Yuanxiang Jin

Oxidative stress response and gene expression with atrazine exposure in adult female zebrafish (Danio rerio).

Atrazine (ATZ) is the most common pesticide of freshwater ecosystems in the world. The effects of ATZ exposure on the induction of oxidative stress and the alteration of gene expression were studied in liver and ovary samples from female zebrafish (Danio rerio). Antioxidant enzyme activities (SOD and CAT), in addition to the GSH and MDA content, in the liver altered significantly; the mRNA levels for the genes encoding these antioxidant proteins, such as Cu/Zn-Sod, Mn-Sod, Cat, and Gpx, were up-regulated significantly in the liver when zebrafish were exposed to various concentrations of ATZ for 14d. However, the mRNA induction patterns were not in accordance with those of antioxidant enzyme changes in the liver and ovary. Moreover, the transcriptional expression of mitochondrial inner membrane genes related to ROS production, such as Ucp-2 and Bcl-2, were altered significantly in high ATZ treatment groups. However, the ATZ treatment did not induce any apparent changes in the transcriptional expression of CoxI, Ndi, or ATPo6, which are related to the mitochondrial respiratory chain and ATP synthesis. To our knowledge, this is the first report to study the transcriptional effects of ATZ stress on zebrafish. The information presented in this study will be helpful in fully understanding the mechanism of oxidative stress induced by ATZ in fish.

Effect of endocrine disrupting chemicals on the transcription of genes related to the innate immune system in the early developmental stage of zebrafish (Danio rerio)

Health concerns regarding the potential interference of endocrine disrupting chemicals (EDCs) in the immune system of wildlife and humans have increased in recent years. However, the effects of EDCs in aquatic systems on the immune system of fish species has only received limited attention. In the present study, we found that the mRNA levels of TNFalpha, IFN, IL-1beta, IL-8, CXCL-Clc, and CC-chemokine, which are closely related to the innate immune system, were affected in newly hatched zebrafish when exposed to EDCs, such as 17beta-estradiol, 17alpha-ethynyestradiol, permethrin, atrazine and nonylphenol at various concentrations (0.1, 0.5, 2.5 and 12.5 microg/l) for three days during the embryo stage. However, the different EDCs displayed different potentials to change innate immune-related gene transcription. Among the selected chemicals, permethrin (PM) and 17beta-estradiol (E2) (12.5 microg/l) significantly increased the mRNA levels of many cytokines, exhibiting their most prominent impacts on the innate immune system of zebrafish. In addition, it was found that the mixture of the above five chemicals (2.5 microg/l each) had a greater effect on innate immune system-related gene transcription in zebrafish than equal amounts of the single compound. Moreover, the genes (such as Bcl2, Ucp2 and iNOS) relating to reactive oxygen species (ROS) and nitrogen reactive free radical production were also influenced by some EDCs and their mixture. We suggest that heavy oxidative stress and the balance of nitric oxide (NO) production lead to death of immune cells. These results may provide an explanation of the possible mode how EDCs influence the innate immune system in zebrafish. Taken together, the results obtained in the present study clearly demonstrate that EDCs and their mixtures in aquatic systems will greatly influence the immune system in fish, suggesting that the effects of EDCs on fish should be associated with immune toxicity.

Cypermethrin has the potential to induce hepatic oxidative stress, DNA damage and apoptosis in adult zebrafish (Danio rerio)

Cypermethrin (CYP), a widely used Type II pyrethroid pesticide, is one of the most common contaminants in the freshwater aquatic system. We studied the effects of CYP exposure on the induction of hepatic oxidative stress, DNA damage and the alteration of gene expression related to apoptosis in adult zebrafish. Hepatic mRNA levels for the genes encoding antioxidant proteins, such as Cu/Zn-Sod, Mn-Sod, Cat, and Gpx, were significantly upregulated when zebrafish were exposed to various concentrations of CYP for 4 or 8 days. In addition, the main genes related to fatty acid β-oxidation and the mitochondrial genes related to respiration and ATP synthesis were also significantly upregulated after exposure to high concentrations (1 and 3 μg L(-1)) of CYP for 4 or 8 days. Moreover, in a comet assay of zebrafish hepatocytes, tail DNA, tail length, tail moment and Olive tail moment increased in a concentration-dependent manner. The significant induction (p<0.01) of all four parameters observed with CYP concentrations of 0.3 μg L(-1) or higher suggests that heavy DNA damage was induced even at low levels. Furthermore, several apoptosis- related genes, such as p53, Apaf1 and Cas3, were significantly upregulated after CYP exposure, and Bcl2/Bax expression ratio decreased, especially in groups treated with 1 and 3 μg L(-1) CYP for 8 days. Taken together, our results suggested that CYP has the potential to induce hepatic oxidative stress, DNA damage and apoptosis in zebrafish. This information will be helpful in fully understanding the mechanism of aquatic toxicology induced by CYP in fish.

Allelochemical stress causes oxidative damage and inhibition of photosynthesis in Chlorella vulgaris.

This study investigated the effects of N-phenyl-2-naphthylamine, an effective allelochemical on aquatic unicellular algae Chlorella vulgaris at physiological gene transcription level. Exposure to 2.5 mg L(-1) of N-phenyl-2-naphthylamine increased the activities of the antioxidant enzymes, superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), which were 2.47, 3.24, and 4.27 times higher than that of the control, however, exposure to 4.0 mg L(-1) N-phenyl-2-naphthylamine decreased the activities of these antioxidant enzymes. An increase in malondialdehyde content and a decrease in chlorophyll content following exposure to N-phenyl-2-naphthylamine suggested that the alga was severely damaged and that cell growth was greatly inhibited. Electron microscopy showed that the plasma membrane was detached from the cell wall, the nucleus was condensed, and the structure of chloroplasts was disrupted, in response to N-phenyl-2-naphthylamine exposure. Real-time PCR showed that N-phenyl-2-naphthylamine reduced the transcript abundance of psaB and psbC to 3% and 1% of the control, respectively. These results demonstrated that N-phenyl-2-naphthylamine not only inhibited photosynthesis, but also triggered the synthesis of reactive oxygen species (ROS) to disrupt the subcellular structure of this aquatic organism.

Embryonic exposure to cypermethrin induces apoptosis and immunotoxicity in zebrafish (Danio rerio)

Cypermethrin (CYP) is widely used for control of indoor and field pests. As a result, CYP is one of the most common contaminants in freshwater aquatic systems. In the present study, we investigated the effects of CYP exposure on the induction of apoptosis and immunotoxicity in zebrafish during the embryo developmental stage. The mRNA levels of some key genes including P53, Puma, Bax, Apaf1, Cas9 and Cas3 on the mitochondrial pathway of cell apoptosis were significantly up-regulated at the concentration of 3 and 10 μg/l CYP. Correspondingly, the activities of Cas3 and Cas9 increased significantly after exposure to 3 or 10 μg/l CYP. In addition, the mRNA levels of iNOS and the total content of NO were also up-regulated significantly after CYP exposure. Moreover, it was also observed that the mRNA levels of IFN, CXCL-Clc, CC-chem and C3, which are closely related to the innate immune system, were affected in newly hatched zebrafish when exposed to 3 and 10 μg/l CYP, exhibiting CYPs prominent impacts on the innate immune system of zebrafish. Taken together, our results suggest that CYP has the potential to induce cell apoptosis and cause innate immune system disruption in zebrafish during the embryo stage. The information presented in this study will help elucidate the mechanism of CYP-induced toxicity in fish.

The toxicity of chlorpyrifos on the early life stage of zebrafish: a survey on the endpoints at development, locomotor behavior, oxidative stress and immunotoxicity.

Chlorpyrifos (CPF) is one of the most toxic pesticides in aquatic ecosystem, but its toxicity mechanisms to fish are still not fully understood. This study examined the toxicity targets of CPF in early life stage of zebrafish on the endpoints at developmental toxicity, neurotoxicity, oxidative stress and immunotoxicity. Firstly, CPF exposure decreased the body length, inhibited the hatchability and heart rate, and resulted in a number of morphological abnormalities, primarily spinal deformities (SD) and pericardial edema (PE), in larval zebrafish. Secondly, the free swimming activities and the swimming behaviors of the larvae in response to the stimulation of light-to-dark photoperiod transition were significantly influenced by the exposure to 100 and 300xa0μg/L CPF. In addition, the activity of acetylcholinesterase (AChE) and the transcription of some genes related to neurotoxicity were also influenced by CPF exposure. Thirdly, CPF exposure induced oxidative stress in the larval zebrafish. The malondialdehyde (MDA) levels increased and the glutathione (GSH) contents decreased significantly in a concentration-dependent manner after the exposure to CPF for 96 hours post fertilization (hpf). CPF affected not only the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glutathione S-transferase (GST), but also the transcriptional levels of their respective genes. Finally, the mRNA levels of the main cytokines including tumor necrosis factor α (Tnfα), interferon (Ifn), interleukin-1 beta (Il-1β), interleukin 6 (Il6), complement factor 4 (C4) in the larvae increased significantly after the exposure to 100 or 300xa0μg/L CPF for 96xa0hpf, suggesting that the innate immune system disturbed by CPF in larvae. Taken together, our results suggested that CPF had the potential to cause developmental toxicity, behavior alterations, oxidative stress and immunotoxicity in the larval zebrafish.

Induction of hepatic estrogen-responsive gene transcription by permethrin enantiomers in male adult zebrafish.

Despite recent studies on enantioselectivity in acute aquatic toxicity and biodegradation of some pyrethroid pesticides, including permethrin (PM), enantiomer-specific estrogenic activity has been the subject of limited research. In this study, real-time quantitative RT-PCR was adopted to investigate induction of hepatic expression of mRNA of selected genes in male adult zebrafish after short-term exposure to PM enantiomers. The PM enantiomers were completely separated by a chiral HPLC column. The in vivo study found that a 2-day exposure to 100 ng/l PM racemate and its enantiomers was sufficient to stimulate transcription of two vitellogenin (vtg) genes, while 250 ng/l exposure significantly induced gene transcription in a pattern and content similar to that of the control (50 ng/l 17beta-estradiol (E2)). Significant differences were detected between the enantiomers in induction of hepatic gene transcription. At exposure level of 500 ng/l, the response to the (-)-trans enantiomer was 2.6 and 1.8 times greater than the (+)-trans enantiomer based on zebrafish vtg1 and vtg2 mRNA induction (p<0.05), respectively. Of the four enantiomers, the (-)-trans enantiomer showed the greatest estrogenic activity, with a relative activity 4-fold higher than the 50 ng/l E2 group. The results strongly suggested the occurrence of significant enantioselectivity in estrogenic activity of PM enantiomers. It would appear from our results and previous studies that using chiral pesticide as a single compound would increase the environmental risk of chronic toxicity, such as endocrine disruption, to humans and wildlife.

Cypermethrin exposure during puberty induces oxidative stress and endocrine disruption in male mice

Cypermethrin (CYP) is one of the most common contaminants in the ecosystem. The effects of CYP exposure on the induction of oxidative stress and endocrine disruption were studied in adolescent male ICR mice. The hepatic activities of antioxidant enzymes including superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT) and total antioxidant capacity (T-AOC) increased significantly after 3 weeks (postnatal day 21-42) of oral administration of 20 mg kg(-1) CYP. In accordance with the enzyme activities, the mRNA levels for the genes encoding these antioxidant proteins, such as Sod1, Sod2, Gpx1 and Gpx2, were also up-regulated significantly in the 10 and 20 mg kg(-1) CYP treatment groups. Furthermore, we also found that the 3-week oral administration of CYP decreased transcription levels of key genes in pathways of cholesterol synthesis and transport and testosterone synthesis including HMG-CoA synthase, steroidogenic acute regulatory protein (StAR) and cytochrome P450 17α-hydroxysteroid dehydrogenase (P450 17α in the liver and testes. Serum testosterone levels also decreased significantly in mice after treatment with 20 mg kg(-1) CYP. Taken together, the results indicated that CYP can induce endocrine disruption in adolescent mice. The findings will be helpful in elucidating the mechanism of toxicity induced by CYP in adolescent mice.

Permethrin exposure during puberty has the potential to enantioselectively induce reproductive toxicity in mice.

Permethrin (PM), as a chiral pesticide, has two asymmetric centers, resulting in two pairs of enantiomers. In China, the commercial formulation of PM contains four enantiomers. The enantiomer-specific activity for endocrine disruption in mice remains unclear. In the present study, the four individual PM enantiomers were separated by preparative HPLC. Then, three week-old male ICR mice (after maternal ablactation) were orally administered (+)-cis, (-)-cis, (+)-trans, and (-)-trans-PM separately daily for 3 weeks at doses of 0, 25, 50 and 100 mg/kg/day, respectively. The results showed that 100 mg/kg of (+)-cis, (-)-cis and (-)-trans-PM treatments resulted in serious testicular histopathological damage, decreases in testis weight and serum testosterone (T) concentrations. Moreover, the transcription status of some key genes involved in cholesterol synthesis and transport as well as T synthesis in the testes were also influenced selectively by PM enantiomers, especially by the (+)-cis-PM. Additionally, peripheral benzodiazepine receptor (PBR) and 17β-hydroxysteroid dehydrogenase (17β-HSD) mRNA levels decreased significantly in the (+)-cis-PM group regardless of the administrated doses, while steroidogenic acute regulatory protein (StAR) levels were significantly down-regulated by (+)-cis and (-)-trans-PM. Moreover, significant differences were mainly found in HMG-CoA reductase, PBR, StAR and 17β-HSD mRNA levels between different enantiomers. Combined with the effects on physiology, histopathology and the expression of genes related to T synthesis, (+)-cis-PM showed the greatest endocrine disruption activities, (-)-cis and (-)-trans-PM were moderate, while (+)-trans-PM exhibited the lowest. These results suggested significant PM enantioselectivity in the reproductive toxicity of mice during puberty exposure.

Inhibitory effects of paraquat on photosynthesis and the response to oxidative stress in Chlorella vulgaris

This study investigated the effects of paraquat, a widely used herbicide, on the aquatic unicellular alga Chlorella vulgaris through short-term toxicity tests at the physiological and gene transcriptional levels. Exposure to 0.5xa0μM paraquat increased the activities of the antioxidant enzymes superoxide dismutase, peroxidase, and catalase to levels 4.93, 3.19, and 3.09 times higher, respectively, than those of the control. Furthermore, exposure to 0.75xa0μM paraquat increased the activities of these antioxidant enzymes to even higher levels. The decrease in chlorophyll content and the increases in reactive oxygen species (ROS) and malondialdehyde content following exposure to paraquat suggest that the alga was severely damaged and cell growth was greatly inhibited. Real-time PCR showed that paraquat reduced the transcript abundance of psaB and rbcL to 7.09 and 29.83% of the control, respectively. Our results demonstrate that paraquat inhibited electron transport and CO2 assimilation, and also triggered the synthesis of ROS that disrupt cellular structure and inhibit cell growth.