Shotgun Proteomic-Based Approach with a Q-Exactive Hybrid Quadrupole-Orbitrap High-Resolution Mass Spectrometer for Protein Adductomics on a 3D Human Brain Tumor Neurospheroid Culture Model: The Identification of Adduct Formation in Calmodulin-Dependent Protein Kinase-2 and Annexin-A1 Induced by Pesticide Mixture.

Pesticides are increasingly used in combinations in crop protection, resulting in enhanced toxicities for various organisms. Although protein adductomics is challenging, it remains a powerful bioanalytical tool to check environmental exposure and characterize xenobiotic adducts as putative toxicity biomarkers with high accuracy, facilitated by recent advances in proteomic methodologies and a mass spectrometry high-throughput technique. The present study aims to predict the potential neurotoxicity effect of imidacloprid and λ-cyhalothrin insecticides on human neural cells. Our protocol consisted first of 3D in vitro developing neurospheroids derived from human brain tumors and then treatment by pesticide mixture. Furthermore, we adopted a bottom-up proteomic-based approach using nanoflow ultraperformance liquid chromatography coupled with a high-resolution mass spectrometer for protein-adduct analysis with prediction of altered sites. Two proteins were selected, namely, calcium-calmodulin-dependent protein kinase-II (CaMK2) and annexin-A1 (ANXA1), as key targets endowed with primordial roles. De novo sequencing revealed several adduct formations in the active site of 82-ANXA1 and 228-CaMK2 as a result of neurotoxicity, predicted by the added mass shifts for the structure of electrophilic precursors. To the best of our knowledge, our study is the first to adopt a proteomic-based approach to investigate in depth pesticide molecular interactions and their potential to adduct proteins which play a crucial role in the neurotoxicity mechanism.

Research of Pesticide Metabolites in Human Brain Tumor Tissues by Chemometrics-Based Gas Chromatography-Mass Spectrometry Analysis for a Hypothetical Correlation between Pesticide Exposure and Risk Factor of Central Nervous System Tumors.

Pesticides are widely used, resulting in continuing human exposure with potential health impacts. Some exposures related to agricultural works have been associated with neurological disorders. Since the 2000s, the hypothesis of the role of pesticides in the occurrence of central nervous system (CNS) tumors has been better documented in the literature. However, the etiology of childhood brain cancers still remains largely unknown. The major objective of this work was to assess the potential role of pesticide exposure as a risk factor for CNS tumors based on questionnaires and statistical analysis of information collected from patients hospitalized in the Neurosurgery Department of the Habib Bourguiba Hospital Medium in Sfax, Tunisia, during the period from January 1, 2022, to May 31, 2023. It also aimed to develop a simple and rapid analytical method by the gas chromatography-mass spectrometry technique for the research traces of pesticide metabolites in some collected human brain tumor tissues in order to more emphasize our hypothesis for such a correlation between pesticide exposure and brain tumor development. Patients with a history of high-risk exposure were selected to conduct further analysis. Chemometric methods were adapted to discern intrinsic variation between pathological and control groups and ascertain effective separation with the identification of differentially expressed metabolites accountable for such variations. Three samples revealed traces of pesticide metabolites that were mostly detected at an early age. The histopathological diagnosis was medulloblastoma for a 10-year-old child and high-grade gliomas for 27- and 35-year-old adults. The bivariate analyses (odds ratio >1 and P value <5%) confirmed the great probability of developing cancer by an exposure case. The Cox proportional hazards model revealed the risk of carcinogenicity beyond the age of 50 as a long-term effect of pesticide toxicity. Our study supports the correlation between pesticide exposure and the risk of development of human brain tumors, suggesting that preconception pesticide exposure, and possibly exposure during pregnancy, is associated with an increased childhood brain tumor risk. This hypothesis was enhanced in identifying traces of metabolites from the carbamate insecticide class known for their neurotoxicity and others from pyridazinone, organochlorines (OCs), triazole fungicide, and N-nitroso compounds known for their carcinogenicity. The 2D-OXYBLOT analysis confirmed the neurotoxicity effect of insecticides to induce oxidative damage in CNS cells. Aldicarb was implicated in brain carcinogenicity confirmed by the identification of oxime metabolites in a stress degradation study. Revealing "aziridine" metabolites from the OC class may better emphasize the theory of detecting traces of pesticide metabolites at an early age. Overall, our findings lead to the recommendation of limiting the residential use of pesticides and the support of public health policies serving this objective that we need to be vigilant in the postmarketing surveillance of human health impacts.

Differential Proteome Profiling Analysis under Pesticide Stress by the Use of a Nano-UHPLC-MS/MS Untargeted Proteomic-Based Approach on a 3D-Developed Neurospheroid Model: Identification of Protein Interactions, Prognostic Biomarkers, and Potential Therapeutic Targets in Human IDH Mutant High-Grade Gliomas.

High-grade gliomas represent the most common group of infiltrative primary brain tumors in adults associated with high invasiveness, agressivity, and resistance to therapy, which highlights the need to develop potent drugs with novel mechanisms of action. The aim of this study is to reveal changes in proteome profiles under stressful conditions to identify prognostic biomarkers and altered apoptogenic pathways involved in the anticancer action of human isocitrate dehydrogenase (IDH) mutant high-grade gliomas. Our protocol consists first of a 3D in vitro developing neurospheroid model and then treatment by a pesticide mixture at relevant concentrations. Furthermore, we adopted an untargeted proteomic-based approach with high-resolution mass spectrometry for a comparative analysis of the differentially expressed proteins between treated and nontreated spheroids. Our analysis revealed that the majority of altered proteins were key members in glioma pathogenesis, implicated in the cellular metabolism, biological regulation, binding, and catalytic and structural activity and linked to many cascading regulatory pathways. Our finding revealed that grade-IV astrocytomas promote the downstream of the mitogen-activated-protein-kinases/extracellular-signal-regulated kinase (MAPK1/ERK2) pathway involving massive calcium influx. The gonadotrophin-releasing-hormone signaling enhances MAKP activity and may serve as a negative feedback compensating regulator. Thus, our study can pave the way for effective new therapeutic and diagnostic strategies to improve the overall survival.

A jojoba (Simmondsia chinensis) seed cake extracts express hepatoprotective activity against paracetamol-induced toxicity in rats.

This study aimed to investigate the hepatoprotective activity of jojoba seed cake extracts against an acute paracetamol (PC) intoxication. Two aqueous extracts from jojoba (Simmondsia chinensis) seed cake, a simmondsin-rich extract (WE), and a simmondsin-hydrolyzed extract (NE) using Viscozyme L enzyme have been prepared and characterized. After enzyme treatment, simmondsin content decreased from 33.0 % to 3.0 % and glucose content increased from 16.2 % to 27.3 % reflecting simmondsin hydrolysis. Both extracts were administered to different rat groups via gavage (0.6 g/kg b.w.) before PC treatment (2 g/kg b.w.) three times a week for 3 weeks. The PC intoxication altered the serum biomarkers, the oxidative status, and the Tumor necrosis factor alpha (TNF-α), Bax and Bcl-2 protein expressions of tested animals. In addition, the histological analysis of liver tissues proved significant injury and hepatocellular necrosis. WE and NE extract showed a relatively high in vitro radical scavenging (ORAC) and averting activities (HORAC) with a polyphenol content of 3.6 % and 2.9 %, respectively. Both extracts showed a powerful in vivo hepatoprotective activity against PC-induced toxicity by improving the hepatocellular antioxidant status and blocking proteins expression (TNF-α, Bax and Bcl-2), involved in inflammation and liver damage. However, the enzymatic treatment improved the hepatoprotective activity of NE despite its lower simmondsin content and lower in vitro antioxidant capacity. This enhancement could be linked to the synergetic effect between the antioxidant components and the new hydrolytic products as glucose, uronic acids, arabinose and simmondsin-aglycons. These results suggest that jojoba waste could be potentially valorized in developing hepatoprotective drugs.

Optimization of microwave assisted extraction of simmondsins and polyphenols from Jojoba (Simmondsia chinensis) seed cake using Box-Behnken statistical design.

A closed-vessel microwave-assisted extraction (MAE) of simmondsins and polyphenols from defatted Jojoba cake using Box-Benkhen design with four independent variables (solvent/cake ratio, ethanol concentration, extraction time and microwave power) was investigated. ANOVA results showed that the obtained models were significant at 95% confidence level. Optimal extraction conditions were found for highest values of microwave power (500 W) and extraction time (15 min) and for moderate values of solvent to cake ratio (41 - 45 mL/g). Optimum simmondsins yield (23.35%) was obtained with pure water as solvent. However, optimum polyphenols yield (2.33%) and ORAC antioxidant activity (656 µmol TE/g) were obtained with 46.79% and 42.04% ethanol in water, respectively. ORAC antioxidant activity was found to be well correlated to polyphenol and simmondsin contents. These results indicate that MAE is an effective technique for recovery of bioactive compounds for food and pharmaceutical industries from Jojoba by-products.

Effect of olive mill wastewaters on Scenedesmus sp. growth, metabolism and polyphenols removal.

BACKGROUND: The three-phase extraction process of olive oil produces highly contaminated wastewater (OMW). The elimination of this toxic by-product is an important environmental issue that requires the development of an appropriate management solution. The cultivation of microalgae using OMW as growth medium was therefore studied using single (the culture medium was formed by 0% to 80% ultrafiltered olive mill wastewater (OMUF) or OMW added to BG11) and two-stage strategies (microalgae were firstly cultivated in the BG11 medium. In the second stage, 40% and 80% of OMUF and OMW were added to the culture). In this work, biodegradation of OMW and subsequent extraction of lipid and antioxidant molecules was investigated as an ecofriendly method for the bioremediation and valorization of OMW. RESULTS: For two-stage cultivation, OMUF and OMW stress enhanced the intracellular amount of polyphenol accumulated in Scenedesmus sp. and exhibited the highest 2, 2-diphenyl-1- picrylhydrazyl radical (DPPH) and 2,2'-azino-bis (3-ethylbenzoline-6-sulfonate) radical (ABTS) scavenging ability compared with single-stage cultivation. Moreover, the lipid profile is dominated by polyunsaturated acids. In the single-stage cultivation, the Ch a, Ch b, carotenoid, carbohydrate and lipid content of 2.57, 7.4, 1.69, 368, and 644 g kg-1 were observed in 40% OMUF added culture, respectively, along with high biomass productivity and 58% of polyphenol removal. Moreover, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the biomass of Scenedesmus sp. cultured on 40% OMUF did not show any toxic effect, making it an efficient strategy. CONCLUSION: The results indicate that Scenedesmus sp. is a promising microalga for the biotreatment of OMW and the extraction of bioactive metabolites. © 2021 Society of Chemical Industry.

Hepatoprotective Effect of Oleuropein-Rich Extract from Olive Leaves against Cadmium-Induced Toxicity in Mice.

Cadmium (Cd) is a harmful pollutant which mainly affects the liver and kidney. In this work, we investigated the hepatoprotective effects of olive leaf extract based on oleuropein against hepatic cadmium toxicity in mice. Three groups of animals were used: the first one served as the control (C); the second one received intraperitoneal injection of cadmium 2 mg/kg b.w. (CD), administered five times during two weeks; and the third group received the same doses of Cd and simultaneously 16 mg/kg b.w. of oleuropein. Results showed that Cd induced a significant increase in liver injury biomarkers coupled with enhanced lipid peroxidation (MDA) and significant depletion of antioxidants (CAT and SOD). Histological and immunohistochemical analysis confirmed these findings. In fact, we observed a severe central lobular apoptosis and inflammation around central veins. Cotreatment with oleuropein significantly reduced the oxidative damage induced by cadmium. Our findings suggest that oleuropein could be used in the prevention of Cd hepatotoxicity.

Oxidative stability of refined olive and sunflower oils supplemented with lycopene-rich oleoresin from tomato peels industrial by-product, during accelerated shelf-life storage.

Tomato peels by-product from a Tunisian industry was used for the extraction of lycopene-rich oleoresin using hexane solvent maceration. Tomato peels oleoresin, TPO, exhibited competitive free radicals scavenging activity with synthetic antioxidants. The efficacy of TPO in stabilizing refined olive (ROO) and sunflower (RSO) oils was investigated for five months, under accelerated shelf-life, compared to the synthetic antioxidant, butylated hydroxytoluene (BHT). TPO was added to ROO and RSO at four different concentrations, namely 250, 500, 1000 and 2000 µg/g and BHT standard at 200 µg/g. Lipid oxidation was tracked by measuring the peroxide value, acidity, conjugated dienes and trienes. Results suggested the highest efficiency of 250 µg/g and 2000 µg/g of TPO, referring to 5 µg/g and 40 µg/g of lycopene, for the oxidative stabilization of ROO and RSO, respectively. The protective effect of TPO against the primary oxidation of these refined oils was significantly correlated to their lycopene contents.