Mechanisms of silver nanoparticles-induced cytotoxicity and apoptosis in rat tracheal epithelial cells.

Silver nanoparticles (AgNPs) are increasingly utilized in a number of applications. This study was designed to investigate AgNPs induced cytotoxicity, oxidative stress and apoptosis in rat tracheal epithelial cells (RTE). The RTE cells were treated with 0, 100 µg/L and 10,000 µg/L of the AgNPs with diameters of 10 nm and 100 nm for 12 hr. The cell inhibition level, apoptosis ratio, reactive oxygen species (ROS), malondialdehyde (MDA) and metallothionein (MT) content were determined. The mRNA expression of cytoc, caspase 3, and caspase 9 was measured by quantitative real-time polymerase chain reaction (qRT-PCR). In addition, we also analyzed the cytoc, caspase 3, pro-caspase 3, caspase 9, and pro-caspase 9 protein expression by western blotting. Electric cell-substrate impedance sensing (ECIS) analysis showed that the growth and proliferation of RTE cells were significantly inhibited in a dose-dependent manner under AgNPs exposure. The cell dynamic changes induced by 10 nm AgNPs were more severe than that of the 100 nm AgNPs exposure group. The intracellular MT, ROS, and MDA content increased when the exposure concentration increased and size reduced, whereas Ca2+-ATPase activity and Na+/K+-ATPase activity changed inversely. The relative expression of protein of cytoc, caspase 3, and caspase 9 were upregulated significantly, which indicated that AgNPs induced apoptosis of RTE cells through the caspase-dependent mitochondrial pathway. Our results demonstrate that AgNPs caused obvious cytotoxicity, oxidative stress, and apoptosis in RTE cells, which promoted the releasing of cytochrome C and pro-apoptotic proteins into the cytoplasm to activate the caspase cascade and finally led to apoptosis.

Effects of Perfluorooctanoic Acid on the Associated Genes Expression of Autophagy Signaling Pathway of Carassius auratus Lymphocytes in vitro.

Perfluorooctanoic acid (PFOA) has been detected in various water bodies and caused harm to aquatic organisms. The aim of this study was to investigate the cytotoxicity and mechanism associated with autophagy and oxidative stress after exposure to PFOA (0, 1, 10, 100 µg/L) for 12 h on lymphocytes, which was isolated from the head kidney of Carassius auratus (C. auratus). Both of autophagy formation, cell activity, and intracellular reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) levels were measured. The relative expression of partial autophagy-related genes autophagy related 5 (Atg 5), autophagy related 7 (Atg 7), and Beclin 1 were also cloned and detected. Homologous relationships analysis showed high identities of genes in C. auratus and other fish by blast. C. auratus lymphocytes growth inhibition rates was increased induced by PFOA. Compared with the control group, the ROS generation and the MDA content were significantly increased in all of the PFOA-treated group. Besides, decreased SOD activity and decrease of GSH activity induced by PFOA further confirmed the occurrence of oxidative stress. The number of autophagosome formations was increased in a dose-dependent manner. Compared with the control group, Atg 7 and Beclin 1 mRNA expression was elevated significantly after PFOA exposed, showing a time-dependent manner, while mRNA expression of Atg 5 was increased remarkably in 100 µg/L PFOA-treated group. Our results indicated that PFOA caused oxidative damage to lymphocytes in C. auratus and caused various autophagy signaling pathway-associated genes imbalances in the lymphocytes. Autophagy signaling pathway-associated genes imbalance could weaken antioxidant capacity and involve in the mechanism of C. auratus lymphocytes oxidative injury caused by PFOA.

Role of the Nrf2-ARE pathway in perfluorooctanoic acid (PFOA)-induced hepatotoxicity in Rana nigromaculata.

Perfluorooctanoic acid (PFOA) is widely distributed in various environmental media and is toxic to organisms. This study demonstrated that PFOA induces hepatotoxicity in the frog and evaluated the role of CYP3A and the Nrf2-ARE signaling pathway in regulating responses to PFOA-induced hepatotoxicity. Rana nigromaculata were exposed to 0, 0.01, 0.1, 0.5, or 1 mg/L PFOA solutions in a static-renewal system for 14 days. Liver tissue samples were collected 24 h after the last treatment. Hepatic histology was observed by HE staining and transmission electron microscopy. The oxidative stress levels in the liver were measured. The expression levels of CYP3A, Nrf2, NQO1, and HO-1 mRNA were measured by quantitative reverse transcription-polymerase chain reaction. PFOA-treated frog liver tissue exhibited diffuse cell borders, cytoplasmic vacuolization, broken nuclei, nuclear chromatin margination, and swollen mitochondria. In addition, the livers of PFOA-treated frogs showed a significantly elevated content of reactive oxygen species, malondialdehyde, glutathione and glutathione S-transferase activity compared to the livers of control frogs. However, the glutathione peroxidase activities concomitantly decreased in PFOA-treated frogs compared to those in the control group. Furthermore, compared with control frogs, the expression levels of CYP3A, Nrf2, and NQO1 mRNA significantly increased in PFOA-treated frogs. HO-1 mRNA expression remarkably increased only in groups treated with 0.5 or 1 mg/L PFOA. Our results indicate that PFOA induces hepatotoxicity in a dose-dependent manner. Furthermore, the results of the comparison analysis between different gender groups illustrated that PFOA is more toxic to female frogs than male frogs. Our results demonstrated that PFOA causes liver damage and that CYP3A enhances PFOA-induced female frogs hepatotoxicity are more virulent than male through biotransformation, and the activation of the Nrf2-ARE pathway is induced to protect against hepatotoxicity in Rana nigromaculata, all of which provide the scientific basis for the protection of amphibians against environmental contaminants.

Effects of MCLR exposure on sex hormone synthesis and reproduction-related genes expression of testis in male Rana nigromaculata.

Microcystin-leucine-arginine (MCLR) is the most popular and toxic variant among microcystins, which can cause severe reproductive toxicity to animals. However, the mechanisms of reproductive toxicity induced by MCLR in amphibians are still not entirely clear. In the current study, toxicity mechanisms of MCLR on the reproductive system of male Rana nigromaculata followed by low concentration (0, 0.1, 1, and 10 µg/L) and short-term (0, 7, and 14 days) MCLR exposure were shown. It was observed that MCLR could be bioaccumulated in the testes of male frogs, and the theoretical bioaccumulation factor values were 0.24 and 0.19 exposed to 1 µg/L and 10 µg/L MCLR for 14 days, respectively. MCLR exposure significantly decreased testosterone (T) concentrations and increased estradiol (E2) concentrations exposed to 1 and 10 µg/L MCLR for 14 days. The mRNA levels of HSD17B3 were downregulated, and HSD17B1 and CYP19A1 mRNA expression levels were upregulated, respectively. Only 10 µg/L MCLR group showed significant induction of follicle-stimulating hormone (FSH) levels and cyclic adenosine monophosphate (cAMP) content. Moreover, AR and ESR1 mRNA expression levels were significantly upregulated exposed to 1 and 10 µg/L MCLR for 14 days, respectively. Our results suggested that low-concentration MCLR induced transcription changes of CYP19A1, HSD17B3, and HSD17B1 led to endocrine disorders, and caused interference of spermatogenesis by the decrease of T and abnormal gene expressions of AR and ESR1 in the testes of R. nigromaculata.