In Vitro and in Vivo Assays of 3,5-Disubstituted-Tetrahydro-2H-1,3,5-Thiadiazin-2-Thione Derivatives against Trypanosoma cruzi

Cytotoxicity assays of 24 new 3,5-disubstituted-tetrahydro-2H-1,3,5-thiadiazin-2-thione derivatives were performed. The 17 compounds with higher anti-epimastigote activity and lower cytotoxicity were, thereafter, screened against amastigote of Trypanosoma cruzi. Out of these 17 derivatives S-2d was selected to be assayed in vivo, because of its remarkable trypanocidal properties. To determine toxicity against J774 macrophages, a method based on quantification of cell damage, after 24 h, was used. Cell respiration, an indicator of cell viability, was assessed by the reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to formazan. Anti-amastigote activity was estimated after 48 h by microscopic counts of May Grünwald-Giemsa-stained monolayers. Nifurtimox and benznidazole were used as reference drugs. For the in vivo experiences, mice were infected with 10(4) blood trypomastigotes and then treated during 15 days with S-2d or nifurtimox by oral route. All of the compounds were highly toxic at 100 µg/ml for macrophages and a few of them maintained this cytotoxicity even at 10 µg/ml. Of the derivatives assayed against amastigotes 3k and S-2d showed an interesting activity, that was held even at 1µg/ml. It is demonstrated that the high anti-epimastigote activity previously reported is mainly due to the non-specific toxicity of these compounds. In vivo assays assessed a reduction of parasitemia after administration of S-2d to infected mice.

In Vitro and in Vivo Anti-Trypanosoma cruzi Activity of a Novel Nitro-derivative

Nitroarylidenemalononitriles and their cyanoacetamide derivatives with remarkable anti-epimastigote properties, were synthesized attempting to obtain new 3,5-diamino-4-(5'-nitroarylidene)-4H-thiadiazine 1,1-dioxide derivatives, which in previous reports had shown anti-Trypanosoma cruzi activity. Tests to evaluate the cytotoxicity of compounds were performed on J774 macrophages. 5-nitro-2-thienyl-malononitrile (5NO2TM), was the only product which maintained a high anti-epimastigote activity at concentrations in which it was no longer cytotoxic, thus it was assayed against intracellular amastigotes. Its anti-amastigote activity was similar to that of nifurtimox. Afterwards in vivo toxicity and anti-chagasic activity were determined. A reduction in parasitemia was observed.

In vitro antiviral activity of antimicrobial peptides against herpes simplex virus 1, adenovirus, and rotavirus

Peptides with broad-spectrum antimicrobial activity, known as antimicrobial peptides, have been isolated from distinct organisms. This paper describes the in vitro evaluation of the cytotoxicity and antiviral activity of nine peptides with different structures and origins against herpes simplex virus type 1, human adenovirus respiratory strain, and rotavirus SA11. Most of the evaluated peptides presented antiviral activity but they were only active near cytotoxic concentrations. Nevertheless, these results seem promising, and further modifications on the peptide's structures may improve their selectivity and reduce their cytotoxicity.

In vitro anti-microbial activity of the Cuban medicinal plants Simarouba glauca DC, Melaleuca leucadendron L and Artemisia absinthium L

In the present study, an extensive in vitro antimicrobial profiling was performed for three medicinal plants grown in Cuba, namely Simarouba glauca, Melaleuca leucadendron and Artemisia absinthium. Ethanol extracts were tested for their antiprotozoal potential against Trypanosoma b. brucei, Trypanosoma cruzi, Leishmania infantum and Plasmodium falciparum. Antifungal activities were evaluated against Microsporum canis and Candida albicans whereas Escherichia coli and Staphylococcus aureus were used as test organisms for antibacterial activity. Cytotoxicity was assessed against human MRC-5 cells. Only M. leucadendron extract showed selective activity against microorganisms tested. Although S. glauca exhibited strong activity against all protozoa, it must be considered non-specific. The value of integrated evaluation of extracts with particular reference to selectivity is discussed.

In vitro antiplasmodial activity and toxicity assessment of plant extracts used in traditional malaria therapy in the Lake Victoria Region

As part of our program screening the flora of the Lake Victoria Region, a total of 54 organic extracts from seven plant families (8 species) were individually tested for antiplasmodial activity against chloroquine-sensitive [Sierra Leone (D-6)] and chloroquine-resistant [Vietnam (W-2)] strains. Only 22% of these extracts exhibited very high in vitro antiplasmodial activity. Six methanol (MeOH) extracts and one chloroform extract showed in vitro antiplasmodial activity against the D-6 Plasmodium falciparum strain, while only three MeOH extracts were active against the W-2 strain. All of the ethyl acetate extracts proved to be inactive against both strains of P. falciparum. A brine shrimp cytotoxicity assay was used to predict the potential toxicity of the extracts. The cytotoxicity to antiplasmodial ratios for the MeOH extracts were found to be greater than 100, which could indicate that the extracts are of low toxicity.

In vitro activity of thienyl-2-nitropropene compounds against Trypanosoma cruzi

The in vitro activity of four 2-nitropropene derivatives, 1-(3-benzothienyl)-2-nitropropene (N1), 1-(3-thienyl)-2-nitropropene (N2), 1-(5-bromo-2-thienyl)-2-nitropropene (N3) and 1-(4-bromo-2-thienyl)-2-nitropropene (N4), were tested against cultures of the parasite Trypanosoma cruzi. Cytotoxicity studies were performed using Vero cells. The blood trypomastigotes, amastigotes and epimastigotes showed differential degrees of sensitivity towards the four tested compounds; the highest activity against the epimastigotes and blood tripomastigotes was exhibited by N1, followed by N3, N4 and finally N2. In contrast, whereas the compounds N1, N3 and N4 exerted similar magnitudes of activity against amastigotes, N2 was found to be a much less potent compound. According to our results, the compound N1 had the highest level of activity (IC50: 0.6 μM) against epimastigotes.

Virucidal activity of Colombian Lippia essential oils on dengue virus replication in vitro

The inhibitory effect of Lippia alba and Lippia citriodora essential oils on dengue virus serotypes replication in vitro was investigated. The cytotoxicity (CC50) was evaluated by the MTT assay and the mode of viral inhibitory effect was investigated with a plaque reduction assay. The virus was treated with the essential oil for 2 h at 37ºC before cell adsorption and experiments were conducted to evaluate inhibition of untreated-virus replication in the presence of oil. Antiviral activity was defined as the concentration of essential oil that caused 50% reduction of the virus plaque number (IC50). L. alba oil resulted in less cytotoxicity than L. citriodora oil (CC50: 139.5 vs. 57.6 μg/mL). Virus plaque reduction for all four dengue serotypes was observed by treatment of the virus before adsorption on cell. The IC50 values for L. alba oil were between 0.4-32.6 μg/mL and between 1.9-33.7 μg/mL for L. citriodora oil. No viral inhibitory effect was observed by addition of the essential oil after virus adsorption. The inhibitory effect of the essential oil seems to cause direct virus inactivation before adsorption on host cell.

In vitro evaluation of new terpenoid derivatives against Leishmania infantum and Leishmania braziliensis

The activity of five (1-5) abietane phenol derivatives against Leishmania infantum and Leishmania braziliensis was studied using promastigotes and axenic and intracellular amastigotes. Infectivity and cytotoxicity tests were performed with J774.2 macrophage cells using Glucantime as a reference drug. The mechanisms of action were analysed by performing metabolite excretion and transmission electron microscopy ultrastructural studies. Compounds 1-5 were more active and less toxic than Glucantime. The infection rates and mean number of parasites per cell observed in amastigote experiments showed that derivatives 2, 4 and 5 were the most effective against both L. infantum and L. braziliensis. The ultrastructural changes observed in the treated promastigote forms confirmed that the greatest cell damage was caused by the most active compound (4). Only compound 5 caused changes in the nature and amounts of catabolites excreted by the parasites, as measured by ¹H nuclear magnetic resonance spectroscopy. All of the assayed compounds were active against the two Leishmania species in vitro and were less toxic in mammalian cells than the reference drug.

In vitro antimicrobial assessment of Cuban propolis extracts

Propolis is a resinous mixture of different plant exudates collected by honeybees. Currently, propolis is widely used as a food supplement and in folk medicine. We have evaluated 20 Cuban propolis extracts of different chemical types, brown (BCP), red and yellow (YCP), with respect to their in vitro antibacterial, antifungal and antiprotozoal properties. The extracts inhibited the growth of Staphylococcus aureus and Trichophyton rubrum at low µg/mL concentrations, whereas they were not active against Escherichia coli and Candida albicans. The major activity of the extracts was found against the protozoa Leishmania, Trypanosoma and Plasmodium, although cytotoxicity against MRC-5 cells was also observed. The BCP-3, YCP-39 and YCP-60 extracts showed the highest activity against P. falciparum, with 50% of microbial growth (IC50) values of 0.2 µg/mL. A positive correlation between the biological activity and the chemical composition was observed for YCP extracts. The most promising antimicrobial activity corresponds to YCP subtype B, which contains acetyl triterpenes as the main constituents. The present in vitro study highlights the potential of propolis against protozoa, but further research is needed to increase selectivity towards the parasite. The observed chemical composition-activity relationship of propolis can contribute to the identification of the active principles and standardisation of this bee product.

Blood shizonticidal activities of phenazines and naphthoquinoidal compounds against Plasmodium falciparum in vitro and in mice malaria studies

Due to the recent advances of atovaquone, a naphthoquinone, through clinical trials as treatment for malarial infection, 19 quinone derivatives with previously reported structures were also evaluated for blood schizonticide activity against the malaria parasite Plasmodium falciparum. These compounds include 2-hydroxy-3-methylamino naphthoquinones (2-9), lapachol (10), nor-lapachol (11), iso-lapachol (12), phthiocol (13) and phenazines (12-20). Their cytotoxicities were also evaluated against human hepatoma and normal monkey kidney cell lines. Compounds 2 and 5 showed the highest activity against P. falciparum chloroquine-resistant blood-stage parasites (clone W2), indicated by their low inhibitory concentration for 50% (IC50) of parasite growth. The therapeutic potential of the active compounds was evaluated according to the selectivity index, which is a ratio of the cytotoxicity minimum lethal dose which eliminates 50% of cells and the in vitro IC50. Naphthoquinones 2 and 5, with activities similar to the reference antimalarial chloroquine, were also active against malaria in mice and suppressed parasitaemia by more than 60% in contrast to compound 11 which was inactive. Based on their in vitro and in vivo activities, compounds 2 and 5 are considered promising molecules for antimalarial treatment and warrant further study.

In vitro activity of 1,3-bisaryloxypropanamines against Trypanosoma cruzi-infected L929 cultures

We describe herein the antitrypanosomal activity of 20 novel 1,3-bis(aryloxy)propan-2-amine derivatives. Compounds 2, 4, 6, 12, 15, 16 and 19 were significantly active against amastigote and trypomastigote forms, with half maximal inhibitory concentrationvalues in the range of 6-18 µM. In the cytotoxicity tests against L929 cells, only compound 4 presented selectivity index value above 10, indicating low toxicity.

Neuronal in vitro models for the estimation of acute systemic toxicity.

The objective of the EU funded integrated project "ACuteTox" is to develop a strategy in which general cytotoxicity, together with organ-specific endpoints and biokinetic features, are taken into consideration in the in vitro prediction of oral acute systemic toxicity. With regard to the nervous system, the effects of 23 reference chemicals were tested with approximately 50 endpoints, using a neuronal cell line, primary neuronal cell cultures, brain slices and aggregated brain cell cultures. Comparison of the in vitro neurotoxicity data with general cytotoxicity data generated in a non-neuronal cell line and with in vivo data such as acute human lethal blood concentration, revealed that GABA(A) receptor function, acetylcholine esterase activity, cell membrane potential, glucose uptake, total RNA expression and altered gene expression of NF-H, GFAP, MBP, HSP32 and caspase-3 were the best endpoints to use for further testing with 36 additional chemicals. The results of the second analysis showed that no single neuronal endpoint could give a perfect improvement in the in vitro-in vivo correlation, indicating that several specific endpoints need to be analysed and combined with biokinetic data to obtain the best correlation with in vivo acute toxicity.

Lead acetate toxicity in vitro: Dependence on the cell composition of the cultures.

It is well known that exposure to low doses of lead causes long-lasting neurobehavioural deficits, but the cellular changes underlying these behavioural changes remain to be elucidated. A protective role of glial cells on neurons through lead sequestration by astrocytes has been proposed. The possible modulation of lead neurotoxicity by neuron-glia interactions was examined in three-dimensional cultures of foetal rat telencephalon. Mixed-brain cell cultures or cultures enriched in either neurons or glial cells were treated for 10 days with lead acetate (10(-6) m), a concentration below the limit of cytotoxicity. Intracellular lead content and cell type-specific enzyme activities were determined. It was found that in enriched cultures neurons stored more lead than glial cells, and each cell type alone stored more lead than in co-culture. Moreover, glial cells but not neurons were more affected by lead in enriched culture than in co-culture. These results show that neuron-glia interactions attenuate the cellular lead uptake and the glial susceptibility to lead, but they do not support the idea of a protective role of astrocytes.

Enhanced generation of specific tumor-reactive CTL in vitro by selected Melan-A/MART-1 immunodominant peptide analogues.

The Melan-A/MART-1 gene, which is expressed by normal melanocytes as well as by most fresh melanoma samples and melanoma cell lines, codes for Ags recognized by tumor-reactive CTL. HLA-A*0201-restricted Melan-A-specific CTL recognize primarily the Melan-A(27-35) (AAGIGILTV) and the Melan-A(26-35) (EAAGIGILTV) peptides. The sequences of these two peptides are not necessarily optimal as far as binding to HLA-A*0201 is concerned, since both lack one of the dominant anchor amino acid residues (leucine or methionine) at position 2. In this study we introduced single amino acid substitutions in either one of the two natural peptide sequences with the aim of improving peptide binding to HLA-A*0201 and/or recognition by specific CTL. Surprisingly, analogues of the Melan-A(27-35) peptide, which bound more efficiently than the natural nonapeptide to HLA-A*0201, were poorly recognized by tumor-reactive CTL. In contrast, among the Melan-A(26-35) peptide analogues tested, the peptide ELAGIGILTV was not only able to display stable binding to HLA-A2.1 but was also recognized more efficiently than the natural peptide by two short-term cultured tumor-infiltrated lymph node cell cultures as well as by five of five tumor-reactive CTL clones. Moreover, in vitro generation of tumor-reactive CTL by stimulation of PBMC from HLA-A*0201 melanoma patients with this particular peptide analogue was much more efficient than that observed with either one of the two natural peptides. These results suggest that the Melan-A(26-35) peptide analogue ELAGIGILTV may be more immunogenic than the natural peptides in HLA-A*0201 melanoma patients and should thus be considered as a candidate for future peptide-based vaccine trials.

Toxic effects of brake wear particles on epithelial lung cells in vitro

Background: Fine particulate matter originating from traffic correlates with increased morbidity and mortality. An important source of traffic particles is brake wear of cars which contributes up to 20% of the total traffic emissions. The aim of this study was to evaluate potential toxicological effects of human epithelial lung cells exposed to freshly generated brake wear particles. Results: An exposure box was mounted around a car's braking system. Lung cells cultured at the air-liquid interface were then exposed to particles emitted from two typical braking behaviours ("full stop" and "normal deceleration"). The particle size distribution as well as the brake emission components like metals and carbons was measured on-line, and the particles deposited on grids for transmission electron microscopy were counted. The tight junction arrangement was observed by laser scanning microscopy. Cellular responses were assessed by measurement of lactate dehydrogenase (cytotoxicity), by investigating the production of reactive oxidative species and the release of the pro-inflammatory mediator interleukin-8. The tight junction protein occludin density decreased significantly (p < 0.05) with increasing concentrations of metals on the particles (iron, copper and manganese, which were all strongly correlated with each other). Occludin was also negatively correlated with the intensity of reactive oxidative species. The concentrations of interleukin-8 were significantly correlated with increasing organic carbon concentrations. No correlation was observed between occludin and interleukin-8, nor between reactive oxidative species and interleukin-8. Conclusion: These findings suggest that the metals on brake wear particles damage tight junctions with a mechanism involving oxidative stress. Brake wear particles also increase pro-inflammatory responses. However, this might be due to another mechanism than via oxidative stress. [Authors]