磷酸三甲苯酯对秀丽隐杆线虫的生殖毒性及机制

Reproductive toxicity and associated mechanism of tricresyl phosphate on Caenorhabditis elegans

[背景]磷酸三甲苯酯(TCP)主要用作阻燃剂。研究证实其具有细胞毒性和神经毒性,但对于其生殖毒性尚不明确。[目的]研究TCP亚急性暴露对秀丽隐杆线虫的生殖毒性以及潜在机制。[方法]将秀丽隐杆线虫分别暴露于溶剂对照以及0.1、1、10、100、1 000μg·L^(-1)TCP的培养基72 h,检测后代数目和子宫内受精卵的数量,评价生殖能力;检测总生殖细胞数和生殖腺臂相对面积,评价生殖腺发育;检测体长和体宽,评价生长发育;检测秀丽隐杆线虫体内活性氧(ROS)水平、超氧化物歧化酶(SOD)活性,利用实时荧光定量PCR检测N2线虫线粒体活性氧代谢基因(mev-1和gas-1)的表达,评价氧化应激水平。WS1433转基因线虫和野生型线虫N2分别暴露于溶剂对照或TCP(0.1、1、10、100、1 000μg·L^(-1))后,检测WS1433转基因线虫生殖细胞DNA损伤情况,实时荧光定量PCR检测N2线虫DNA损伤相关基因(hus-1、clk-2、cep-1和egl-1)的相对表达,评价TCP暴露对秀丽隐杆线虫遗传损伤的影响。[结果]与溶剂对照组相比(217.00±12.20),100μg·L^(-1)和1 000μg·L^(-1)TCP组N2线虫的后代数目减少(170.80±11.51、169.60±10.52,P <0.05)。与溶剂对照组相比(18.43±1.69),100μg·L^(-1)和1 000μg·L^(-1)TCP组N2线虫的子宫内受精卵数目减少(13.47±0.81、11.95±0.90,P <0.05)。与溶剂对照组相比(312.46±77.4),100μg·L^(-1)和1 000μg·L^(-1)TCP组N2线虫的总生殖细胞数减少(281.80±12.98、273.50±8.53,P <0.05)。与溶剂对照组相比,100μg·L^(-1)和1 000μg·L^(-1)TCP组N2线虫的生殖腺相对面积分别减少13.83%、17.25%(P <0.05)。与溶剂对照组相比[(1 058.10±80.12)μm、(78.21±14.69)μm],100μg·L^(-1)和1 000μg·L^(-1)TCP组N2线虫的体长、体宽均减少(P <0.05)。与溶剂对照组相比,10、100和1 000μg·L^(-1)TCP组线虫体内ROS相对荧光强度增加(为107.60%±1.02%、105.90%±1.40%、106.40%±1.85%,P <0.05),SOD活性分别降低(降低20.66%、15.88%、16.44%,P <0.05)。与溶剂对照组相比(1.3±1.3),100和1 000μg·L^(-1)TCP组WS1433线虫DNA损伤的生殖细胞数目上升(2.4±0.3、2.7±0.3,P <0.05);10、100和1 000μg·L^(-1)TCP组N2线虫mev-1和gas-1基因表达降低(P <0.05);0.1~1 000μg·L^(-1)TCP组线虫hus-1基因表达增加(P <0.05);100和1 000μg·L^(-1)TCP组线虫clk-2和egl-1基因表达增加(P <0.05);1、10和100μg·L^(-1)TCP组线虫cep-1基因表达增加(P <0.05)。[结论] TCP可能通过氧化应激和生殖细胞DNA损伤对线虫造成生殖损伤。

[Background] Tricresyl phosphate(TCP) is mainly used as a flame retardant. Studies have confirmed that it has cytotoxicity and neurotoxicity, but its reproductive toxicity is not clear.[Objective] To investigate the reproductive toxicity and potential mechanism of TCP subacute exposure on Caenorhabditis elegans.[Methods] Caenorhabditis elegans were exposed to solvent control and 0.1, 1, 10, 100, and1 000 μg·L^(-1)TCP respectively for 72 h. Brood size and number of fertilized eggs in the uterus were detected to evaluate reproductive ability. The number of total germline cells and the relative area of gonad arm were measured to evaluate the development of gonads. The body length and body width of Caenorhabditis elegans were detected to evaluate growth and development. The activities of reactive oxygen species(ROS) and superoxide dismutase(SOD) in Caenorhabditis elegans, and the mitochondrial active oxygen metabolism genes(mev-1 and gas-1) of N2 nematodes were detected by real-time fluorescence quantitative polymerase chain reaction(q RT-PCR) to evaluate oxidative stress. WS1433 transgenic nematodes and wild-type nematodes N2 were exposed to solvent control or TCP(0.1, 1, 10, 100, and 1 000 μg·L^(-1)) respectively. DNA damage in germ cells of WS1433 transgenic nematodes was detected, the relative expressions of DNA damage-related genes(hus-1, clk-2, cep-1, and egl-1) in N2 nematodes were detected by q RT-PCR to evaluate the effect of TCP exposure on genetic damage.[Results] Compared with the solvent control group(217.00 ± 12.20), the brood size of N2 nematodes in the 100 μg·L^(-1)and 1 000 μg·L^(-1)TCP groups decreased(170.80 ± 11.51, 169.60 ± 10.52, P < 0.05). Compared with the solvent control group(18.43 ± 1.69), the number of fertilized eggs of N2 nematodes in the 100 μg·L^(-1)and 1 000 μg·L^(-1)TCP groups decreased(13.47 ± 0.81, 11.95 ± 0.90, P < 0.05). Compared with the solvent control group(312.46 ± 77.4), the number of total germline cells of N2 nematodes in the 100 μg·L^(-1)and 1 000 μg·L^(-1)TCP groups decreased(281.80 ± 12.98, 273.50 ± 8.53, P < 0.05). Compared with the solvent control group, the relative area of gonads of N2 nematodes in the 100 μg·L^(-1)and 1 000 μg·L^(-1)TCP groups decreased by 13.83% and 17.25% respectively(P < 0.05). Compared with the solvent control group [(1 058.10±80.12) μm,(78.21±14.69) μm], the body length and body width of N2 nematodes in the 100 μg·L^(-1)and1 000 μg·L^(-1)TCP groups decreased(P < 0.05). Compared with the solvent control group, the relative fluorescence intensity of ROS in nematodes in the 10, 100, and 1 000 μg·L^(-1)TCP groups increased significantly(107.60%±1.02%, 105.90%±1.40%, and 106.40%±1.85%,respectively, P < 0.05), and the activities of SOD were reduced(by 20.66%, 15.88%, and 16.44%, respectively, P < 0.05). Compared with the solvent control group(1.3±1.3), the number of DNA-damaged germ cells of WS1433 nematodes in the 100 and 1 000 μg·L^(-1)TCP groups increased significantly(2.4±0.3, 2.7±0.3, P < 0.05);the expressions of mev-1 and gas-1 genes in N2 nematodes in the 10, 100 and 1 000 μg·L^(-1)TCP groups decreased significantly(P < 0.05);the expressions of hus-1 in the 0.1-1 000 μg·L^(-1)TCP groups significantly increased(P < 0.05);the expressions of clk-2 and egl-1 in the 100 and 1 000 μg·L^(-1)TCP groups increased significantly(P < 0.05);the expressions of cep-1 in the 1, 10, and 100 μg·L^(-1)TCP groups increased significantly(P < 0.05).[Conclusion] TCP may cause reproductive damage to nematodes through oxidative stress and germ cell DNA damage.