 1 mg/L and 50 mg/L, and clearly decreased at 100 mg/L, compared with the control. TCEP did not affect on the morphological change of HEK293 cells from 0.01 mg/L to 100 mg/L, but slightly decreased cell number at 100 mg/L, whereas did not change cell number at 0.01 mg/L 1 mg/L and 50 mg/L, compared with the control. In the TCPP treated cells, both morphological and numerical changes were clearly found at 50 mg/L and 100 mg/L, compared with the control, and
more number of shrunken cells was found at 100 mg/L than at 50 mg/L. Not clear change of cell number was found at 0.01 mg/L and 1 mg/L, compared with the control.
2.3 Effect of three chemicals on cell cyclic regulatory protein expression
In the results of mechanism study, TDCP at 0.01 mg/L significantly inhibited the expression of CDK4, one of typical cell cycle regulatory proteins, but slightly decreased the expression at 1 mg/L and 50 mg/L, compared with the control (Fig.2). However, TDCP slightly increased the expression of CDK2 at 1 mg/L and 50 mg/L, whereas no effect was found at 0.01 mg/L, compared with the control. The expression of cyclin E was induced by 1 mg/L and 50 mg/L of TDCP, but no effect by 0.01 mg/L, compared with the control.
The expression of cell cycle regulatory proteins treated by TCEP was also introduced in Fig.3.TCEP significantly inhibited the expression of CDK4 at tested three concentrations, but slightly increased the expression of CDK2 at 1 mg/L and 50 mg/L. The expression of cyclin E was significantly induced at 0.01 mg/L, and slightly increased at 1 mg/L, however, significantly decreased at 50 mg/L, compared with the control.
Table 2 Morpholog ical analysis of HEK293 cells (20× magnification) treated with three chemicals after 48 h of exposure
β-actin was used as the internal standard; the lower panels denote the means ± standard errorFig.2 Effect of TDCP-treated HEK293 cells on cell cycle regulatory protein expression
TCPP slightly increased the expression of CDK4 at 0.01 mg/L, but significantly inhibited the expression at 1 mg/L and 50 mg/L (Fig.4. The expression of CDK2 was increased at all tested concentrations. TCPP significantly increased the expression of cyclin E at 0.01 mg/L, but significantly decreased the expression at 50 mg/L, compared with the control.
3 Discussions
In the cytotoxic results conducted from this research, TDCP slightly decreased cell viability at 0.001 mg/L and 0.01 mg/L, but significantly inhibited from 50 mog/L on HEK293 cells, and Liu et al[18] reported similar results that less than 80% cell viability of H295R was obtained at 10 mg/L and higher concentrations. Dishaw et al[12] also investigated the effect of cell vi ability only at 50 μmol/L (21.6 mg/L) TDCP using PC12 cells, and no adverse effect was found. However, Ta et al[19] reported cytotoxic results using PC12 cells that TDCP significantly decreased the cell viability from 5 μmol/L (2.2 mg/L) to 75 μmol/L (32.3 mg/L). TCEP in this study did not affect cell viability until 75 mg/L, but significantly decreased from 100
mg/L and higher concentrations, and Liu et al[18] also reported similar results from this research that less than 80% cell viability of H295R cells was achieved at 100 mg/L and greater. Ta et al[19] indicated that TCEP slightly decreased viability of PC12 cells at 15 μmol/L (4.3 mg/L) and 20
μmol/L (5.7 mg/L), whereas significantly inhibited from 40 μmol/L (11.4 mg/L) and higher concentrations. Ren et al[13] also resulted that TCEP did not affect cell viability at 0.01 mg/L, but significantly decreased from 10
mg/L using sensitive primary cultured cells. Even detection method of cytotoxicity was different from this research, Föllmann and Wober[11] als o indicated that moderate cytotoxicity was found above 10 μmol/L (2.9 mg/L) in V79 cells with the presence of S9-mix. TCPP in this research significantly decreased cell viability from 50 mg/L and upper concentrations, whereas moderate cytotoxicity was reported above 1 mmol/L (327.6 mg/L) with the presence of S9-mix[11]. Liu et al [18] could not find cytotoxic effects of TCPP even at 100 mg/L.
Fig.3 Effect of TCEP-treated HEK293 cells on cell cycle regulatory protein expression
Fig.4 Effect of TCPP-treated HEK293 cells on cell cycle regulatory protein expression
Depend on the results of EC50 conducted from this research, TCPP had four times lower EC50 value than TCEP, 58.4 mg/L and 276.8 mg/L, respectively. After using primary cultured rabbit renal proximal tubule cells, the EC50 value of TCEP was 2.9 mg/L[13]. No EC50 value of TDCP was inducted in this study due to similar percentage of cell viability from 50
mg/L to 1 000 mg/L, showing 82.3%～87.4% of the control, however, Ta et al[19] could have an EC50 value of TDCP from their results of PC12 cell viability even not mentioned in their report.
The results of cell cycle regulatory protein expression as a typical tool for studying molecular toxicity of cell growth in this research indicated that decrease of cell viability treated by TDCP might be inducted through decrease of CDK4 expression because the increase of CDK2 and cyclin E
expressions might activate DNA synthesis[20—22]. The results of cell cycle regulatory protein expressions treated by TCEP and TCPP in this study also indicated that the decrease of CDK4 expression might inhibit cell viability, showing significant decrease of the expression at 1 mg/L and 50 mg/L, whereas CDK2 and cyclin E expression showed self-discrepancy results, and further more experiments will be required. In the case of Ren et al[13], TCEP decreased cell growth via inhibiting the expression of CDK4, CDK2 and cyclin E in the primary cultured rabbit renal proximal tubule cells. Practically, it was found that decrease of cell viability might induce the decrease of cell number treated by TDCP, TCEP and TCPP, and might cause morphological changes on TCPP treated cells.
4 Conclusion
(1) TDCP, TCEP and TCPP significantly inhibited cell viability at high concentrations, but slight decrease of cell viability was only found at environmental concentrations on TDCP treated cells.
(2) The EC50 value of TCPP was four times lower than TCEP, 58.4 mg/L and 276.8 mg/L, respectively, whereas no EC50 value of TDCP was found on HEK293 cells.
(3) Morphological change was only found at 50 mg/L and 100 mg/L on TCPP treated cells, and numerical decrease was all found on TDCP, TCEP and TCPP treated cells.
(4) The three tested chemicals induced cytotoxicity might through inhibition of CDK4 regulatory protein expression.
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