Cholesterol catalyzes unfolding in membrane-inserted motifs of the pore forming protein cytolysin A.

Plasma membrane-induced protein folding and conformational transitions play a central role in cellular homeostasis. Several transmembrane proteins are folded in the complex lipid milieu to acquire a specific structure and function. Bacterial pore forming toxins (PFTs) are proteins expressed by a large class of pathogenic bacteria that exploit the plasma membrane environment to efficiently undergo secondary structure changes, oligomerize, and form transmembrane pores. Unregulated pore formation causes ion imbalance, leading to cell death and infection. Determining the free energy landscape of these membrane-driven-driven transitions remains a challenging problem. Although cholesterol recognition is required for lytic activity of several proteins in the PFT family of toxins, the regulatory role of cholesterol for the α-PFT, cytolysin A expressed by Escherichia coli remains unexplained. In a recent free energy computation, we showed that the ß tongue, a critical membrane-inserted motif of the ClyA toxin, has an on-pathway partially unfolded intermediate that refolds into the helix-turn-helix motif of the pore state. To understand the molecular role played by cholesterol, we carry out string-method-based computations in membranes devoid of cholesterol, which reveals an increase of ∼30 times in the free energy barrier for the loss of ß sheet secondary structure when compared with membranes containing cholesterol. Specifically, the tyrosine-cholesterol interaction was found to be critical to creating the unfolded intermediate. Cholesterol also increases the packing and hydrophobicity of the bilayer, resulting in enhanced interactions of the bound protein before complete membrane insertion. Our study illustrates that cholesterol is critical to catalyzing and stabilizing the membrane-inserted unfolded state of the ß tongue motif of ClyA, opening up fresh insights into cholesterol-assisted unfolding of membrane proteins.

Bioactive properties of Persea lingue Ness (Lauraceae) fruit and leaf extracts / Propriedades bioativas de extratos de folhas e frutos de Persea lingue Ness (Lauraceae)

Persea lingue Ness is a tree species that lives mainly in temperate forests of south-central Chile. Its leaves are used in ethnomedicine, the fruit is a drupe similar to that of the avocado and has not been studied. The aim of this study was to determine the cytotoxicity in leukemia cell and antibacterial activity, along with some chemical content characteristics of P. lingue fruit and leaf extracts. The antibacterial activity was determined by the inhibition of bacterial growth in liquid medium assay against Gram-positive and Gram-negative bacteria. The leukemia cell lines Kasumi-1 and Jurkat were used to evaluate the cytotoxic activity by using propidium iodide and AlamarBlue assays. Total phenolic, flavonoid, condensed tannin, alkaloid and lipid contents were evaluated in the fruit and in the leaf extracts. The antioxidant activity of both extracts were also elavaluated. Leaf extract presented the highest content of total phenols, condensed tannins and flavonoids, and also the highest antioxidant activity. While the fruit extract has a higher amount of lipids and alkaloids and the high antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus megaterium and Micrococcus luteus. The leaf extract only showed activity against M. luteus. Concerning the cytotoxic activity, only the fruit extract showed cytotoxicity against the cell lines Jurkat and Kasumi-1. P. lingue fruit extract is a potential source of biologically active molecules for the development of new drugs to be used in some types of leukemia, as well as antibacterial agent.

Persea lingue Ness é uma árvore que vive principalmente na floresta temperada do centro-sul do Chile. As folhas são usadas na etnomedicina. O fruto é uma drupa similar ao abacate e que nunca foi pesquisada anteriormente. O objetivo deste estudo foi o de avaliar a citotoxicidade em células leucêmicas e as atividades antibacterianas, assim como algumas características químicas do extrato de fruto e da folha do P. lingue. As atividades antibacterianas foram determinadas pelo método da inibição do crescimento bacteriano em meio líquido empregando-se bactérias Gram-positivas e Gram-negativas. As linhagens celulares leucêmicas, Kasumi-1 e Jurkat foram usadas para avaliar a atividade citotóxica em ensaios empregando-se iodeto de propídio e AlamarBlue. Foram avaliados os teores totais de fenóis, flavonóides, taninos condensados, alcalóides e lipídeos presentes nos extratos das folhas e dos frutos. As atividades antioxidantes de ambos os extratos também foram avaliadas. O extrato das folhas foi o que apresentou o maior conteúdo de fenóis, taninos condensados e flavonóides totais e a maior atividade antioxidante. Já o extrato de fruto apresentou a maior quantidade de lipídios e alcaloides e a melhor atividade antibacteriana contra Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus megaterium e Micrococcus luteus. Já o extrato das folhas apresentou apenas atividade contra M. luteus. Em relação à atividade citotóxica, apenas o extrato do fruto apresentou citotoxicidade contra as linhagens celulares Jurkat e Kasumi-1. Em resumo, o extrato do fruto de P. lingue é uma potencial fonte de moléculas com atividade biológica para o desenvolvimento de novos fármacos a serem utilizados em alguns tipos de leucemia, bem como agente antibacteriano.

Local Cytotoxic Effects in Cobra Envenoming: A Pilot Study.

The cobra (genus Naja (N.)) is one of the most common venomous snakes. Due to its frequency and deadly complications of muscle paralysis, local necrosis, and chronic musculoskeletal disability, it should not be ignored. The pathology of devastating tissue destruction, even though specific antivenoms exist, is not fully clear. Here, we attempted to dig in envenomed tissues to study the clinical toxicology of cobra venom. Four cases of N. atra snake envenomation, in which the subjects developed advanced tissue injury, were involved in this study. We used enzyme-ligand sandwich immunoassay (ELISA) to assay the whole venom, cytotoxin A3 and short-chain neurotoxin (sNTX) in blood, bullae, wound discharge, and debrided tissue. We found that persistently high concentrations of venom and toxins, especially cytotoxin A3, were detected in bullae, wound discharge fluid and necrotic tissue of these patients even after large doses of specific antivenom treatment, and wide excision and advanced debridement could largely remove these toxins, lessen the size of necrosis, and promote wound healing. We also found that the point-of-care apparatus, ICT-Cobra kit, might be used to promptly monitor the wound condition and as one of the indicators of surgical intervention in cases of cobra envenomation in Taiwan.

Reprogramming cholesterol metabolism in macrophages and its role in host defense against cholesterol-dependent cytolysins.

Cholesterol is a critical lipid for all mammalian cells, ensuring proper membrane integrity, fluidity, and biochemical function. Accumulating evidence indicates that macrophages rapidly and profoundly reprogram their cholesterol metabolism in response to activation signals to support host defense processes. However, our understanding of the molecular details underlying how and why cholesterol homeostasis is specifically reshaped during immune responses remains less well understood. This review discusses our current knowledge of cellular cholesterol homeostatic machinery and introduces emerging concepts regarding how plasma membrane cholesterol is partitioned into distinct pools. We then discuss how proinflammatory signals can markedly reshape the cholesterol metabolism of macrophages, with a focus on the differences between MyD88-dependent pattern recognition receptors and the interferon signaling pathway. We also discuss recent work investigating the capacity of these proinflammatory signals to selectively reshape plasma membrane cholesterol homeostasis. We examine how these changes in plasma membrane cholesterol metabolism influence sensitivity to a set of microbial pore-forming toxins known as cholesterol-dependent cytolysins that specifically target cholesterol for their effector functions. We also discuss whether lipid metabolic reprogramming can be leveraged for therapy to mitigate tissue damage mediated by cholesterol-dependent cytolysins in necrotizing fasciitis and other related infections. We expect that advancing our understanding of the crosstalk between metabolism and innate immunity will help explain how inflammation underlies metabolic diseases and highlight pathways that could be targeted to normalize metabolic homeostasis in disease states.

Fish Cytolysins in All Their Complexity.

The majority of the effects observed upon envenomation by scorpaenoid fish species can be reproduced by the cytolysins present in their venoms. Fish cytolysins are multifunctional proteins that elicit lethal, cytolytic, cardiovascular, inflammatory, nociceptive, and neuromuscular activities, representing a novel class of protein toxins. These large proteins (MW 150-320 kDa) are composed by two different subunits, termed α and ß, with about 700 amino acid residues each, being usually active in oligomeric form. There is a high degree of similarity between the primary sequences of cytolysins from different fish species. This suggests these molecules share similar mechanisms of action, which, at least regarding the cytolytic activity, has been proved to involve pore formation. Although the remaining components of fish venoms have interesting biological activities, fish cytolysins stand out because of their multifunctional nature and their ability to reproduce the main events of envenomation on their own. Considerable knowledge about fish cytolysins has been accumulated over the years, although there remains much to be unveiled. In this review, we compiled and compared the current information on the biochemical aspects and pharmacological activities of fish cytolysins, going over their structures, activities, mechanisms of action, and perspectives for the future.

Cytotoxin antibody-based colourimetric sensor for field-level differential detection of elapid among big four snake venom.

Development of a rapid, on-site detection tool for snakebite is highly sought after, owing to its clinically and forensically relevant medicolegal significance. Polyvalent antivenom therapy in the management of such envenomation cases is finite due to its poor venom neutralization capabilities as well as diagnostic ramifications manifested as untoward immunological reactions. For precise molecular diagnosis of elapid venoms of the big four snakes, we have developed a lateral flow kit using a monoclonal antibody (AB1; IgG1 - κ chain; Kd: 31 nM) generated against recombinant cytotoxin-7 (rCTX-7; 7.7 kDa) protein of the elapid venom. The monoclonal antibody specifically detected the venoms of Naja naja (p < 0.0001) and Bungarus caeruleus (p<0.0001), without showing any immunoreactivity against the viperidae snakes in big four venomous snakes. The kit developed attained the limit of quantitation of 170 pg/µL and 2.1 ng/µL in spiked buffer samples and 28.7 ng/µL and 110 ng/µL in spiked serum samples for detection of N. naja and B. caeruleus venoms, respectively. This kit holds enormous potential in identification of elapid venom of the big four snakes for effective prognosis of an envenomation; as per the existing medical guidelines.

Cytotoxicity of Mycotoxins Frequently Present in Aquafeeds to the Fish Cell Line RTGill-W1.

In the last decades, the aquaculture industry has introduced plant-based ingredients as a source of protein in aquafeeds. This has led to mycotoxin contaminations, representing an ecological, health and economic problem. The aim of this study was to determine in the RTgill-W1 fish cell line the toxicity of fifteen mycotoxins of common occurrence in aquafeeds. To identify the most sensitive endpoint of toxicity, the triple assay was used. It consisted of three assays: alamarBlue, Neutral Red Uptake and CFDA-AM, which revealed the mitochondrial activity, the lysosomal integrity and the plasma membrane integrity, respectively. Most of the assayed mycotoxins were toxic predominantly at lysosomal level (enniatins, beauvericin, zearalenone, ochratoxin A, deoxynivalenol (DON) and its acetylated metabolites 15-O-acetyl-DON and 3-acetyl-DON). Aflatoxins B1 and B2 exerted the greatest effects at mitochondrial level, while fumonisins B1 and B2 and nivalenol were not toxic up to 100 µg/mL. In general, low toxicity was observed at plasma membrane level. The vast majority of the mycotoxins assayed exerted a pronounced acute effect in the fish RTgill-W1 cell line, emphasizing the need for further studies to ascertain the impact of mycotoxin contamination of fish feeds in the aquaculture industry and to establish safe limits in aquafeeds.

Comparative Study of Phytochemical, Antioxidant, and Cytotoxic Activities and Phenolic Content of Syzygium aqueum (Burm. f. Alston f.) Extracts Growing in West Sumatera Indonesia.

Syzygium aqueum, consisting of various fruit colors, is one of the plants that have been used as traditional medicine. This study aims to evaluate and compare phytochemical, antioxidant, and cytotoxic activities and total phenolic content of leaves and stem bark extracts of S. aqueum with pink and red fruits, in order to identify the best extract that can be used as a natural antioxidant. Phytochemical constituents were evaluated qualitatively using chemicals, while cytotoxic activities were identified using the brine shrimp lethality test. Total phenolic content was determined via the Folin-Ciocalteu method. Leaves and stem bark of S. aqueum contained flavonoids, phenolics, and triterpenoids, but the stem bark also contained saponins and alkaloids. Methanol and ethyl acetate extracts of leaves and stem bark were categorized as very powerful antioxidants to DPPH (IC50 9.71-38.69 µg/mL) and hydrogen peroxide (IC50 16.44-44.02 µg/mL), while hexane extracts were inactive. Methanol, ethyl acetate, and hexane extracts of leaves and stem bark were categorized as moderately cytotoxic to A. salina larvae (LC50 104.04-440.65 µg/mL). Comparing leaves and stem barks, antioxidant and cytotoxic activities of stem bark extracts were higher than those of leaves extracts. Total phenolic content of leaves extracts was higher than that of stem bark extracts where the order of total phenolic content progressed from methanol extracts > ethyl acetate extracts > hexane extracts. Therefore, the stem bark of S. aqueum was identified as the better source of natural antioxidant compared with the leaves.

Melittin-induced metabolic changes on the Madin-Darby Bovine Kidney cell line / [Mudanças metabólicas induzidas por melitina em células da linhagem Madin-Darby Bovine Kidney]

In this study, the toxic effects of melittin on Madin-Darby Bovine Kidney cells (MDBK) were analyzed with respect to mitochondrial functionality by reduction of MTT and flow cytometry, apoptosis potential, necrosis, oxygen reactive species (ROS) production, lipid peroxidation, and DNA fragmentation using flow cytometry and cell membrane destabilization by confocal microscopy. The toxicity presented dose-dependent characteristics and mitochondrial activity was inhibited by up to 78.24 ±3.59% (P<0.01, n = 6) in MDBK cells exposed to melittin (10µg/mL). Flow cytometry analysis revealed that melittin at 2µg/mL had the highest necrosis rate (P<0.05) for the cells. The lipoperoxidation of the membranes was also higher at 2µg/mL of melittin (P<0.05), which was further confirmed by the microphotographs obtained by confocal microscopy. The highest ROS production occurred when the cells were exposed to 2.5µg/mL melittin (P<0.05), and this concentration also increased DNA fragmentation (P<0.05). There was a significative and positive correlation between the lipoperoxidation of membranes with ROS (R=0.4158), mitochondrial functionality (R=0.4149), and apoptosis (R=0.4978). Thus, the oxidative stress generated by melittin culminates in the elevation of intracellular ROS that initiates a cascade of toxic events in MDBK cells.(AU)

Neste estudo, os efeitos tóxicos da melitina em células Madin-Darby Bovine Kidney (MDBK) foram analisados quanto à funcionalidade mitocondrial, por redução de MTT e citometria de fluxo, potencial de apoptose, necrose, produção de espécies reativas de oxigênio (ROS), peroxidação lipídica e fragmentação de DNA, utilizando-se citometria de fluxo e desestabilização da membrana celular, por microscopia confocal. A toxicidade apresentou características dose-dependentes e a atividade mitocondrial foi inibida até 78,24±3,59% (P<0,01, n = 6) em células MDBK expostas à melitina (10µg/mL). Análises por citometria de fluxo revelaram que a melitina a 2µg/mL apresentou o maior índice necrótico celular (P<0,05). A maior lipoperoxidação de membranas também foi na concentração de 2µg/mL de melitina (P<0,05), o que foi posteriormente confirmado por microscopia confocal. A maior produção de ROS aconteceu quando as células foram expostas a 2,5µg/mL de melitina (P<0,05), e essa concentração também aumentou a fragmentação de DNA (P<0,05). Houve uma significativa correlação positiva entre a lipoperoxidação de membranas e a produção de ROS (R=0,4158), funcionalidade mitocondrial (R=0,4149) e apoptose (R=0,4978). Portanto, o estresse oxidativo gerado pela melitina culminou na elevação de ROS intracelular, que inicia uma cascata de eventos tóxicos nas células MDBK.(AU)

Silver nanoparticles: Correlating particle size and ionic Ag release with cytotoxicity, genotoxicity, and inflammatory responses in human cell lines.

The widespread use of silver nanoparticles (AgNPs), their many sources for human exposure, and the ability of AgNPs to enter organisms and induce general toxicological responses have raised concerns regarding their public health and environmental safety. To elucidate the differential toxic effects of polyvinylpyrrolidone-capped AgNPs with different primary particle sizes (i.e. 5, 50, and 75 nm), we performed a battery of cytotoxicity and genotoxicity assays and examined the inflammatory responses in two human cell lines (i.e. HepG2 and A549). Concentration-dependent decreases in cell proliferation and mitochondrial membrane potential and increases in cytokine (i.e. interleukin-6 and interleukin-8) excretion indicated disruption of mitochondrial function and inflammation as the main mediating factors of AgNPs-induced cytotoxicity. An incremental increase in genotoxicity with decreasing AgNPs diameter was noted in HepG2 cells, which was associated with S and G2/M accumulation and transcriptional activation of the GADD45α promoter as reflected by luciferase activity. Dose-related genetic damage, as indicated by Olive tail moment and micronucleus formation, was also observed in A549 cells, but these effects as well as the AgNPs-induced cytotoxicity were more associated with ionic Ag release from nanoparticles (NPs). In summary, the present study addressed different toxicity mechanisms of AgNPs, depending on the cell model, toxicological endpoint, particle size, and degree of Ag+ release from NPs. The results suggest that the GADD45α promoter-driven luciferase reporter cell system provided a rapid screening tool for the identification of genotoxic properties of NPs across a range of different sizes and concentrations.

Decrease of the DXR-induced genotoxicity and nongenotoxic effects of Theobroma cacao revealed by micronucleus assay / Diminuição da genotoxicidade induzida por DXR e efeitos não genotóxicos de Theobroma cacao revelados pelo ensaio de micronúcleo

This study evaluated the genotoxicity of lyophilized glycolic extract of Theobroma cacao Linné seeds (TCL), using the micronucleus assay in bone marrow of mice. The interaction between TCL and doxorubicin (DXR) was also analyzed. Experimental groups were evaluated 24-48 h after treatment with N-Nitroso-N-ethylurea (NEU: 50 mg/kg), DXR (5 mg/kg), NaCl (145 mM), TCL (0.5-2 g/kg), and TCL (2 g/kg) in combination with DXR (antigenotoxic assays). Analysis of micronucleated polychromatic erythrocytes (MNPCEs) showed no significant differences between all the treatment doses of TCL and NaCl control. Mice experimentally treated with DXR and NEU significantly induced MNPCEs. However, a significant reduction of MNPCEs was also observed when TCL was administered in combination with the chemotherapeutic agent DXR. The analysis of the PCE/NCE ratio revealed no significant differences between the NaCl control, all doses of TCL, and DXR. However, there were significant differences in the PCE/NCE ratio between positive NEU control and all other treatments. The PCE/NCE ratio observed after treatment with TCL and DXR showed significant differences and intermediate values to controls (NaCl and NEU). This study suggests absence of genotoxicity and cytotoxicity of TCL, regardless of dose, sex, and time. TCL reduced genotoxic effects induced by DXR, suggesting potential antigenotoxic effects.

Este estudo avaliou a genotoxicidade do extrato glicólico liofilizado de sementes de Theobroma cacao Linné (TCL), usando o ensaio do micronúcleo em medula óssea de camundongos. A interação entre TCL e doxorrubicina (DXR) foi também analisada. Grupos experimentais foram avaliados 24-48 h após tratamento com N-Nitroso-N-etilureia (NEU: 50 mg/kg), DXR (5 mg/kg), NaCl (145 mM), TCL (0,5-2 g/kg), e TCL (2 g/kg) em combinação com DXR (ensaio antigenotóxico). As análises de eritrócitos policromáticos micronucleados (EPCMNs) não mostraram diferenças significantes entre todas as doses de tratamento do TCL e o controle NaCl. Camundongos experimentalmente tratados com DXR e NEU induziram significativamente EPCMNs. Contudo, uma redução significante de EPCMNs foi também observada quando TCL foi administrada em combinação com o agente quimioterapêutico DXR. As análises da relação EPC/ENC (eritrócito policromático/eritrócito normocromático) revelaram ausência de diferenças significantes entre o controle NaCl, todas as doses de TCL e DXR. Contudo, houve diferenças significantes na relação EPC/ENC entre o controle positivo NEU e todos os outros tratamento. A relação ECP/ENC observada após o tratamento com TCL e DXR mostrou diferenças significantes e valores intermediários aos controles (NaCl e NEU). Este estudo sugere ausência de genotoxicidade e citotoxicidade de TCL, independentemente da dose, sexo e tempo. TCL reduziu os efeitos genotóxicos induzidos por DXR, sugerindo potencial efeitos antigenotóxicos.

Preformulation studies of l-glutathione: physicochemical properties, degradation kinetics, and in vitro cytotoxicity investigations.

Objectives: l-Glutathione (GSH) is an endogenous tripeptide with super antioxidant properties. In this study, preformulation parameters of GSH and its degradation products were fully investigated.Significance: To date, no experimental preformulation data is available for GSH. Therefore, to the author's knowledge, this is the first study to experimentally determine the preformulation parameters of GSH, which can be considered more reliable for further studies.Methods: An HPLC method for GSH was optimized and validated to accurately quantify the GSH amount in solution, used to investigate GSH's solubility and Log P. Differential Scanning Calorimeter and Thermogravimetric Analyzer were used to evaluate the thermal properties of GSH. Polarized microscope and Fourier-transform Infrared Spectroscopy were used to determine GSH's crystal habits and functional groups, respectively. Forced degradation kinetics and the degradation products were investigated and identified by LC-MS, respectively. GSH's cellular cytotoxicity on fibroblasts was investigated by MTT assay.Results: It was determined that GSH has high aqueous solubility (252.7 mg/mL), low Log P (-3.1), a melting endotherm of 195 °C and decomposition at 210°C, negligible moisture content, and a rectangular/cylindrical-shaped crystalline form. Seven degradation products were identified; one of the major degradation products of GSH under different conditions is first order kinetic oxidation into glutathione disulfide. No cytotoxicity was observed when fibroblasts were treated with GSH (0.005-10.000 mg/mL).Conclusions: Precise preformulation parameters of GSH were obtained, and these are imperative for the development and optimization of advanced GSH formulations.

Does selenium fortification of kale and kohlrabi sprouts change significantly their biochemical and cytotoxic properties?

BACKGROUND: The sprouts of Brassica vegetables are known from their nutritional and chemopreventive values. Moreover, sprouts fortification with some trace elements, like selenium, may increase their importance in human diet. Thus, the aim of our study was to examine if selenium enrichment of kale and kohlrabi sprouts may influence their biochemical properties (phenolic acids and L-tryptophan content, antioxidant potential) or cytotoxic activity. Additional aim of the study was to evaluate the profile of selenium compounds and to describe the multidimensional interactions between the mentioned parameters. METHODS: Selenium content in the sprouts was evaluated by double-channel atomic fluorescence spectrometer AFS-230 with the flow hydride-generation system. Separation of selenium species in water soluble fraction was performed by size-exclusion LC-ICP-MS. The identification and quantification of phenolic acids and L-tryptophan was performed by HPLC. For antioxidant activity DPPH and FRAP methods were used. Cytotoxic activity of the sprouts extracts on a panel of human metastatic carcinoma cells was evaluated by MTT test. RESULTS: Selenium content in the fortified sprouts was several orders of magnitude higher than in the unfortified ones. Only small percentage of supplemented selenium (ca. 10 %) was incorporated into the sprouts as seleno-L-methionine, while the other detected selenium species remained unidentified. Selenium fortification differently stimulated the production of phenolic acids (sinapic, chlorogenic, isochlorogenic and caffeic acid) in the tested sprouts, depending on the particular species, selenium dose and the investigated compound. PCA analysis revealed strong correlation between antioxidant parameters and phenolic acids and L-tryptophan, while Se correlated only with caffeic acid. The sprouts extracts (≥1 mg/mL) showed cytotoxic potency to all the studied cancer cell lines (SW480, SW620, HepG2, SiHa), regardless the selenium supplementation. CONCLUSION: Se-fortified kale and kohlrabi sprouts are good candidates for functional food ingredients. Moreover, these results indicate that the sprouts enriched with sodium selenite show higher nutritional value, without significant changes in their cytotoxic activity.

Integration of dry-column flash chromatography with NMR and FTIR metabolomics to reveal cytotoxic metabolites from Amphoricarpos autariatus.

Metabolomics generate a profile of small molecules from plant extracts, which could be directly responsible for bioactivity effects. Using dry-column flash chromatography enabled a rapid and inexpensive method for the very efficient separation of plant extract with a high resolution. This separation method coupled to NMR and FTIR-based metabolomics is applied to identify bioactive natural products. OPLS multivariate analysis method, was used for correlation the chemical composition of the plant extracts, Amphoricarpos autariatus, with the results of cytotoxic activity against Human cervical adenocarcinoma cell line (HeLa) and epithelial lung cancer cell line (A549). In this way, the highest contribution to the cytotoxic activity was recorded for the guaianolide sesquiterpene lactones named amphoricarpolides. The compounds indicated as bioactive after metabolomics analysis were tested, and their cytotoxic activity were confirmed.

Could combustion-generated nanoparticles induce cytotoxicity also at the extremely low doses typical of indoor environments?

SUMMARY: The presence of nanoparticles in the environment is mainly attributed to outdoor sources but sub-10 nm particles may also form indoor as effect of domestic activities such as cooking, heating, air freshening. Today, due to the COVID-19 pandemic, people are staying home for longer periods of times, thus being exposed to a poor indoor air quality. Due to elevated numerical concentration and large surface area, the health effect of sub-10 nm particles can go beyond what expected from their low mass concentration in the atmosphere. The objective of this study is to find, based on analysis of recent in vitro studies, a dose-effect correlation based on particle size/surface more than on particle mass. Such a correlation cold be useful to assess the health effects of people exposed to very low mass doses of nanoparticles either indoor or outdoor.

A fully validated simple new method for environmental monitoring by surface sampling for cytotoxics.

A wipe sampling procedure followed by a simple ultra-performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for simultaneous quantification of six cytotoxic drugs: 5-fluorouracil (5FU), doxorubicin (DOXO), epirubicin (EPI), ifosfamide (IF), cyclophosphamide (CP) and gemcitabine (GEM), as surrogate markers for occupational exposure. After a solid-phase extraction of wiping filter on 10 × 10 cm surface, the separation was performed within 6.5 min, using a gradient mobile phase and the analytes were detected by mass spectrometry in the multiple reaction ion monitoring mode. The method was validated according to the recommendations of the US Food and Drug Administration. The method was linear (r2 > 0.9912) between 2.5 and 200 ng per wiping sample (25 to 2000 pg/cm2) for 5FU, doxorubicin and epirubicin and between 0.2 and 40 ng per wiping sample (2 to 400 pg/cm2) for cyclophosphamide, ifosfamide and gemcitabine. The lower limits of quantification were 2.5 ng (25 pg/ cm2) for 5FU, doxorubicin and epirubicin, and 0.2 ng (2 pg/cm2) for CP, IF and GEM. Within-day and between-day imprecisions were <14.0, 10.6, 11.1, 8.7, 11.2 and 10.9% for 5-fluorouracil, doxorubicin, epirubicin, ifosfamide cyclophosphamide and gemcitabine, respectively. The inaccuracies did not exceed 2.7, 10.9, 1.1, 4.5, 1.6 and 2.9% for the studied molecules, respectively. This new sensitive validated method for surface contamination studies of cytotoxics was successfully applied on different localizations in hospital. This approach is particularly suitable to assess occupational exposure risk to cytotoxic drugs.

Cytotoxic and genotoxic potential of industrialized powdered milk for infants and young children

This study aimed to evaluate the cytotoxicity and genotoxicity and determine the LC50 concentration of powdered infant formulas widely marketed in South American countries. To this, milk samples, called as A, B, C and D, were analyzed in root meristem cells of Allium cepa, at concentrations of 0.075; 0.15 and 0.30 g mL-1, for 24 and 48 hours; and through cell viability in culture of normal line cells, via MTT test, for 24 hours, in the concentrations 0.018; 0.0375; 0.075 and 0.15 g mL-1. In A. cepa, all dairy products in the three concentrations caused significant inhibition of cell division in the meristems within the first 24 hours of exposure. In the in vitro evaluation, all milk formulas at 0.15 g mL-1, as well as milk A at a concentration of 0.037 g mL-1, C at 0.075 g mL-1 and D at 0.037 g mL-1, significantly reduced the cellular viability of the cell culture exposed to the foods studied, being potentially toxic. The milk A was considered the most toxic, with LC50 of 0.031 g mL-1, and B as the least toxic, with LC50 of 0.15 g mL-1. Therefore, the milk evaluated caused significant instability in cells of the test systems used and were characterized as cytotoxic.

Cytotoxic, clastogenic and genotoxic effects of cis-tetraammine(oxalato)ruthenium(III) dithionate on human peripheral blood lymphocytes

There is a concern about stablishing the clinical risk of drugs used for cancer treatment. In this study, the cytotoxic, clastogenic and genotoxic properties of cis-tetraammine(oxalato)ruthenium(III) dithionite - cis-[Ru(C2O4)(NH3)4]2(S2O6), were evaluated in vitro in human lymphocytes. The mitotic index (MI), chromosomal aberrations (CA) and DNA damage by comet assay were also analyzed. The MTT test revealed that the ruthenium compound showed a slight cytotoxic effect at the highest concentration tested. The IC50 value for the compound after 24 hours of exposure was 185.4 µM. The MI values of human peripheral blood lymphocytes treated with 0.015, 0.15, 1.5 and 150 µM of cis-[Ru(C2O4)(NH3)4]2(S2O6) were 6.1, 3.9, 3.2 and 0.2%, respectively. The lowest concentration, 0.015 µM, did not show any cytotoxic activity. The CA values for the 0.015, 0.15 and 1.5 µM concentrations presented low frequency (1.5, 1.6 and 2.3%, respectively), and did not express clastogenic activity when compared to the negative control, although it was observed clastogenic activity in the highest concentration tested (150 µM). The results obtained by the comet assay suggest that this compound does not present genotoxic activity at lower concentrations. The results show that cis-[Ru(C2O4)(NH3)4]2(S2O6) has no cytotoxic, clastogenic or genotoxic in vitro effects at concentrations less than or equal to 0.015 µM. This information proves as promising in the treatment of cancer and is crucial for future trials.

Cytotoxicity and oxidative stress induced by nickel and titanium ions from dental alloys on cells of gastrointestinal tract.

The aim was to explore the biological effect of nickel (Ni) and titanium (Ti) ions released from dental alloys. NiTi alloy were exposed to 40 mL of artificial saliva (pH = 4.8, t = 37 °C). The dynamics of Ni and Ti ions release during corrosion were recorded on the 3th, 7th and 14th day. Biological effect of Ni and Ti ions released from alloy was explored on cell lines of human tongue CAL 27, liver Hep G2 and colon Caco-2. Neutral Red uptake assay for the estimation of cell viability/cytotoxicity and 2',7'-dichlorofluorescein diacetate fluorimetric assay for reactive oxygen species were used. Cells were exposed to the following concentration of corrosion products: 5.0×, 1.0×, 0.5 and 0.1× during the period of 24, 48 and 72 h. To check the effect of each metal separately, cells were exposed to nickel-chloride and titanium-dioxide of corresponding concentration. The release of Ni is higher than of Ti (15.1-30.4 µg/L for Ni and 9.0-17.3 µg/L for Ti, respectively) and 5× higher concentrations are needed to induce cytotoxic effect. Ni and Ti ions alone do not induce a major cytotoxic effect, but their combination does indicating their synergistic effect. Increase in concentration of Ni and Ti tends to increase cytotoxicity, Ti more than Ni. Cytotoxicity and induction of free radicals are in strong positive linear correlation. Ions released from NiTi alloy during 14 days do not induce significant cytotoxic effect and would not have a clinically important impact. Cytotoxic effect is largely the result of the induction of free radicals.

Evaluation of antimutagenic and cytotoxic activity of skin secretion extract of Rhinella marina and Rhaebo guttatus (Anura, Bufonidae) / Avaliação da atividade antimutagênica e citotóxica da secreção cutânea de Rhinella marina e Rhaebo guttatus (Anura, Bufonidae)

The skin secretion from toads of the Bufonidae family has great potential in the search for new active compounds to be used as drug candidates in treating some diseases, among them cancer. In this context, this study aimed to evaluate the cytotoxic and antimutagenic activity of the parotoid gland secretion extracts of Rhinella marina and Rhaebo guttatus, as well as biochemically analyze transaminases and serum creatinine for liver and renal damage, respectively. Cytotoxicity was performed by the colorimetric method based on MTT (3- [4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide) with different concentrations of the extracts in Walker or splenic tumor cell cultures from rats and mice. The micronucleus test was performed with male Swiss mice treated orally with the extracts for 15 days, and then intraperitoneally with N-ethyl-N-nitrosurea (50 mg kg-1). Micronucleated polychromatic erythrocytes (MNPCE) were evaluated in bone marrow. The extracts showed cytotoxic activity in the evaluated cells. There was a significant reduction in the frequency of MNPCE (R. marina = 56% and R. guttatus = 75%, p < 0.001), indicating antimutagenic potential of the extracts. The groups treated only with extract showed an increase in MNPCE frequency, evidencing mutagenic potential. Biochemical analyzes showed no significant difference between treatments. Thus, under our experimental conditions, the extracts of R. marina and R. guttatus skin secretions presented chemopreventive potential for cancer. (AU)

A secreção cutânea de anuros da família Bufonidae tem grande potencial na busca de novos compostos ativos para utilização como fármacos candidatos no tratamento de algumas doenças, entre elas o câncer. Neste contexto, este estudo teve como objetivo avaliar a atividade citotóxica e antimutagênica dos extratos da secreção da glândula parótida de Rhinella marina e Rhaebo guttatus, bem como a análise bioquímica de transaminases e creatinina séricas, para avaliar dano hepático e renal, respectivamente. A avaliação de citotoxicidade foi realizada pelo método colorimétrico baseado no MTT (3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide), com diferentes concentrações dos extratos em culturas de células do Tumor de Walker ou células esplênicas de rato e camundongo. O teste do micronúcleo foi realizado com camundongos Swiss machos que receberam tratamento oral com os extratos durante 15 dias, seguido de tratamento intraperitoneal com N-etil-N-nitrosuréia (50 mg kg-1). A frequência de eritrócitos policromáticos micronucleados (PCEMN) foi determinada em medula óssea. Os extratos apresentaram ação citotóxica nas células avaliadas. Houve uma redução significativa na frequência de PCEMN (R. marina = 56% e R. guttatus = 75%, p < 0,001), observando-se um potencial antimutagênico dos extratos. Os grupos tratados somente com os extratos apresentaram um aumento na frequência de PCEMNs, evidenciando um potencial mutagênico. As análises bioquímicas não apresentaram diferença significativa entre os tratamentos. Assim, nas condições experimentais testadas, as secreções cutâneas de R. marina e R. guttatus apresentaram potencial quimiopreventivo para câncer.(AU)