Xiuying Jia

Cadmium-induced oxidative stress and apoptosis in the testes of frog Rana limnocharis.

This study explored the genetic damage induced by cadmium exposure in the testes of Rana limnocharis. Healthy adult frogs were exposed to 2.5, 5, 7.5, or 10 mg/L of cadmium solution for 14 days. The results showed that exposure to these concentrations increased the levels of reactive oxygen species and malondialdehyde content in the testes, clearly indicating a dose-effect relationship. Moreover, the same dosages of Cd(2+) solution increased glutathione (reduced) content, with the values being significantly different from those observed in the control group (P<0.01). The comet assay results demonstrated that the DNA damage rate, tail length, and tail moment of samples obtained from frogs exposed to 2.5-7.5 mg/L of cadmium solution significantly increased compared with those of samples obtained from the control group (P<0.01). These findings suggest that cadmium can induce free radical generation, followed by lipid peroxidation and DNA damage. Ultrastructural observation revealed vacuoles in the spermatogenic cells, cell dispersion, incomplete cell structures, and deformed nucleoli. Moreover, cadmium exposure induced significant down-regulation of Bcl-2 expression and up-regulation of Bax and caspase-3 expressions. Taken together, these data indicate that cadmium can induce testicular cell apoptosis in R. limnocharis. Exploring the effects of cadmium on the mechanism of reproductive toxicity in amphibians will help provide a scientific basis accounting for the global population decline in amphibian species.

Oxidative damage and apoptosis induced by microcystin-LR in the liver of Rana nigromaculata in vivo

Microcystins (MCs) are hepatotoxins with potent inhibitor activity of protein phosphatases PP1 and PP2A. The present study shows that MC-LR can induce severe oxidative damage and apoptosis in the livers of frogs (Rana nigromaculata) exposed to 1μg/L MC-LR for 7 and 14d in vivo. Ultrastructural observation showed the apoptotic morphology of perinuclear chromatin margination and swollen mitochondria, indicating that MC-LR can significantly damage frog liver. Reactive oxygen species (ROS) production and malondialdehyde (MDA) content were positively correlated with exposure time. Meanwhile, reduced glutathione (GSH) content and GSH peroxidase (GPx) activity rapidly decreased after prolonged exposure to 1μg/L MC-LR in a time-dependent manner. These results imply that the antioxidant defense systems of the liver were damaged. Enhanced apoptosis of cells in the livers of MC-treated frogs was detected by terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling (TUNEL) associated with up-regulation of the mitochondrial system. MC-LR significantly stimulated the livers to release cytochrome c, which improved the protein expressions of Bax, caspase-3, and caspase-9 (p<0.01) and inhibited the protein expression of Bcl-2 with prolonged exposure (p<0.01) via the mitochondrial pathway. These results imply that the mitochondrial pathway has a key function in toxin-induced liver cell apoptosis. The expression of caspase-8 was induced significantly (p<0.01), which illustrates the mechanism that the death receptor pathway is also involved in apoptosis. The present findings show that MC-LR can induce apoptosis in frog liver, which may be related with the decline of amphibian populations. The World Health Organization-recommended drinking water limit for MC-LR in water may be not safe for amphibians.

Apoptotic responses of Carassius auratus lymphocytes to nodularin exposure in vitro

Nodularin, a metabolite of Nodularin spumigena, is widely detected in water blooms worldwide and causes serious negative effects on fish. The apoptosis-related cytotoxic effects and mechanisms of nodularin on Carassius auratus lymphocytes were investigated. Transmission electron microscopy results showed that nodularin-treated lymphocytes display a series of morphological changes, including condensed cytoplasm, nuclear chromatin agglutination and marginalization. DNA fragmentation was verified by the DNA-ladder and formation of sub-G1 DNA peaks. These cell characteristics confirmed the occurrence of apoptosis in lymphocytes. Flow cytometric results showed that the percentages of apoptotic cells incubated with 1, 5, 10, and 100 μg/L nodularin for 12 h reached 15.76%, 17.36%, 20.34% and 44.21%, respectively; controls showed low rates of apoptosis (2.4%). The mechanism of apoptosis induced by nodularin was determined, and results showed that nodularin exposure caused a significant increase in intracellular reactive oxygen species (ROS), loss of mitochondrial transmembrane potential in a dose-dependent manner, upregulation of intracellular Ca²⁺, downregulation of Bcl-2 and upregulation of Bax expression at the mRNA and protein levels, and activation of caspase-3 and caspase-9 without caspase-8. In summary, all the results suggest that nodularin induces lymphocyte apoptosis via the mitochondrial apoptotic pathway and destroys the immune response of fish.

Mitochondrial and endoplasmic reticulum pathways involved in microcystin-LR-induced apoptosis of the testes of male frog (Rana nigromaculata) in vivo

Previous studies have shown that toxins produced by toxic cyanobacterial blooms are hazardous materials. In the present study, 1 μg/L microcystin-LR (MC-LR) was observed to induce apoptosis in the testes of male Rana nigromaculata via the mitochondrial and endoplasmic reticulum (ER) pathways at exposure times ranging from 7 d to 14 d. The results showed that reactive oxygen species production and malondialdehyde content were positively correlated with exposure time. Antioxidant enzyme contents, such as reduced glutathione and glutathione peroxidase rapidly decreased, implying that the defense system of the testes induces oxidative damage. MC-LR significantly stimulated the release of cytochrome c in the testes, thereby improving the protein expressions of Bax and caspases-3, 8, and 9 (p<0.01) and inhibiting the protein expression of Bcl-2 with prolonged exposure (p<0.01). Ultrastructural observations showed distention of the mitochondria and endoplasmic reticulum and deformation of the nucleolus. Moreover, prolonged exposure times strengthened and weakened the relative expression levels of C/EBP homologous protein and GRP78, respectively. These results indicate that MC-LR-induced apoptosis of the testes in male frogs in vivo may occur through the mitochondrial and ER pathways. It also further proves our previous findings that MC-LR can induce toxicity in the male reproductive system of R. nigromaculata in vitro. The findings show that MC-LR is highly hazardous to frogs and that the accepted drinking water limit of 1 μg/L MC-LR exerts significant toxicity to amphibians.

Endocrine-disrupting effects and reproductive toxicity of low dose MCLR on male frogs (Rana nigromaculata) in vivo.

Toxic cyanobacterial blooms are potential global threats to aquatic ecosystems and human health. The World Health Organization has set a provisional guideline limit of 1 μg/L microcystin-LR (MCLR) in freshwater. However, MCLR concentrations in several water bodies have exceeded this level. Despite this recommended human safety standard, MCLR-induced endocrine-disrupting effects and reproductive toxicity on male frog (Rana nigromaculata) were demonstrated in this study. Results showed that sperm motility and sperm count were significantly and negatively correlated with exposure time and concentration. By contrast, abnormal sperm rate was positively correlated with both parameters. Ultrastructural observation results revealed abnormal sperm morphologies, vacuoles in spermatogenic cells, cell dispersion, incomplete cell structures, and deformed nucleoli. These results indicated that MCLR could induce toxic effects on the reproductive system of frogs, significantly decrease testosterone content, and rapidly increase estradiol content. Prolonged exposure and increased concentration enhanced the relative expression levels of P450 aromatase and steroidogenic factor 1; thus, endocrine function in frogs was disrupted. This study is the first to demonstrate in vivo MCLR toxicity in the reproductive system of male R. nigromaculata. This study provided a scientific basis of the global decline in amphibian populations.

Removal of microcystin-LR from drinking water using a bamboo-based charcoal adsorbent modified with chitosan

A new kind of low-cost syntactic adsorbent from bamboo charcoal and chitosan was developed for the removal of microcystin-LR from drinking water. Removal efficiency was higher for the syntactic adsorbent when the amount of bamboo charcoal was increased. The optimum dose ratio of bamboo charcoal to chitosan was 6:4, and the optimum amount was 15 mg/L; equilibrium time was 6 hr. The adsorption isotherm was non-linear and could be simulated by the Freundlich model (R2 = 0.9337). Adsorption efficiency was strongly affected by pH and natural organic matter (NOM). Removal efficiency was 16% higher at pH 3 than at pH 9. Efficiency rate was reduced by 15% with 25 mg/L NOM (UV254 = 0.089 cm(-1)) in drinking water. This study demonstrated that the bamboo charcoal modified with chitosan can effectively remove microcystin-LR from drinking water.

Toxic effects of microcystin-LR on the reproductive system of male Rana nigromaculata in vitro.

This study aims to demonstrate that microcystin-LR (MC-LR) has toxic effects on the reproductive system of male Rana nigromaculata in vitro. R. nigromaculata were treated with 0, 0.1, 1, 10, and 100 nmol/L of MC-LR for 6 h. Results show that exposure to 1 nmol/L to 100 nmol/L of MC-LR decreased sperm motility and number of sperm cells and increased the sperm abnormality rate, whose values were significantly different from those of the control (P<0.01). Moreover, the same dosage of MC-LR increased reactive oxygen species production and malondialdehyde content. At the same time, antioxidant enzyme (catalase and glutathione S-transferase) activity and glutathione reduced content rapidly increased, whereas antioxidant enzyme superoxide dismutase activity significantly decreased. These results imply that the defense system of the testes quickly responds to oxidative stress. Ultrastructural observation shows distention of the mitochondria, endoplasmic reticulum, and Golgi apparatus and changes in the mitochondrial matrix color, cristae number, and morphology. Moreover, using real-time PCR, increased relative expressions of P450 aromatase and SF-1 genes were observed. The results demonstrate for the first time that MC-LR could induce toxicity in the male reproductive system of R. nigromaculata. The findings in this research will provide more insights into the relationships between aquatic microcystins and amphibians.

Protective role of oligomeric proanthocyanidin complex against hazardous nodularin-induced oxidative toxicity in Carassius auratus lymphocytes

Nodularin (NOD) is a hazardous material widely detected in water blooms. Fish immune cells are extremely vulnerable to NOD-induced oxidative stress. Oligomeric proanthocyanidin complex (OPC), extracted from grapeseed, was used as an antioxidant to eliminate reactive oxygen species and prevent apoptotic effects. Carassius auratus lymphocytes were treated with different concentrations (0, 10, 100, and 1,000 μg/L) of OPC and a constant dose (100 μg/L) of NOD for 12h in vitro. OPC inhibited mitosis by decreasing intracellular levels of oxidative stress, regulating antioxidant enzymes (CAT, SOD, GPx, GR, and GST), mediating bcl-2 family proteins, and deactivating caspase-3. Glutathione (GSH) levels in group V (NOD 100 μg/L; OPC 1,000 μg/L) showed a twofold increase compared with corresponding levels in group II (NOD 100 μg/L). Structure parameters of NOD and NOD-GSH were calculated using SYBYL 7.1 software. ClogP and HINK logP values of NOD-GSH decreased by 10.4- and 2.3-fold, respectively, compared with corresponding values of NOD. OPC-stimulated GSH can lower the lipophilicity and polarity of NOD. OPC, as a protective agent, can alleviate NOD-induced toxicity in C. auratus lymphocytes by regulating oxidative stress and inducing NOD-GSH detoxification.

Atrazine Triggers the Extrinsic Apoptosis Pathway in Lymphocytes of the Frog Pelophylax nigromaculata in Vivo.

Atrazine (ATR) is extensively used worldwide as an herbicide, with a global ecological influence. The widespread distribution of herbicides may be one of the possible reasons for the decline in the global amphibian population. The acute toxicity and potential toxicological mechanisms of ATR on the immune system of frogs are not well-understood. In this study, Pelophylax nigromaculata was used as an experimental carrier and exposed to 0, 1, 10, 100, and 1000 μg/L ATR solutions for 14 days, resulting in a significant decrease in the viability of their lymphocytes. The characteristics of apoptosis, such as DNA damage, percentage of apoptotic cells, DNA laddering, and morphological features, were measured in lymphocytes from the ATR-exposed groups, and the increase in apoptosis observed appears to be the result of the alterated expression of some key proteins in the extrinsic apoptosis pathway. The expression of the key apoptosis proteins Fas, Fas-L, c-FLIP, caspase-8, Bid, and caspase-3 was significantly modulated in a dose-dependent manner. Moreover, c-FLIP was shown to modulate the Fas-dependent apoptosis of the lymphocytes. In summary, acute ATR exposure damaged the lymphocytes, resulting in their apoptosis via an extrinsic signaling pathway. This study provides novel insights into the immunological and toxicological responses of amphibians exposed to triazine herbicides.

Lipid accumulation responses in the liver of Rana nigromaculata induced by perfluorooctanoic acid (PFOA)

Perfluorooctanoic acid (PFOA) is a perfluorinated compound that is widely distributed, is persistent in the environment, and has a low-level chronic exposure effect on human health. The aim of this study was to investigate the peroxisome proliferator activated receptors γ (PPARγ) and the sterol regulatory element-binding protein 2 (SREBP2) signaling pathways in regulating the lipid damage response to PFOA in the livers of amphibians. Male and female frogs (Rana nigromaculata) were exposed to 0, 0.01, 0.1, 0.5 and 1 mg/L PFOA. After treatment, we evaluated the pathological changes in the liver by Oil Red O, staining and examined the total cholesterol (T-CHO) and triglyceride (TG) contents. The mRNA expression levels of PPARγ, Fatty acid synthase (FAS), Acetyl-CoA carboxylase (ACC), Glycerol-3-phosphate acyltransferase (GPAT), SREBP2 and 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The administration of PFOA caused marked lipid accumulation damage in the amphibian livers. The T-CHO contents were elevated significantly after PFOA treatment; these results show a dose-dependent manner in both sexes. The TG content showed a significant increase in male livers, while it was elevated significantly in female livers. The RT-PCR results showed that the mRNA expression levels of PPARγ, ACC, FAS, GPAT, SREBP2 and HMG-CoA were significantly dose-dependently increased in the PFOA-treated groups compared with those of the control group. Our results demonstrated that PFOA-induced lipid accumulation also affected the expression levels of genes FAS, ACC, GPAT and HMG-CoA in the PPARγ and SREBP2 signaling pathways in the liver. These finding will provide a scientific theoretical basis for the protection of Rana nigromaculata against PFOA effects.