Oxidant and Antioxidant Effects of Gentisic Acid in a 177Lu-Labelled Methionine-Containing Minigastrin Analogue.

BACKGROUND: The radiolabelling of receptor-binding peptides for therapy is a challenge since the peptide itself is exposed (during labelling, storage and transport) to radiation-induced damage, directly or indirectly, in aqueous solution. Hence, the use of radiostabilizers seems to be mandatory, especially in peptide molecules that contain radiation-sensitive amino acids. OBJECTIVE: The aim of this study was to investigate the effect of two stabilizers, gentisic acid and methionine, to delve into how each of them affects the radiolabelling and stability of the minigastrin analogue [177Lu]Lu-DOTA-His-His-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 through the analysis of the 22 species distinguished over time by an optimized HPLC system. METHODS: The stabilizers, in different combinations, were present from the beginning of the labelling process carried out at 96 °C for 15 min. The stability was studied for up to 7 days. RESULTS: The unexpected selective oxidation of the methionine residue of the radiolabelled peptide, promoted by gentisic acid, led to studying the effect of pH, from 3.5 to 6.0, in the presence of only this stabilizer. A pH-dependent antioxidant behaviour was revealed, showing a decrease in peptide impurities but an increase in the selective oxidation as the pH was increased. CONCLUSION: The selective oxidation of the methionine residue could be induced by oxidizing species probably produced in the reaction between gentisic acid and free radicals of water, during the protection of the radiolabelled peptide from the attack of these harmful species. Therefore, the addition of methionine becomes necessary to effectively decrease this selective oxidation in the methioninecontaining peptide.

Utilization of naproxen by Amycolatopsis sp. Poz 14 and detection of the enzymes involved in the degradation metabolic pathway.

The pollution of aquatic environments by drugs is a problem for which scarce research has been conducted in regards of their removal. Amycolatopsis sp. Poz 14 presents the ability to biotransformation naphthalene at high efficiency, therefore, in this work this bacterium was proposed as an assimilator of naproxen and carbamazepine. Growth curves at different concentrations of naproxen and carbamazepine showed that Amycolatopsis sp. Poz 14 is able to utilize these drugs at a concentration of 50 mg L-1 as a source of carbon and energy. At higher concentrations, the bacterial growth was inhibited. The transformation kinetics of naproxen showed the total elimination of the compound in 18 days, but carbamazepine was only eliminated in 19.9%. The supplementation with cometabolites such as yeast extract and naphthalene (structure similar to naproxen) at 50 mg L-1, showed that the yeast extract shortened the naproxen elimination to 6 days and reached a higher global consumption rate compared to the naphthalene cometabolite. The biotransformation of carbamazepine was not improved by the addition of cometabolites. The partial sequencing of the genome of Amycolatopsis sp. Poz 14 detected genes encoding putative enzymes for the degradation of cyclic aromatic compounds and the activities of aromatic monooxygenase, catechol 1,2-dioxygenase and gentisate 1,2-dioxygenase exhibited their involving in the naproxen biodegradation. The HPLC-MS analysis detected the 5-methoxysalicylic acid at the end of the biotransformation kinetics. This work demonstrates that Amycolatopsis sp. Poz 14 utilizes naproxen and transforms it to 5-methoxysalicylic acid which is the initial compound for the catechol and gentisic acid metabolic pathway.

Cytotoxicity, mutagenicity, and antimutagenicity of the gentisic acid on HTC cells.

Gentisic acid (GA) exhibits antioxidant, anti-inflammatory, and antibiotic activities. This substance can be found in citrus fruits, grapes, olive oil, and peas. Considering that there are few studies in the literature on the toxicity of GA, the present work aimed to investigate its cytotoxic, mutagenic, and antimutagenic activities on HTC cells. GA was diluted in culture medium at the final concentration of 0.08, 0.16, 0.8, 1.6, and 8 µg/mL. The cytotoxicity was determined by the MTT assay and Trypan Blue exclusion method, with methyl methanesulfonate and doxorubicin as positive controls, respectively. The cytokinesis-block micronucleus assay determined the mutagenic/antimutagenic activity with benzo[a]pyrene as positive control. Negative control received culture medium only. GA (0.08-8 µg/mL) was not cytotoxic to HTC cells by the MTT assay nor the Trypan Blue exclusion method as no statistical difference was observed when compared to the control. Concentration of 0.08 and 0.8 µg/mL showed no mutagenic or clastogenic effects, as no significant micronuclei inductions were observed, different from 8 µg/mL, that was mutagenic. Furthermore, none of the concentrations presented an antiproliferative activity. The antimutagenic activity of GA (0.08 µg/mL) was observed at the simultaneous treatment, as it reduced the frequency of micronuclei by 76% (24 h) and 79% (48 h). Although pre- and post-treatments were not statistically different from the mutagen, they reduced the induced-damage by 11% and 21%, respectively. The present study indicated the absence of cytotoxicity and antiproliferative activities of GA, in addition to their antimutagenic/protective effects that may contribute to human health.