Antioxidant activity of melatonin in human erythrocytes, exposed to oxidative stress by cumene hydroperoxide (cumOOH), was investigated. CumOOH at 300 microM progressively oxidized a 1% suspension of red blood cells (RBCs), leading to 100% hemolysis in 180 min. Malondialdehyde and protein carbonyls in the membrane showed a progressive increase, as a result of the oxidative damage to membrane lipids and proteins, reaching peak values after 30 and 40 min, respectively. The membrane antioxidant vitamin E and the cytosolic reduced glutathione (GSH) were totally depleted in 20 min. As a consequence of the irreversible oxidative damage to hemoglobin (Hb), hemin accumulated into the RBC membrane during 40 min. Sodium dodecyl sulfate (SDS) gel electrophoresis of membrane proteins showed a progressive loss of the cytoskeleton proteins and formation of low molecular weight bands and protein aggregates, with an increment of the intensity of the Hb band. Melatonin at 50 microM strongly enhanced the RBC resistance to oxidative lysis, leading to a 100% hemolysis in 330 min. Melatonin had no effect on the membrane lipid peroxidation, nor prevented the consumption of glutathione (GSH) or vitamin E. However, it completely inhibited the formation of membrane protein carbonyls for 20 min and hemin precipitation for 10 min. The electrophoretic pattern provided further evidence that melatonin delayed modifications to the membrane proteins and to Hb. In addition, RBCs incubated for 15 min with 300 microM cumOOH in the presence of 50 microM melatonin were less susceptible, when submitted to osmotic lysis, than cells incubated in its absence. Extraction and high-performance liquid chromatography (HPLC) analysis showed a much more rapid consumption of melatonin during the first 10 min of incubation, then melatonin slowly decreased up to 30 min and remained stable thereafter. Equilibrium partition experiments showed that 15% of the melatonin in the incubation mixture was recovered in the RBC cytosol, and no melatonin was extracted from RBC membrane. However, 35% of the added melatonin was consumed during RBC oxidation. Hydroxyl radical trapping agents, such as dimethylsulfoxide or mannitol, added into the assay in a 1,000 times molar excess, did not vary melatonin consumption, suggesting that hydroxyl radicals were not involved in the indole consumption. Our results indicate that melatonin is actively taken up into erythrocytes under oxidative stress, and is consumed in the defence of the cell, delaying Hb denaturation and release of hemin. RBCs are highly exposed to oxygen and can be a site for radical formation, under pathological conditions, which results in their destruction. A protective role of melatonin should be explored in hemolytic diseases.

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A total of 205 bluefin and yellowfin tuna samples were examined for mercury detection in order to verify possible differences and have a detailed risk assessment of the two tuna species. The results showed significant higher mercury concentration in muscle tissue of bluefin tuna respect yellowfin tuna (p < 0.001) with mean concentration of 0.84 mg/kg and maximum value of 1.94 mg/kg. These differences can be due the different biological and ecological aspects of the two tuna species and to different oceanographic aspects between Atlantic Ocean and Mediterranean sea. The results obtained in this study suggest an advisable containment of the sources of pollution and further studies on the closed-loop farming of bluefin tuna, in order to ensure the product safety.

A sensitive LC-ESI-MS/MS method was developed for the determination of 165 pesticides in 50 citrus fruit samples collected in Sicily. Moreover, an evaluation of pesticides levels in the citrus layers (peel, albedo, and pulp) was carried out. The method presented acceptable trueness, precision, and linearity with LOQ of 5 lg/kg. The results obtained showed a high frequency of fungicides class pesticides in all the citrus samples examined (>95%) with the highest concentrations in the peel (4468 mg/Kg). A significant difference of concentrations was found between the layers of the citrus fruits analysed (p < 0.05). In particular, the peel and albedo present higher pesticides significantly higher than the pulp. Our findings confirming the widespread use of these substances in citrus cultivation and suggesting the importance of pesticides analysis in all the citrus fruit layers separately, considering the different interactions between the physicochemical characteristics of the matrices and the pesticides.

The reliability of a rapid LC-HRMS method was studied in order to find a sensitive and accurate, simple, cheap, and rapid method for screening and quantitative determination of malachite green (MG), leucomalachite green (LMG), brilliant green (BG), crystal violet (CV), and leucocrystal violet (LCV) in fish muscle. All the analytes were extracted using a mixture of acetonitrile and citrate buffer, while the cleanup phase was carried out by Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method. All the data analyzed were acquired using both full MS and dd-MS2 modes. Good specificity, precision (relative standard deviation <11% for each sample tested), recovery (10–60%), decision limit (CCα between 0.55 and 0.62 μg kg−1), detection capabilities (CCβ between 0.65 and 0.78 μg kg−1), and ruggedness were achieved for the reliability of the method. Satisfactory results were obtained during the linearity test in the range of 0.10–2 ng mL−1 (r2 > 0.999). Best recoveries were obtained by the QuEChERS cleanup method for all the analytes examined, presenting values between 70 and 104%. The application of 70,000 FWHM mass resolution and narrow mass windows significantly improved the selectivity of the method, leading to simultaneous screening and quantification of dye residues in comparison to other methods proposed in literature. The optimized method proposed in this work enables a simple and fast preparation; it offers exceptional sensitivity and selectivity and maximizes efficiency and reproducibility with a low consumption of reagents. Finally, the present method was successfully employed to detect dye residues in 73 fish samples, as provided for the national residue control plan