The toxic effects of Tris-（2,3-dibromopropyl） isocyanurate（TBC） on genes expression of bmp2b and bmp4 of zebrafish embryos

JIA+Wan-jun

【Abstract】We exposed zebrafish embryos to Tris-（2,3-dibromopropyl）isocyanurate（TBC） at the concentration of 20ppb, 100ppb, 400ppb, 1000ppb for 120h and 0.1%DMSO was set as the control group. Bmp2b and bmp4 were chosen perform RT-PCR to determine their genes expression level. The results showed that, TBC influenced their genes expression level in some extent and it significantly raised the genes expression level at the concentration of 20ppb.

【Key words】Bmp; Gene expression; RT-PCR; Zebrafish

0Introduction

Tris-（2,3-dibromopropyl） isocyanurate（TBC） is a new type of brominated flame retardants. It is stable and has high octanol-water partition coefficient. Since TBC was first detected from Liuyang River in Hunan province, China, in 1997[1], people started to study on its environmental toxicology. According to preliminary studies, TBC can damage zebrafishs gill, liver, intestines, gonad and swimming bladder[1-2].

Bmp signal pathway plays important role in the development of zebrafish. It involved in many aspects about vital actions, therein, Bmp2b and bmp4 are one kind of bmp subfamilies and are all expressed at early embryos[3-4]. In this study, we exposed zebrafish embryos to different concentration of TBC and through RT-PCR to determine the expression level of bmp2b and bmp4 to explore the influence of TBC on bmp signal pathway.

１Materials and methods

1.1Chemicals and animals

Tris-(2,3-dibromopropyl） isocyanurate（TBC, 97% purity） was purchased from Sigma, USA. The zebrafish（about 6 months） were bought from Nanshan market, Qingdao. Then, the fish were bred fresh Artemia nauplii twice a day in constant temperature room at 28℃ with the photoperiod about 14h light:10h dark.

1.2Methods

1.2.1Preparation of the solutions

The TBC stock solutions（20ppm, 100ppm, 400ppm,1000ppm） were prepared and then used zebrafish embryo culture medium to dilute 1000 times when used. The zebrafish embryo culture medium consists of 5mM NaCl、0.17mM KCl、0.33mM CaCl2、0.33mM MgSO4、0.00001%（W/V）methylene blue.

1.2.2Exposure experiment

The night before gathering embryos, we set the adult fish in the hatchery with♂2:♀2. Next morning, diaphragms were all pumped away. Then, the male fish chased the female fish drastically and lay eggs. After about 30min, the eggs were all transferred and cultured in a illumination incubator at 28.5℃. The TBC exposure experiment was initiated at two hour post-fertilization（hpf） and implemented at 6-well plates with 8ml exposure solutions to 120hpf. The exposure solution concentration were 20ppb, 100ppb, 400ppb and 1000ppb, 0.1%DMSO was set as the control group. Each group had three replicates.

1.2.3RNA extraction and cDNA synthesis

120hpf zebrafish embryos were collected and cleaned. Then, we used RNAiso Plus（Takara）, followed the manuscript protocol, to extract the total RNA. The quality and concentration of total RNA were determined by using ultraviolet-uisible spectrophotometer and agarose gel electrophoresis.

1μg total RNA was reverse transcribed by using PrimeScriptTM RT reagent Kit With gDNA Eraser（Perfect Real Time）（Takara）, and cDNA was stocked at -20℃.

1.2.4Gene expression analysis

Primers of bmp2b, bmp4 and actb were designed by using primer premier 5（given in tab.1）. RT-PCR were operated on 7500 Real Time System, Applied Biosystems, using Fast Start Universal SYBR Green Master（ROX）（Roche）. Operating programs were 95℃, 10min; 95℃, 15s; 61℃, 60s, by 40 cycles. Each gene has three repetitions. The gene relative expression level was analyzed by 2^-△△CT method.

Tab.1Primers used for RT-PCR

2Results and discussion

Fig.1.The relative gene expression level of bmp2b under different concentration of TBC

The relative gene expression level of bmp2b and bmp4 were respectively shown in fig.1. After 120h exposure of TBC, the genes expressive quantity were all changed, especially at 20ppb, the genes expression level of bmp2b and bmp4 are all increased significantly with about 3.3-fold up-regulation. At the concentration of 100ppb and 400ppb, the genes expression level of bmp2b and bmp4 had not changed a lot, but the genes expression level has a little increase at the concentration of 1000ppb. It is not a monotonicity curve between the relative genes expression level and the exposure concentration of TBC. For this phenomenon, we inferred that it was one kind of excitatory compensation performance by the organisms. Bmp signal pathway plays important role in vital movement and we should pay more attention to explore the mechanism of toxicity on bmp signal pathwayin the future.

【Reference】

［１］Ruan T, Wang Y, Wang C, Wang P ,et al. Identification and Evaluation of a novel heterocyclic brominated flame, retardant tris(2,3-dibromopropyl) isocyanurate in environmental matrices near a manufacturing plant in southern China[J].Environmental science & technology, 2009, 43(9):3080-3086.

［2］Zhang Xu, Li Juan, Chen Minjie, et al. Toxicity of the brominated flame retardant tris(2,3-dibromopropyl) isoeyanurate in zebrafish (Danio rerio)[J].Chinese Science Bulletin, 2011, 56(15):1548-155.

［3］JP Martínez-Barberá, H Toresson, RS Da, S Krauss. (1997). Cloning and expression of three members of the zebrafish Bmp family: Bmp2a, Bmp2b and Bmp4[J].Gene, 1997,198(1-2):53-59.

［4］M Nikaido, M Tada, T Saji, N Ueno. Conservation of BMP signaling in zebrafish mesoderm Patterning[J]. Mechanisms of Development, 1997,61(1-2):75-88.

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