Protocol for generating mutant zebrafish using CRISPR-Cas9 followed by quantitative evaluation of vascular formation.

The use of vascular-specific transgenic zebrafish provides advantages for identifying new mutations affecting angiogenesis and vascular development. Here, we present a protocol for establishing, screening, and phenotyping CRISPR-Cas9-based mutagenesis in fluorescently labeled transgenic zebrafish. We describe steps for designing single-guide RNA (sgRNA) oligos, synthesizing sgRNA and Cas9 mRNA, and microinjection and generation of mutant lines. We then detail procedures for visualizing dynamic vasculature and quantitatively evaluating vascular formation in transgenic zebrafish. For complete details on the use and execution of this protocol, please refer to Luo et al.1.

Silver Nanoparticles Cause Neural and Vascular Disruption by Affecting Key Neuroactive Ligand-Receptor Interaction and VEGF Signaling Pathways.

Introduction: Silver nanoparticles (AgNP) are widely used as coating materials. However, the potential risks of AgNP to human health, especially for neural and vascular systems, are still poorly understood. Methods: The vascular and neurotoxicity of various concentrations of AgNP in zebrafish were examined using fluorescence microscopy. In addition, Illumina high-throughput global transcriptome analysis was performed to explore the transcriptome profiles of zebrafish embryos after exposure to AgNP. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to elucidate the top 3000 differentially expressed genes (DEGs) between AgNP-exposed and control groups. Results: We systematically investigated the neural and vascular developmental toxicities of AgNP exposure in zebrafish. The results demonstrated that AgNP exposure could cause neurodevelopmental anomalies, including a small-eye phenotype, neuronal morphology defects, and inhibition of athletic abilities. In addition, we found that AgNP exposure induces angiogenesis malformation in zebrafish embryos. Further RNA-seq revealed that DEGs were mainly enriched in the neuroactive ligand-receptor interaction and vascular endothelial growth factor (Vegf) signaling pathways in AgNP-treated zebrafish embryos. Specifically, the mRNA levels of the neuroactive ligand-receptor interaction pathway and Vegf signaling pathway-related genes, including si:ch73-55i23.1, nfatc2a, prkcg, si:ch211-132p1.2, lepa, mchr1b, pla2g4aa, rac1b, p2ry6, adrb2, chrnb1, and chrm1b, were significantly regulated in AgNP-treated zebrafish embryos. Conclusion: Our findings indicate that AgNP exposure transcriptionally induces developmental toxicity in neural and vascular development by disturbing neuroactive ligand-receptor interactions and the Vegf signaling pathway in zebrafish embryos.

Nuclear translocation of cGAS orchestrates VEGF-A-mediated angiogenesis.

Cyclic GMP-AMP synthase (cGAS) senses cytosolic incoming DNA and consequently activates stimulator of interferon response cGAMP interactor 1 (STING) to mount immune response. Here, we show nuclear cGAS could regulate VEGF-A-mediated angiogenesis in an immune-independent manner. We found VEGF-A stimulation induces cGAS nuclear translocation via importin-ß pathway. Moreover, nuclear cGAS subsequently regulates miR-212-5p-ARPC3 cascade to modulate VEGF-A-mediated angiogenesis through affecting cytoskeletal dynamics and VEGFR2 trafficking from trans-Golgi network (TGN) to plasma membrane via a regulatory feedback loop. In contrast, cGAS deficiency remarkably impairs VEGF-A-mediated angiogenesis in vivo and in vitro. Furthermore, we found strong association between the expression of nuclear cGAS and VEGF-A, and the malignancy and prognosis in malignant glioma, suggesting that nuclear cGAS might play important roles in human pathology. Collectively, our findings illustrated the function of cGAS in angiogenesis other than immune surveillance, which might be a potential therapeutic target for pathological angiogenesis-related diseases.

L1CAM promotes vasculogenic mimicry formation by miR-143-3p-induced expression of hexokinase 2 in glioma.

In recent decades, antiangiogenic therapy, which blocks the supply of oxygen and nutrition to tumor cells, has become a promising clinical strategy for the treatment of patients with tumors. However, recent studies revealed that vasculogenic mimicry (VM), which is the process by which vascular morphological structures are formed by highly invasive tumor cells, has been considered a potential factor for the failure of antiangiogenic therapy in patients with tumors. Thus, inhibition of VM formation might be a potential target for improving the outcome of antiangiogenic strategies. However, the mechanism underlying VM formation is still incompletely elucidated. Herein, we report that L1CAM might be a critical regulator of VM formation in glioma, and might be associated with the resistance of glioma to antiangiogenic therapy. We found that the tumor-invasion and tube-formation capabilities of L1CAM-overexpressing cells were significantly enhanced in vitro and in vivo. In addition, the results indicated that miR-143-3p, which might directly target the 3'UTR of the hexokinase 2 (HK2) gene to regulate its protein expression, was subsequently involved in L1CAM-mediated VM formation by glioma cells. Further study revealed that the regulation of MMP2, MMP9, and VEGFA expression was involved in this process. Moreover, we identified that activation of the downstream PI3K/AKT signaling pathway of the L1CAM/HK2 cascade is critical for VM formation by glioma cells. Furthermore, we found that the combined treatment of anti-L1CAM neutralizing monoclonal antibody and bevacizumab increases efficacy beyond that of bevacizumab alone, and suppresses glioma growth in vivo, indicating that the inhibition of L1CAM-mediated VM formation might efficiently improve the effect of antiangiogenic treatment for glioma patients. Together, our findings demonstrated a critical role of L1CAM in regulating VM formation in glioma, and that L1CAM might be a potential target for ameliorating tumor resistance to antiangiogenic therapy in glioma patients.

Par6 Enhances Glioma Invasion by Activating MEK/ERK Pathway Through a LIN28/let-7d Positive Feedback Loop.

The invasion of glioblastoma usually results in the recurrence and poor prognosis in patients with glioma. However, the underlying mechanisms involved in glioma invasion remains undefined. In this study, immunohistochemistry analyses of glioma specimens demonstrated that high expression of Par6 was positively correlated with malignancy and poor prognosis of patients with glioma. Par6-overexpressing glioma cells showed much more fibroblast-like morphology, suggesting that regulation of Par6 expression might be associated with tumor invasion in glioma cells. Further study indicated that Par6 overexpression subsequently increased CD44 and N-cadherin expression to enhance glioma invasion through activating MEK/ERK/STAT3 pathway, in vivo and in vitro. Moreover, we found that LIN28/let-7d axis was involved in this process via a positive feedback loop, suggesting that MEK/ERK/LIN28/let-7d/STAT3 cascade might be essential for Par6-mediated glioma invasion. Therefore, these data highlight the roles of Par6 in glioma invasion, and Par6 may serve as a potential therapeutic target for patients with glioma.

Polystyrene Nanoplastic Exposure Induces Developmental Toxicity by Activating the Oxidative Stress Response and Base Excision Repair Pathway in Zebrafish (Danio rerio).

The widespread accumulation of nanoplastics is a growing concern for the environmental and human health. However, studies on the mechanisms of nanoplastic-induced developmental toxicity are still limited. Here, we systematically investigated the potential biological roles of nanoplastic exposure in zebrafish during the early developmental stage. The zebrafish embryos were subjected to exposure to 100 nm polystyrene nanoplastics with different concentrations (0, 100, 200, and 400 mg/L). The results indicated that nanoplastic exposure could decrease the hatching and survival rates of zebrafish embryos. In addition, the developmental toxicity test indicated that nanoplastic exposure exhibits developmental toxicity via the inhibition of the heart rate and body length in zebrafish embryos. Besides, behavioral activity was also significantly suppressed after 96 h of nanoplastic exposure in zebrafish larvae. Further biochemical assays revealed that nanoplastic-induced activation of the oxidative stress responses, including reactive oxygen species accumulation and enhanced superoxide dismutase and catalase activities, might affect developmental toxicity in zebrafish embryos. Furthermore, a quantitative polymerase chain reaction assay demonstrated that the mRNA levels of the base excision repair (BER) pathway-related genes, including lig1, lig3, polb, parp1, pold, fen1, nthl1, apex, xrcc1, and ogg1, were altered in zebrafish embryos for 24 h after nanoplastic exposure, indicating that the activation of the BER pathway would be stimulated after nanoplastic exposure in zebrafish embryos. Therefore, our findings illustrated that nanoplastics could induce developmental toxicity through activation of the oxidative stress response and BER pathways in zebrafish.

Silver nanoparticles induce developmental toxicity via oxidative stress and mitochondrial dysfunction in zebrafish (Danio rerio).

Sliver nanoparticles (AgNPs) are widely used in industry, agriculture, and medicine, potentially resulting in adverse effects on human health and aquatic environments. Here, we investigated the developmental toxicity of zebrafish embryos with acute exposure to AgNPs. Our results demonstrated developmental defects in 4 hpf zebrafish embryos after exposure to different concentrations of AgNPs for 72 h. In addition, RNA-seq profiling of zebrafish embryos after AgNPs treatment. Further Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that the differentially expressed genes (DEGs) were enriched in DNA replication initiation, oxidoreductase activity, DNA replication, cellular senescence, and oxidative phosphorylation signaling pathways in the AgNPs-treated group. Notably, we also found that AgNPs exposure could result in the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), the inhibition of superoxide dismutase (SOD), catalase (CAT), and mitochondrial complex I-V activities, and the downregulated expression of SOD, CAT, and mitochondrial complex I-IV chain-related genes. Moreover, the expression of mitochondrion-mediated apoptosis signaling pathway-related genes, such as bax, bcl2, caspase-3, and caspase-9, was significantly regulated after AgNPs exposure in zebrafish. Therefore, these findings demonstrated that AgNPs exposure could cause oxidative stress, induce mitochondrial dysfunction, and ultimately lead to developmental toxicity.

Molecular Spectrum, Ethnic and Geographical Distribution of Thalassemia in the Southern Area of Hainan, China.

Background: Thalassemia is one of the most common genetic diseases in southern China. Accurate population frequency data regarding the occurrence and distribution of thalassemia are important for designing appropriate prevention strategies for thalassemia. This study aims to reveal the molecular spectrum, ethnic and geographical distribution of thalassemia in the southern area of Hainan Province, China. Methods: A total of 9813 suspected carriers of thalassemia were screened for genetic analysis by using the PCR-reverse dot blot hybridization method targeting three known deletions of α-thalassemias (--SEA, -α3.7, and -α4.2), three nondeletional mutations of α-thalassaemias (αCS, αQS, and αWS) and the 17 most common mutations of ß-thalassaemias in the Chinese population. Results: Approximately 6,924 subjects were genetically diagnosed as thalassemia carriers or patients, including 5812 cases of α-thalassemia (83.9%), 369 cases of ß-thalassemia (5.3%), and 743 cases of α-composite ß-thalassemia (10.7%). A total of 21 distinct genotypes were identified among the 5,812 α-thalassemia carriers, -α4.2/αα, -α3.7/αα, and -α3.7/-α4.2 were the most common α-thalassemia genotypes. The most frequent ß-thalassemia genotype was ßCD41-42/ßN, with a notable proportion of 69.6%, followed by the ß-28M /ßN, ßIVS-II-654/ßN, ßCD71-72/ßN, ßE/ßN, and ßCD17/ßN genotypes. In addition, 37 genotypes were detected among the 743 cases of both α- and ß-thalassemia mutations. The α-thalassemia genotypes were most commonly found in the Li people, who accounted for 73.5% of α-thalassemia carriers. The ß-thalassemia genotypes were most commonly identified in the Han people, who accounted for 59.4% of ß-thalassemia carriers. Among the subjects carrying both α- and ß-thalassemia variations, only three ethnic minorities were identified, including the Li, Han, and Miao people, accounting for 82.0, 17.4, and 0.7%, respectively. Conclusions: Our study indicates that there is high genetic heterogeneity, geographical and ethnic differences in thalassemia in populations in the southern area of Hainan Province. These findings will be helpful in guiding genetic counseling and prenatal diagnosis of thalassemia in Hainan Province.

Cooperation between liver-specific mutations of pten and tp53 genetically induces hepatocarcinogenesis in zebrafish.

BACKGROUND: Liver cancer, mainly hepatocellular carcinoma, is one of the deadliest cancers worldwide and has a poor prognosis due to insufficient understanding of hepatocarcinogenesis. Previous studies have revealed that the mutations in PTEN and TP53 are the two most common genetic events in hepatocarcinogenesis. Here, we illustrated the crosstalk between aberrant Pten and Tp53 pathways during hepatocarcinogenesis in zebrafish. METHODS: We used the CRISPR/Cas9 system to establish several transgenic zebrafish lines with single or double tissue-specific mutations of pten and tp53 to genetically induce liver tumorigenesis. Next, the morphological and histological determination were performed to investigate the roles of Pten and Tp53 signalling pathways in hepatocarcinogenesis in zebrafish. RESULTS: We demonstrated that Pten loss alone induces hepatocarcinogenesis with only low efficiency, whereas single mutation of tp53 failed to induce tumour formation in liver tissue in zebrafish. Moreover, zebrafish with double mutations of pten and tp53 exhibits a much higher tumour incidence, higher-grade histology, and a shorter survival time than single-mutant zebrafish, indicating that these two signalling pathways play important roles in dynamic biological events critical for the initiation and progression of hepatocarcinogenesis in zebrafish. Further histological and pathological analyses showed significant similarity between the tumours generated from liver tissues of zebrafish and humans. Furthermore, the treatment with MK-2206, a specific Akt inhibitor, effectively suppressed hepatocarcinogenesis in zebrafish. CONCLUSION: Our findings will offer a preclinical animal model for genetically investigating hepatocarcinogenesis and provide a useful platform for high-throughput anticancer drug screening.

Genetic variation and gene expression of anthocyanin synthesis and transport related enzymes in Oryza sativa against thiocyanate.

Plants exposed to environmental contaminants often synthesize anthocyanins (ATHs) as an approach to safeguard themselves from adverse impact. However, the overload of ATHs in plant cells can threaten their growth and development through proteins oxidization and intercalating with DNAs inside cells. In the present study, a microcosm hydroponic experiment was conducted using rice seedlings to investigate the molecular signaling pathways involved in regulating and controlling ATHs synthesis and transport exposed to thiocyanate (SCN-). Our results indicated that SCN- exposure significantly (p < 0.05) increased the expression of ATHs synthesis related genes (i.e., PAL, CHS, ANS, UFGT genes) in rice tissues, altered the activities of these ATHs synthesis related enzymes, and consequently elevated the ATHs content. However, SCN- exposure significantly decreased the expression of ATHs transport related genes (i.e., GST, ABC, MATE genes) in rice seedlings, suggesting that SCN- exposure have restrained ATHs transport from cytosol to vacuole in cells, eventually posing a significant adverse effect on cells survival. Our findings highlight on one of the plant aspects in managing the toxicity triggered by secondary metabolites under stress conditions.

Stepwise crosstalk between aberrant Nf1, Tp53 and Rb signalling pathways induces gliomagenesis in zebrafish.

The molecular pathogenesis of glioblastoma indicates that RTK/Ras/PI3K, RB and TP53 pathways are critical for human gliomagenesis. Here, several transgenic zebrafish lines with single or multiple deletions of nf1, tp53 and rb1 in astrocytes, were established to genetically induce gliomagenesis in zebrafish. In the mutant with a single deletion, we found only the nf1 mutation low-efficiently induced tumour incidence, suggesting that the Nf1 pathway is critical for the initiation of gliomagenesis in zebrafish. Combination of mutations, nf1;tp53 and rb1;tp53 combined knockout fish, showed much higher tumour incidences, high-grade histology, increased invasiveness, and shortened survival time. Further bioinformatics analyses demonstrated the alterations in RTK/Ras/PI3K, cell cycle, and focal adhesion pathways, induced by abrogated nf1, tp53, or rb1, were probably the critical stepwise biological events for the initiation and development of gliomagenesis in zebrafish. Gene expression profiling and histological analyses showed the tumours derived from zebrafish have significant similarities to the subgroups of human gliomas. Furthermore, temozolomide treatment effectively suppressed gliomagenesis in these glioma zebrafish models, and the histological responses in temozolomide-treated zebrafish were similar to those observed in clinically treated glioma patients. Thus, our findings will offer a potential tool for genetically investigating gliomagenesis and screening potential targeted anti-tumour compounds for glioma treatment.

The oxidative stress responses caused by phthalate acid esters increases mRNA abundance of base excision repair (BER) genes in vivo and in vitro.

The base excision repair (BER) pathway is an important defense response to oxidative DNA damage. It is known that exposures to phthalate esters (PAEs), including Dibutyl phthalate (DBP), Mono-(2-ethylhexyl) phthalate (MEHP), and Di-(2-ethylhexyl) phthalate (DEHP), cause reactive oxygen species-induced DNA damage and oxidative stress. Here, we determined the mRNA levels of BER pathway-related genes (ogg1, nthl1, apex1, parp1, xrcc1, lig3, ung, pcna, polb, pold, fen1, and lig1), pro-apoptotic gene (bax), and apoptotic suppressor gene (bcl2) in different PAEs-exposed zebrafish larvae and HEK293T cells. Further investigations were performed to examine reactive oxygen species (ROS) accumulation, superoxide dismutase (SOD) activity, developmental toxicity, and cell viability after PAEs exposure in vivo and in vitro. The results showed that PAEs exposure can induce developmental abnormalities in zebrafish larvae, and inhibit cell viability in HEK293T cells. Additionally, we found that PAEs exposure results in the accumulation of ROS and the inhibition of SOD activation in vivo and in vitro. Notably, the mRNA levels of BER pathway-related genes (OGG1, NTHL1, APEX1, XRCC1, UNG, POLB, POLD, FEN1) were significantly upregulated after DBP or MEHP exposure, whereas the mRNA levels of NTHL1, UNG, POLB, POLD, and FEN1 were significantly altered in DEHP-treated HEK293T cells. In zebrafish, the mRNA levels of ogg1, pcna, fen1 and lig1 genes were increased after DBP or DEHP exposure, whereas the mRNA levels of nthl1, apex1, parp1, lig3, pcna and polb were decreased after MEHP exposure, respectively. Thus, our findings indicated that PAEs exposure can induce developmental toxicity, cytotoxicity, and oxidative stress, as well as activate BER pathway in vivo and in vitro, suggesting that BER pathway might play critical roles in PAEs-induced oxidative stress through repairing oxidative DNA damage.

Transcriptomic analysis of cytochrome P450 genes and pathways involved in chromium toxicity in Oryza sativa.

In plants, cytochrome P450 monooxygenase (CYP) plays an important role in detoxifying xenobiotic chemicals and coordinating abiotic stresses. Agilent 44 K rice microarray has been used to focus on the transcriptional profile of osCYP genes in rice seedling exposed to Cr solution containing K2CrO4 or Cr(NO3)3. Our study showed that expression profiles of 264 osCYP genes identified were tissue, dose and stimulus specific in rice seedlings. Comparative genomics analysis revealed that more differentially expressed osCYP genes were discovered in roots than in shoots under both Cr exposures. Results from Venn diagram analysis of differentially expressed osCYP genes demonstrated that there were common osCYP genes and unique osCYP genes present in different rice tissue as well as in different Cr treatments, which may control and/or regulate involvement of different CYP isoenzymes under Cr exposure individually or combinedly. KEGG analysis indicated that significant up- and down-regulated osCYP genes in rice tissues were chiefly related to "biosynthesis of secondary metabolites". However, involvements of osCYP genes mapped in the "biosynthesis of secondary metabolites" were tissue and dose specific, implying their distinctly responsive and adaptive mechanisms during Cr exposure. Overall, our findings are evident to describe and clarify their individual roles of specific osCYP genes in regulating involvement of CYP isoforms in Cr detoxification by rice seedlings.

Regulation Network of Sucrose Metabolism in Response to Trivalent and Hexavalent Chromium in Oryza sativa.

The presence of chromium (Cr) in cultivated fields affects carbohydrate metabolism of rice (Oryza sativa L.) and weakens its productivity. Little is known about the molecular mechanism of sucrose metabolism underlying Cr stress response in rice plants. In the present study, the transcriptome map of sucrose metabolism in rice seedlings exposed to both trivalent and hexavalent chromium was investigated using Agilent 4 × 44K rice microarray analysis. Results indicated that Cr exposure (3 days) significantly (p < 0.05) improved sucrose accumulation, and altered the activities of sucrose synthetase, sucrose phosphate phosphatase, and amylosynthease in rice tissues. We identified 119 differentially regulated genes involved in 17 sucrose metabolizing enzymes and found that gene responses in roots were significantly (p < 0.05) stronger than in shoots under both Cr(III) and Cr(VI) treatment. The network maps of gene regulation responsible for sucrose metabolism in rice plants provide a theoretical basis for further cultivating Cr-resistant rice cultivars through molecular genetic improvement.

Impact of water quality on the microbial diversity in the surface water along the Three Gorge Reservoir (TGR), China.

The Three Gorges Reservoir (TGR), one of the world's largest reservoirs, has crucial roles in flood control, power generation, and navigation. The TGR is contaminated because of the human activities, and how the contaminated water influences the distribution of the microbial community have not been well studied. In this study, we collected 41 freshwater samples from 13 main dwelling districts along the TGR to investigate the water quality, the distribution of the microbial community, and how water quality affects the microbial community structure. The sampling sites cover the whole TGR along the stream, with 670 km distance. Our results show that both water quality and the compositions of bacterial community vary along the TGR. The distribution of bacterial community is closely related to the local water quality. There is the highest concentration of chemical oxygen demand (COD), the highest relative abundance of Firmicutes, and the highest relative abundance of Bacillus in the upstream, compared to the middle and down streams. Redundancy analysis (RDA) showed that PO43- and COD were the main environmental factors influencing on the structure of bacterial community. The relative abundance of nitrification and denitrification functional genes also altered along the streams. These findings provide the basic data for water quality, the distribution of bacterial community, the link of environmental factors, and the bacterial community structure along the TGR, which guides the local environmental protection agency to launch protection strategy for maintaining the ecosystem health of the TGR.

Nanotoxicity of Silver Nanoparticles on HEK293T Cells: A Combined Study Using Biomechanical and Biological Techniques.

Human embryonic kidney 293T cells (HEK293T cells) before and after treatment with silver nanoparticles (AgNPs) were measured using advanced atomic force microscopy (AFM) force measurement technique, and the biomechanical property of cells was analyzed using a theoretical model. The biomechanical results showed that the factor of viscosity of untreated HEK293T cells reduced from 0.65 to 0.40 for cells exposure to 40 µg/mL of AgNPs. Comet assay indicated that significant DNA damage occurred in the treated cells, measured as tail DNA% and tail moment. Furthermore, gene expression analysis showed that for the cells treated with 40 µg/mL of AgNPs, the antiapoptosis genes Bcl2-t and Bclw were, respectively, downregulated to 0.65- and 0.66-fold of control, and that the proapoptosis gene Bid was upregulated to 1.55-fold of control, which indicates that apoptosis occurred in cells exposed to AgNPs. Interestingly, excellent negative correlations were found between the factor of viscosity and tail DNA%, and tail moment, which suggest that the biomechanical property can be correlated with genotoxicity of nanoparticles on the cells. Based on the above results, we conclude that (1) AgNPs can lead to biomechanical changes in HEK293T cells, concomitantly with biological changes including cell viability, DNA damage, and cell apoptosis; (2) the factor of viscosity can be exploited as a promising label-free biomechanical marker to assess the nanotoxicity of nanoparticles on the cells; and (3) the combination of AFM-based mechanical technique with conventional biological methods can provide more comprehensive understanding of the nanotoxicity of nanoparticles than merely by using the biological techniques.

Role of cytochrome c in modulating chromium-induced oxidative stress in Oryza sativa.

Cytochrome c (CYTc) is one of the redox-active molecules responsible for electron transport in plant mitochondria. It is important in the interaction of metal ions with mitochondrial proteome, it is also essential for insights into electron transport during oxidative stress. This study focused on the clarification of interactions of CYTc with metal accumulation and associated modulation of CYTc with reactive oxygen species (ROS) generation in rice seedlings exposed to chromium either hexavalent Cr(VI) or trivalent Cr(III). The result indicates that exposures to both Cr species resulted in significant accumulation of Cr in rice tissues. ROS-dependent growth inhibition of rice seedlings was also evident from both Cr treatments. Changes of CYTc content in roots and shoots maintained almost the same pattern after both Cr exposures. However, dynamically transcriptional levels of rice CYTc genes conducted by real-time quantitative RT-PCR were inconsistent in rice tissues. The results presented here demonstrate that oxidative stresses induced by ROS accumulation do not equally and/or detrimentally influence the perturbations of CYTc content and expression patterns of CYTc genes in rice tissues after Cr exposure, suggesting that the decrease of CYTc levels in roots did not originate ROS accumulation, it may be due to metal-binding properties, while enhancement of CYTc content in shoots was probably a signal of severe growth inhibition owing to ROS accumulation.

Potential adverse outcome pathway (AOP) of silver nanoparticles mediated reproductive toxicity in zebrafish.

Recently, the augmented utilization of silver nanoparticles (AgNPs) resulted in increasingrates of its release to aquatic environment, which potentially caused adverse effects to aquatic organisms. Therefore, this study investigated - reproductive toxicity and associated potential adverse outcome pathway (AOP) in zebrafish after chronic exposure to AgNPs. To serve the purpose, three-month-old adult zebrafish were exposed to different concentrations (0, 10, 33 and 100 µg/L) of AgNPs for five weeks. Exposure to 33 and 100 µg/L of AgNPs significantly decreased the fecundity in female zebrafish, accompanied by increasing apoptotic cells in the ovarian and testicular tissue using TUNEL assay. Increasing tissue burdens of AgNPs and reactive oxygen species (ROS) production were also found in both ovary and testis after five-week exposure to AgNPs. To explore the mechanism of the apoptotic pathway, the transcription levels of various genes (bax, bcl-2, caspase-3, and caspase-9) associated with the mitochondrion-mediated apoptosis pathway were examined in zebrafish after exposure to AgNPs. The results showed that the expression patterns of all the investigated genes were altered to some extent. These findings demonstrated that AgNPs exposure caused oxidative stress, induced germ cells apoptosis via mitochondrial-dependent pathway, and ultimately impaired the reproduction in zebrafish.

Microarray-based expression analysis of phytohormone-related genes in rice seedlings during cyanide metabolism.

Plants exhibit highly coordinated, dynamic reactions to various abiotic stressors. As cyanide is a non-essential element for plant growth, entry inside plants can exert toxicity at multiple levels. In plant, hormone plays a pivot role under stress conditions. The fluctuations of stress-responsive hormones help in altering cellular dynamics and hence play a central role in coordination and adaptation growth responses under stress. This study focusses on uptake of cyanide in Oryza sativa seedlings and its effect on physiological and on genetic level. Microarray approach has been focused on transcriptional profiling of genes which are involved in systemic acquired resistance for cyanide. Our study shows that the change in different hormonal contents maintained almost the same pattern in roots and shoots upon CN exposure, except for SA. However, the hormone-related gene expression pattern conducted by microarray analysis was inconsistent in both plant materials (root/shoots). Comparison of gene expression between root/shoots showed a total of 29 in roots and 16 DEGs, respectively, indicating that hormone-related genes in roots were more responsive than those in shoots during exogenous CN metabolism. These results showed a remarkable change at transcript level of plant hormone-related genes, including biosynthesis, degradation, induction, and signal transduction under cyanide stress.

Reproductive effects linked to DNA methylation in male zebrafish chronically exposed to environmentally relevant concentrations of di-(2-ethylhexyl) phthalate.

Di-(2-ethylhexyl) phthalate (DEHP) possesses the potential to interfere with the male reproductive endocrine system in mammals; however, its reproductive toxicity in male zebrafish and associated epigenetic studies have not been explored. In this study, three-month-old male zebrafish were exposed to environmentally relevant concentrations of DEHP (0, 10, 33 and 100 µg/L) for 3 months, and then the impact on the reproduction of males and the underlying mechanism were investigated. Histological testing showed that an exposure concentration of 100 µg/L DEHP significantly inhibited spermatogenesis, with an associated decline in capability to fertilize untreated oocytes. Electron microscopic examinations also revealed noticeable damage to the testicular ultrastructure at the 100 µg/L DEHP exposure level. In addition, exposure to 33 and 100 µg/L of DEHP resulted in a decline of circulating testosterone (T) and an increase in the level of 17ß-estradiol (E2), both of which were possibly derived from the downregulation of cyp17a1 and hsd17b3 genes and the upregulation of the cyp19a1a gene in the gonads. The DNA methylation statuses of these genes were altered within their promoter regions. A significant increase in global DNA methylation in both the male testes and their offspring larvae was observed at higher exposure concentration of DEHP. Our findings demonstrate that exposure to environmentally relevant concentrations of DEHP can damage the testes, disturbe the sex hormones production, and inhibite spermatogenesis, which ultimately impairs the reproduction of male zebrafish.