Silver nanoparticles induce liver inflammation through ferroptosis in zebrafish.

As the most widely employed artificial nanomaterials, silver nanoparticles (AgNPs) have been implicated in oxidative stress-induced liver injury. Despite these observations, the precise mechanisms underpinning AgNPs-induced hepatotoxicity have yet to be fully elucidated. This study embarked on an intersectional analysis of the GEO dataset (GSE139560), which encompassed murine liver tissues subjected to AgNPs, alongside datasets related to ferroptosis. Through this approach, three pivotal ferroptosis-associated genes (Arrdc3, Txnip, and Egfr) were identified. Further integration with disease model analysis from GSE111407 and GSE183158 unveiled a significant association between AgNPs exposure and alterations in glucose metabolism and insulin signaling pathways, intricately linked with the identified key ferroptosis genes. This correlation fostered the hypothesis that ferroptosis significantly contributed to the hepatotoxicity triggered by AgNPs. Subsequent Gene Set Enrichment Analysis (GSEA) pointed to the activation of ferroptosis-associated pathways, specifically MAPK and PPAR, under AgNPs exposure. Examination of the miRNA-mRNA interaction network revealed co-regulated upstream miRNAs targeting these pivotal genes, establishing a nexus to ferroptosis and heightened liver susceptibility. Experimental validation employing an adult zebrafish model exposed to AgNPs from 90 to 120 dpf demonstrated elevated levels of Fe2+ and MDA in the zebrafish livers, along with conspicuous mitochondrial morphological alterations, thereby reinforcing the notion that AgNPs precipitate liver dysfunction predominantly through the induction of ferroptosis. These insights collectively underscore the role of ferroptosis in mediating the adverse effects of AgNPs on liver glucose metabolism and insulin sensitivity, culminating in liver dysfunction. Overall, these results enhance the understanding of nanomaterial-induced hepatotoxicity and inform strategies to mitigate such health risks.

Oncogenic Alterations in Histologically Negative Lymph Nodes Are Associated with Prognosis of Patients with Stage I Lung Adenocarcinoma.

Background: Survival of patients with stage I non-small cell lung cancer (NSCLC) varies greatly. We sought to explore whether presence of oncogenic alterations in histologically-negative lymph nodes (LNs) can be of prognostic significance in stage I lung adenocarcinoma (LUAD). Methods: Genomic analysis of oncogenic alterations was applied to 123 stage I LUAD tumors. The same genomic variants identified in primary tumors were examined in corresponding histologically-negative LNs. Results: A total of 102 (82.9%) patients had at least one canonical oncogenic alteration detected in primary tumors, and 57 LNs from 12 patients (11.8%) were found to carry the identical oncogenic alterations detected in the corresponding primary tumor tissues, including EGFR mutations (six cases), KRAS mutations (three cases), ALK fusion (one case), BRAF mutation (one case) and HER2 & NRAS co-mutations (one case). None of these LNs was found to have occult tumor cells by routine pathological assessment or immunohistochemistry staining using antibodies against pan-cytokeratins (AE1/AE3) and the epithelial marker Ber-EP4. The detection rate of oncogenenic alterations in LN was significantly higher in RAS-mutant tumors than EGFR mutant tumors (36.36% verse 7.41%, p = 0.017). Patients with oncogenic alterations in LN showed inferior disease-free survival (DFS, p = 0.025) and overall survival (OS, p = 0.027). Furthermore, patients with RAS-mutations detected in LN had the worst DFS and OS (p = 0.001). Among the 11 patients with RAS mutation in primary tumors, DFS and OS in the four patients with mutations detected in LN were significantly shorter than the remaining seven patients without mutations LN (DFS, p = 0.001, OS, p = 0.002). Conclusions: Genomic analysis has the potential to detect oncogenic alterations in regional LNs for localized LUAD and presence of oncogenic alterations in regional LN may be associated with inferior clinical outcome of stage I LUAD, particularly for certain molecular subgroups. ClinicalTrials.gov ID NCT04266691.

Lipid metabolism disorder induced by up-regulation of miR-125b and miR-144 following ß-diketone antibiotic exposure to F0-zebrafish (Danio rerio).

ß-Diketone antibiotics (DKAs) are widely used in human and veterinary medicine to prevent and treat a large variety of infectious diseases. Long-term DKA exposure to zebrafish can result in lipid metabolism disorders and liver function abnormalities. Based on our previous miRNA-seq analyses, miR-144 and miR-125b were identified as target genes regulating lipid metabolism. DKA-exposure at 12.5 and 25 mg/L significantly increased the expressions of miR-144 and miR-125b. The expression levels for the two miRNAs exhibited an inverse relationship with their lipid-metabolism-related target genes (ppardb, bcl2a, pparaa and pparda). Over-expression and inhibition of miR-144 and miR-125b were observed by micro-injection of agomir-144, agomir-125b, antagomir-144 and antagomir-125b. The over-expression of miR-144 and miR-125b enhanced lipid accumulation and further induced lipid-metabolism-disorder syndrome in F1-zebrafish. The expression of ppardb and bcl2a in whole-mount in situ hybridization was in general agreement with results from qRT-PCR and was concentration-dependent. Oil red O and H&E staining, as well as related physiological and biochemical indexes, showed that chronic DKA exposure resulted in lipid-metabolism-disorder in F0-adults, and in F1-larvae fat accumulation, increased lipid content, abnormal liver function and obesity. The abnormal levels of triglyceride (TG) and total cholesterol (TCH) in DKA-exposed zebrafish increased the risk of hyperlipidemia, atherosclerosis and coronary heart disease. These observations improve our understanding of mechanisms leading to liver disease from exposure to environmental pollution, thereby having relevant practical significance in health prevention, early intervention, and gene therapy for drug-induced diseases.

Effects of ß-diketone antibiotics on F1-zebrafish (Danio rerio) based on high throughput miRNA sequencing under exposure to parents.

The toxicity of ß-diketone antibiotics (DKAs), a class of ''pseudo-persistent'' environmental pollutants, to F0-zebrafish (Danio rerio) was investigated using 7-dpf F1-zebrafish miRNA sequencing and bioinformatics analyses. Based on relative expression, 47, 134 and 118 of 193 mature miRNAs were differentially expressed between control vs 6.25 mg/L, control vs 12.5 mg/L and 6.25 vs 12.5 mg/L treatments, respectively. Utilizing three databases, 2523 potential target genes were predicted, and they were assigned to 19 high-abundance KEGG pathways and 20 functional categories by COG analysis. Among 11 significantly differential expression and high-abundance miRNAs, the expression levels for 7 miRNAs (miR-144, -124, -499, -125b, -430b, -430c and -152) assessed by qRT-PCR were consistent with those determined by sRNA-seq. A potential network was plotted between 11 miRNAs and their target genes based on differential expression and binding effectiveness. The high degree of connectivity between miRNA-gene pairs suggests that these miRNAs play critical roles in zebrafish development. The expression of miR-124 and miR-499 in whole-mount in situ hybridization was in general agreement with those from qRT-PCR and miRNA-seq and were DKA concentration-dependent. DKA exposure induced severe histopathological changes and damage in F0-zebrafish ovary tissue, as reflected by an increased number of early developmental oocytes, irregular cell distribution, decreased yolk granules, cytoplasmic shrinkage, cell lysis in mature oocytes, and dissolution of internal corona radiata. Chronic DKA exposure affected reproduction of F0-zebrafish and development of F1-zebrafish. These observations demonstrate the toxic effect transfer relation across parent and their offspring, and enhance our understanding of drug-induced diseases.

Effects of ß-diketone antibiotic mixtures on behavior of zebrafish (Danio rerio).

To date, few data are available on neurotoxicity of ß-diketone antibiotics (DKAs) from the perspective of animal behavior. Herein, the effects of long-term DKAs exposure on zebrafish (Danio rerio) behavior were assessed for locomotor activity, anxiety, social interaction and their related molecular mechanisms. DKAs exposure to zebrafish consisted of six DKA species, including ofloxacin, ciprofloxacin, enrofloxacin, doxycycline, chlortetracycline and oxytetracycline, with equal weight concentration and equal volume. DKAs at 6.25 mg/L significantly increased the time spent in the upper portion of the test tank (+40%) and the number of line crossings (±42%), indicating occurrence of anxiolytic behavior. For conditioned place preference test, long-term DKAs exposure at 6.25 mg/L increased the number of motionless positions in the non-preferred white side (+31%), number of transitions to the white side (+221%) and time spent in the white side (+35%) in relation to the control. DKAs at 6.25 mg/L significantly increased zebrafish shoaling behavior (+38%) resulting from an anxiety-like state, but 25 mg/L DKAs exposure decreased zebrafish social cohesion (-41%) possibly due to an autism-like state. With increasing DKAs-exposure concentration, the signal intensity of (1)O2 gradually decreased, leading to insufficient energy supply and movement functional disorders. Based on GO functional annotation and metabolic pathway analysis, 11 genes closely associated with locomotor behavior were identified. Using qRT-PCR, we confirmed that DKAs exposure led to changes in the transcriptional levels of 11 locomotor-related genes. These results suggest that behavior is a potential strategy for evaluating mechanisms underlying the neurochemical basis triggered by stress in zebrafish.