Luciana Grazziotin Rossato

Piperazine designer drugs induce toxicity in cardiomyoblast h9c2 cells through mitochondrial impairment

Abuse of synthetic drugs is widespread among young people worldwide. In this context, piperazine derived drugs recently appeared in the recreational drug market. Clinical studies and case-reports describe sympathomimetic effects including hypertension, tachycardia, and increased heart rate. Our aim was to investigate the cytotoxicity of N-benzylpiperazine (BZP), 1-(3-trifluoromethylphenyl) piperazine (TFMPP), 1-(4-methoxyphenyl) piperazine (MeOPP), and 1-(3,4-methylenedioxybenzyl) piperazine (MDBP) in the H9c2 rat cardiac cell line. Complete cytotoxicity curves were obtained at a 0-20 mM concentration range after 24 h incubations with each drug. The EC50 values (μM) were 343.9, 59.6, 570.1, and 702.5 for BZP, TFMPP, MeOPP, and MDBP, respectively. There was no change in oxidative stress markers. However, a decrease in total GSH content was noted for MDBP, probably due to metabolic conjugation reactions. All drugs caused significant decreases in intracellular ATP, accompanied by increased intracellular calcium levels and a decrease in mitochondrial membrane potential that seems to involve the mitochondrial permeability transition pore. The cell death mode revealed early apoptotic cells and high number of cells undergoing secondary necrosis. Among the tested drugs, TFMPP seems to be the most potent cytotoxic compound. Overall, piperazine designer drugs are potentially cardiotoxic and support concerns on risks associated with the intake of these drugs.

Development and validation of a GC/IT-MS method for simultaneous quantitation of para and meta-synephrine in biological samples.

After the FDAs ban of ephedrine-containing supplements, Citrus aurantium appeared as an alternative to ephedra in herbal weight loss products. Synephrine, the most abundant active component of C. aurantium, exists in three different structural or positional isomeric forms (ortho-o-, meta-m-, and para-p-). Dietary supplements contain m- and p-synephrine, both alpha-adrenergic agonists,while the m-isoform is the most potent at alpha(1)-adrenoreceptors. In spite of the pharmacokinetic and toxicological interest in the study of these compounds, adequate methods for their quantification in biological samples are yet to be developed. Thus, in the present study, a sensitive gas chromatography-ion trap mass spectrometry (GC/IT-MS) method was developed and validated for the simultaneous quantitation of m- and p-synephrine in a cellular matrix after solid phase extraction (SPE). The validation of the method was performed through the evaluation of the following parameters: selectivity, linearity, specificity, precision, accuracy, limit of detection, limit of quantification, and recovery. The methods applicability was studied in two different biological matrices by evaluating p- and m-synephrine uptake in Caco-2 cells and also in freshly isolated cardiomyocytes from adult rat. The developed GC/IT-MS method was shown to be selective, accurate, precise, and valid for simultaneous determination of p- and m-synephrine in biological samples.

Cumulative Mitoxantrone‐Induced Haematological and Hepatic Adverse Effects in a Subchronic In vivo Study

Mitoxantrone (MTX) is an antineoplastic agent that can induce hepato‐ and haematotoxicity. This work aimed to investigate the occurrence of cumulative early and late MTX‐induced hepatic and haematological disturbances in an vivo model. A control group and two groups treated with three cycles of 2.5 mg/kg MTX at days 0, 10 and 20 were formed. One of the treated groups suffered euthanasia on day 22 (MTX22) to evaluate early MTX toxic effects, while the other suffered euthanasia on day 48 (MTX48), to allow the evaluation of MTX late effects. An early immunosuppression with a drop in the IgG levels was observed, causing a slight decrease in the plasma total protein content. The early bone marrow depression was followed by signs of recovery in MTX48. The genotoxic potential of MTX was demonstrated by the presence of several micronuclei in MTX22 leucocytes. Increases in plasma iron and cholesterol levels in the MTX22 rats were observed, while in both groups increases in the unconjugated bilirubin, C4 complement, and decreases in the triglycerides, alanine aminotransferase, alkaline phosphatase and transferrin were found in plasma samples. On MTX 48, the liver histology showed more hepatotoxic signs, the hepatic levels of reduced and oxidized glutathione were increased, and ATP hepatic levels were decreased. However, the hepatic total protein levels were decreased only in the livers of MTX22 group. Results demonstrated the MTX genotoxic effects, haemato‐ and direct hepatotoxicity. While the haematological toxicity is ameliorated with time, the same was not observed in the hepatic injury.

Microbiological Environmental Monitoring After the Use of Air Purifier Ozone Generator

The work aimed to evaluate the indoor microbiological air quality after using the purifier ozone generator (Brizzamar®), by counting the total viable microorganisms. The plates containing the culture media were exposed on pre-defined locations for 10 min at the times 0 h (without purifier), 1, 2 and 3 h (with purifier). The results showed significant decreases in the microorganisms after using the purifier, reaching a reduction up to 80% of the fungi and bacteria in the environment after two hours. The amount of ozone in the atmosphere was kept at 0,01 ppm, which is considered non-toxic to human exposure. The purifier significantly improved the air quality in indoors.

In Vitro Inactivation of Herpes Virus by Ozone

Viruses represent a major threat to human health and might be transmitted by direct and indirect contact. Reducing the viral load, either in the host or in the environment greatly reduces virus spreading. In this work we aimed to evaluate the virucidal activity of ozone against herpes virus of human (Herpes Simplex Virus 1 – HSV-1) and bovine (Bovine Herpes Virus 1 – BoHV-1) origin. The virucidal activity was measured by tittering aliquots of HSV-1 and BoHV-1 exposed for 1, 2, and 3 h to ozone generated by a domestic device. In addition, the possible cytotoxic effect of ozone to cultured MDBK cells was also assessed using the MTT method. MDBK cells exposed to ozone for 3 h and tested immediately after exposure, or after culturing for 24 h, had viability similar to non-exposed cells, indicating that ozone per se was not cytotoxic to the cells. Furthermore, a significant reduction in BoHV-1 (99.62%) and HSV-1 (90.0%) titer was observed after 3 h exposure to ozone. Our results indicate that ozone might be safely used to reduce environmental load of herpes virus.