Metabolomics Study with Gas Chromatography-Mass Spectrometry of Hepatotoxicity on Adult Zebrafish (Brachydanio Rerio) after Exposure to Diquat

Although diquat is a widely used water-soluble herbicide in the world, its toxicity to freshwater fish has not been well characterized. In this study, gas chromatography-mass spectrometry (GC-MS) based metabolomics approach combined with histopathological examination and biochemical assays was applied to comprehensively assess the hepatotoxicity in zebrafish (Brachydanio rerio) after diquat exposure at two dosages of 0.34 and 1.69 mg·L−1 for 35 days. The results indicated that 1.69 mg·L−1 diquat exposure cause serious cellular swell and vacuolization with increased nuclear abnormality, and lead to obvious disturbance of antioxidative system and dysfunction in liver; while no obvious pathological injury could be detected, and changes in liver biochemistry were less pronounced at the dose level of 0.34 mg·L−1. Multivariate statistical analysis and pattern recognition showed different GC-MS profiles of zebrafish liver following exposure to diquat, the cluster of the treated groups were both clearly separated from the control samples. The differentially abundant metabolites mainly include carbohydrates, amino acids, lipids, nucleotides, and their derivatives. In the exposure group of 1.69 mg·L−1 diquat, severe disturbances of amino acid metabolism played important biological roles associated with inhibition of energy metabolism, reduced immunity, and disorders in neurotransmitters as pathway analysis revealed. Additionally, fluctuation of inositol, creatine, and pantothenic acid, substances associated with stress regulation and signal transduction, participating in metabolic abnormalities in zebrafish with diquat-triggered hepatic damage. Energy metabolism of zebrafish exposed on 0.34 mg·L−1 diquat more inclined to rely on anaerobic glycolysis than the normal ones. Amino acid metabolism responses were less affected, but obvious interference effects on lipid metabolism were observed with 0.34 mg·L−1 diquat exposure. These results imply increased sensitivity of metabolomics versus histopathology and clinical chemistry in recognizing liver toxicity of diquat. This study will contribute to explore possible mechanism of hepatic damages on nontarget freshwater fish induced by diquat and provide important basis for its environmental risk assessment.

Gas chromatography-mass spectrometry chemical profile investigation and biological activities of ethylacetate fraction of Baobab (Adansonia digitata L.) pulp used in the treatment of urinary tract infections

Background: As a result of high percentage of women infected with urinary tract infection (UTI) annually, many rural dwellers use Adansonia digitata fruit pulp as herbal cure. Adansonia digitata L. (Malvaceae) is one of the great trees idolised in Africa because of its therapeutic properties.Aim: This study aimed at identifying chemical compounds in the ethylacetate fraction of the baobab fruit pulp and indicated their biological activities to justify its use for the folkloric treatment of UTI.Methods: The crude extract from Baobab fruit pulp was partitioned and ethylacetate extract was used for assay. Gas chromatography-mass spectrometry (GC–MS) analysis was performed to identify the bioactive compounds in the ethylacetate fraction, antioxidant activity was evaluated using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays whilst the total phenolic and flavonoid contents were determined by already adopted protocols. The antibacterial properties were tested against some urinary tract pathogens using macro broth dilution method.Results: A total of 36 bioactive compounds were identified by GC–MS analysis, most of them have been reported as antibacterial, antioxidant, antidiabetic, antiasthma, antifungal, anti-inflammatory and anti-cancer agents. The extract exhibited highest activity against Pseudomonas aeruginosa American Type Culture Collection (ATCC) 19582 (1.22 mg/mL) whilst Escherichia coli ATCC 8739 (˃ 8.6 mg/mL) displayed high level of resistance amongst the five bacterial isolates used in this study.Conclusion: These findings indicated that the extract contained bioactive compounds of therapeutic importance with significant antioxidant and antibacterial potentials and justify the folkloric use of this fruit in the treatment of UTIs by many African dwellers.

Evaluation of a human urine barbiturate test to screen for pentobarbital euthanasia of dogs and cats

Pentobarbital is used commonly to euthanize animals. Occasionally during a death investigation, it is necessary to determine whether a cat or dog was euthanized via pentobarbital overdose. Screening for the detection of barbiturates including pentobarbital can be performed using commercial immunochromatographic tests. We used a commercial immunochromatographic test for barbiturates in humans to screen for barbiturates in urine collected postmortem from 20 dogs and 20 cats to determine whether they had been euthanized with pentobarbital. Additionally, we analyzed the urine for pentobarbital using liquid chromatography–mass spectrometry as a confirmatory test. Screening and confirmation testing revealed 100% agreement between the tests and with the euthanasia status of each animal. Our results support the use of the immunochromatographic test for the screening of urine collected postmortem to assess for the presence of barbiturates, specifically pentobarbital, used for euthanasia.

Different metabolic pathways involved in anthracene biodegradation by Brevibacillus, Pseudomonas and Methylocystis Species

Background Polycyclic aromatic hydrocarbons (PAHs) such as anthracene are one of the most toxic contaminants to our environment. Microbial biodegradation of these xenobiotics is a cost-effective technological solution. The present study aimed to recover some bacterial isolates from Beni-Suef Governorate in Egypt with high capabilities of anthracene biodegradation. The selected isolates were molecularly characterized by 16S rRNA gene sequencing, the degree of anthracene biodegradation was monitored using optical density (OD) and high-performance liquid chromatography (HPLC), PCR amplification of some selected genes encoding biodegradation of PAHs was monitored, and gas chromatography–mass spectrometry (GC–MS) analysis was applied for detecting the resulted metabolites. Result Three bacterial isolates were studied, the 16s rRNA sequences of the isolates showed homology of the first isolate to Brevibacillus sp. (94.58 %), the second isolates showed homology to Pseudomonas sp. (94.53%) and the third isolate showed homology to Methylocystis sp. (99.61 %), all isolates showed the ability to degrade anthracene. PCR amplification of some selected genes encoding biodegradation of PAHs revealed the presence of many biodegrading genes in the selected strains. Gas chromatography-mass spectrometry (GC–MS) analysis of the metabolites resulted from anthracene biodegradation in the present study suggested that more than one biodegradation pathway was followed by the selected isolates. Conclusions The selected strains could represent a potential bioremediation tool in solving the PAHs problem in the Egyptian environment with a clean and cost-effective technique. Graphical Abstract

Metabolic Reprogramming and Risk Stratification of Hepatocellular Carcinoma Studied by Using Gas Chromatography–Mass Spectrometry-Based Metabolomics

Hepatocellular carcinoma (HCC) displays a high degree of metabolic and phenotypic heterogeneity and has dismal prognosis in most patients. Here, a gas chromatography–mass spectrometry (GC-MS)-based nontargeted metabolomics method was applied to analyze the metabolic profiling of 130 pairs of hepatocellular tumor tissues and matched adjacent noncancerous tissues from HCC patients. A total of 81 differential metabolites were identified by paired nonparametric test with false discovery rate correction to compare tumor tissues with adjacent noncancerous tissues. Results demonstrated that the metabolic reprogramming of HCC was mainly characterized by highly active glycolysis, enhanced fatty acid metabolism and inhibited tricarboxylic acid cycle, which satisfied the energy and biomass demands for tumor initiation and progression, meanwhile reducing apoptosis by counteracting oxidative stress. Risk stratification was performed based on the differential metabolites between tumor and adjacent noncancerous tissues by using nonnegative matrix factorization clustering. Three metabolic clusters displaying different characteristics were identified, and the cluster with higher levels of free fatty acids (FFAs) in tumors showed a worse prognosis. Finally, a metabolite classifier composed of six FFAs was further verified in a dependent sample set to have potential to define the patients with poor prognosis. Together, our results offered insights into the molecular pathological characteristics of HCC.