| China has been one of the main producers and consumers of textiles and clothing in the world, with annual yield of 1/3 of the global total output. Dye is one of the indispensable materials in the production of textiles and clothing, whose annual consumption reaches one million tons approximately. Besides the dyeing and printing of textiles, dye is also applied in leather, paper, plastic, rubber, paint and food industries. However, with social development, the requirements for safety and environmental protection become more and more strict.The use of some dyes have been prohibited or confined for being proved to be toxic. Nine dyes including Acid Red 26, Basic Red 9, Basic Violet 14, Direct Black 38, Direct Blue 6, Direct Red 28, Disperse Blue 1, Disperse Orange 11 and Disperse Yellow 3 were classified as carcinogenic in the European ecological textile standard "Oeko-Tex Standard 100". But, most countries except European countries have not listed these 9 dyes into banned substance inventory. The toxicity profiles of Basic Violet 14, Direct Red 28 and Acid Red 26 have not been reported by now. In this dissertation, the acute toxicities of these 9 dyes were assessed and compared using zebrafish model;The developmental toxicities of Basic Violet 14, Direct Red 28 and Acid Red 26 were studied, and Acid Red 26 was further studied for its cardiovascular toxicity and effect on gene expressions. At last, Basic Violet 14, the dye with the largest toxicity among Basic Violet 14, Direct Red 28 and Acid Red 26, was selected to study its hepatotoxicity and molecular mechanism.1. The acute toxicities of these 9 dyes were assessed and compared. The MNLC (Maximum non-lethal concentration), LC10(10% lethal concentration) and LC50(50% lethal concentration) of these 9 dyes were obtained in acute toxicity experiments using zebrafish model. The LC50 of Acid Red 26, Basic Red 9, Basic Violet 14, Direct Black 38, Direct Blue 6, Direct Red 28, Disperse Blue 1, Disperse Orange 11 and Disperse Yellow 3 were calculated as (2500～2800) μg/mL,53.69 μg/mL,60.63 μg/mL, (2000～2500) μ/mL, (1500～2000) μg/mL,476.84μg/mL,224.43 μg/mL,210.29 μg/mL and 755.72 μg/mL, respectively. Therefore, the toxicity in LC50 arrangement is: Basic Red 9> Basic Violet 14> Disperse Orange 11> Disperse Blue 1> Direct Red 28> Disperse Yellow 3> Direct Blue 6> Direct Black 38> Acid Red 26.2. The developmental toxicities of Basic Violet 14, Direct Red 28 and Acid Red 26 were studied, and Acid Red 26 was further studied for its cardiovascular toxicity and effect on gene expressions. The developmental toxicities of Basic Violet 14 were observed as:yolk sac absorption delay (showed larger yolk sac than that of the vehicle control), abnormal swimming bladder development (showed smaller swimming bladder because of agensis or ateliosis of swimming bladder), abnormal intestinal development (showed lack or less intestinal folds than that of the vehicle control), hepatotoxicity (showed smaller liver size and liver nigrescence), cardiovascular toxicity at low incidence rate (showed cardiac edema); The developmental toxicities of Direct Red 28 were observed as:yolk sac absorption delay, abnormal swimming bladder development, abnormal intestinal development, hepatotoxicity and cardiovascular toxicity at low incidence rate; The developmental toxicities of Acid Red 26 were observed as:yolk sac absorption delay, abnormal swimming bladder and cardiovascular toxicity at low incidence rate.The incidence rates of cardiovascular toxicity induced by Acid Red 26 increased obviously at concentrations higher than 450 μg/mL, in spite of the low incidence rates at lower concentrations. The higher the concentration and the longer the exposure time, the higher the incidence rate. Experiment results revealed that groups treated with Acid Red 26 at concentrations of 3000μg/mL and 3500 μg/mL induced cardiovascular toxicity, shown by cardiac edema, decreased heart rate and decreased perfusion, while those in 4000 μg/mL all died at the end of treatment (3dpf). Treatment with Acid Red 26 also resulted in an obvious up-regulation of NKX2.5 and down-regulation of GATA4.3. The hepatotoxicity of Basic Violet 14 and its molecular mechanism were investigated. Experiment results revealed that Basic Violet 14 induced hepatotoxicity, shown by yolk sac absorption delay, hepatatrophy (decreased liver size) and liver nigrescence (decreased transparency), in a dose-dependent manner. Histopathological analysis of liver tissue sections in Basic Violet 14 treated zebrafish showed severe tissue damages, such as inflammation, necrosis, reactive nuclear enlargement and fibrogenesis. Treatment with Basic Violet 14 also resulted in up-regulation of GCLC, HSP70 and NQO-1 expressions. However, the dye had no effect on the expression of VTG.Experiment results revealed that the relative ROS levels of those exposed to 1/10 MNLC,1/3 MNLC, MNLC and LC10 of Basic Violet 14 were 119%,118%,187% and 308% of the vehicle control, respectively. The GSH contents of those exposed to 1/10 MNLC,1/3 MNLC, MNLC and LC10 of Basic Violet 14 were 1.06,1.04,0.92 and 0.90 μmol per zebrafish, respectively (the GSH content of the vehicle control was 1.02 μmol per zebrafish). The increased ROS level and decreased GSH content indicated that Basic Violet 14 induced oxidative stress in zebrafish. Since Basic Violet 14 increased the ROS level in zebrafish, GCLC, HSP70 and NQO-1 were up-regulated to adjust the ROS level. The DNA olive tail moment values of those exposed to 1/10 MNLC,1/3 MNLC, MNLC and LC10 of Basic Violet 14 were 0.76, 1.40,3.39 and 5.70, respectively. The remarkable elongation of the DNA olive tail moment indicated that Basic Violet 14 induced breakage of DNA double strands. The relative Caspase-9 activity of those exposed to 1/10 MNLC,1/3 MNLC, MNLC and LC10 of Basic Violet 14 were 107%,106%,129% and 148% of the vehicle control, while the relative Caspase-3/7 activity were 99%,85%,128% and 274% of the vehicle control, respectively. The increased relative activity of Caspase-9 and Caspase-3/7 indicated that Basic Violet 14 activated intracellular (mitochondrial) apoptotic pathway and induced cell apoptosis. In vitro experimental results showed that Basic Violet 14 caused mitochondria damage and membrane potential decrease of human hepatoma cell line Hep G2, which further evidenced the mitochondrial apoptotic pathway of the hepatotoxicity induced by Basic Violet 14. In conclusion, the molecular mechanism of the hepatotoxicity may be that Basic Violet 14 induces oxidative stress and breakage of DNA double strands, activates intracellular (mitochondrial) apoptotic pathway and induces cell apoptosis. |
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