Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part II: experimental studies withAspidosperma ramiflorum in vivo and in vitro

Several species of Aspidosperma plants are used to treat diseases in the tropics, including Aspidosperma ramiflorum, which acts against leishmaniasis, an activity that is experimentally confirmed. The species, known as guatambu-yellow, yellowperoba, coffee-peroba andmatiambu, grows in the Atlantic Forest of Brazil in the South to the Southeast regions. Through a guided biofractionation of A. ramiflorum extracts, the plant activity against Plasmodium falciparum was evaluated in vitro for toxicity towards human hepatoma G2 cells, normal monkey kidney cells and nonimmortalised human monocytes isolated from peripheral blood. Six of the seven extracts tested were active at low doses (half-maximal drug inhibitory concentration < 3.8 µg/mL); the aqueous extract was inactive. Overall, the plant extracts and the purified compounds displayed low toxicity in vitro. A nonsoluble extract fraction and one purified alkaloid isositsirikine (compound 5) displayed high selectivity indexes (SI) (= 56 and 113, respectively), whereas compounds 2 and 3 were toxic (SI < 10). The structure, activity and low toxicity of isositsirikine in vitro are described here for the first time in A. ramiflorum, but only the neutral and precipitate plant fractions were tested for activity, which caused up to 53% parasitaemia inhibition of Plasmodium bergheiin mice with blood-induced malaria. This plant species is likely to be useful in the further development of an antimalarial drug, but its pharmacological evaluation is still required.

Estrogenic activity assessment of environmental chemicals using in vitro assays: identification of two new estrogenic compounds.

Environmental chemicals with estrogenic activities have been suggested to be associated with deleterious effects in animals and humans. To characterize estrogenic chemicals and their mechanisms of action, we established in vitro and cell culture assays that detect human estrogen receptor [alpha] (hER[alpha])-mediated estrogenicity. First, we assayed chemicals to determine their ability to modulate direct interaction between the hER[alpha] and the steroid receptor coactivator-1 (SRC-1) and in a competition binding assay to displace 17ss-estradiol (E(2)). Second, we tested the chemicals for estrogen-associated transcriptional activity in the yeast estrogen screen and in the estrogen-responsive MCF-7 human breast cancer cell line. The chemicals investigated in this study were o,p'-DDT (racemic mixture and enantiomers), nonylphenol mixture (NPm), and two poorly analyzed compounds in the environment, namely, tris-4-(chlorophenyl)methane (Tris-H) and tris-4-(chlorophenyl)methanol (Tris-OH). In both yeast and MCF-7 cells, we determined estrogenic activity via the estrogen receptor (ER) for o,p'-DDT, NPm, and for the very first time, Tris-H and Tris-OH. However, unlike estrogens, none of these xenobiotics seemed to be able to induce ER/SRC-1 interactions, most likely because the conformation of the activated receptor would not allow direct contacts with this coactivator. However, these compounds were able to inhibit [(3)H]-E(2) binding to hER, which reveals a direct interaction with the receptor. In conclusion, the test compounds are estrogen mimics, but their molecular mechanism of action appears to be different from that of the natural hormone as revealed by the receptor/coactivator interaction analysis.

Cytotoxic effects of FGF2-saporin on bovine epithelial lens cells in vitro.

PURPOSE: To test the ability of two preparations of FGF2-saporin, either FGF2 chemically conjugated to saporin (FGF2-SAP) or genetically engineered FGF2-saporin (rFGF2-SAP) to inhibit the growth of bovine epithelial lens (BEL) cells in vitro when in solution and when immobilized on heparin surface-modified (HSM) polymethylmethacrylate (PMMA) intraocular lenses (IOLs). METHOD: Bovine epithelial lens cells were incubated with various concentrations FGF2-saporin for as long as 4 days. The number of surviving cells was determined by counting the number of nuclei. Because FGF2 binds to heparin, FGF2-saporin was incubated with HSM PMMA IOLs; excess toxin was washed off, and the BEL cells were grown on the FGF2-saporin-treated IOLs (HSM and non-HSM) for 4 days. Cell density was determined by image analysis. RESULTS: Both FGF2-SAP and rFGF2-SAP were highly cytotoxic (nM range), with rFGF2-SAP 10 times less active than FGF2-SAP. FGF2-saporin bound to the surface of HSM IOLs and eluted by 2M NaCl retained its activity. Toxin bound to HSM IOLs killed more than 90% of the BEL cells placed on the IOL surface within 4 days. The ability of FGF2-saporin to prevent the growth of cells on the IOL surface was strictly dependent on the presence of heparin on the IOL. CONCLUSIONS: FGF2-saporin is bound to HSM PMMA IOLs and prevents the growth of epithelial cells on the surface of the lens.

In vitro and in vivo tumor models for studies of distribution of radiolabelled monoclonal antibodies and fragments.

Colon carcinoma multicellular spheroids were incubated in vitro with radiolabelled MAbs. The more rapid penetration of fragments as compared to intact MAbs was clearly demonstrated. For the study of antibody localization in tumors in vivo, the model of nude mice with ligated kidneys was used. Although very artificial, this model allowed to demonstrate that, without urinary excretion, Fab fragments accumulated more rapidly into the tumor than intact MAbs and disappeared faster from the blood. This difference was less striking for F(ab')2 fragments. In the liver a decreased accumulation of both types of fragments as compared to intact MAbs was observed. Concerning radioimmunotherapy we think that Fab fragments are not useful because of their too short half-life in the circulation and in tumor and because they will probably be too toxic for the kidneys. Intact MAbs and F(ab')2 fragments have each their advantages. Intact MAbs show highest tumor accumulation in mice without ligated kidney, however, they remain mostly on the periphery of tumor nodules, as shown by autoradiography. F(ab')2 fragments have been found to penetrate deeper into the tumor and to accumulate less in the liver. It might be therefore an advantage to combine intact MAbs with F(ab')2 fragments, so that in the tumor two different regions could be attacked whereas in normal tissues toxicity could be distributed to different organs such as to the liver with intact MAbs and to the kidney with F(ab')2 fragments.

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]

Contribution of in vitro neurotoxicology studies to the elucidation of neurodegenerative processes.

Only a small percentage of neurodegenerative diseases like Alzheimer's disease and Parkinson's disease is directly related to familial forms. The etiology of the most abundant, sporadic forms seems to involve both genetic and environmental factors. Environmental compounds are now extensively studied for their possible contribution to neurodegeneration. Chemicals were found which were able to reproduce symptoms of known neurodegenerative diseases, others may either predispose to the onset of neurodegeneration, or exacerbate distinct pathogenic processes of these diseases. In any case, in vitro studies performed with models presenting various degrees of complexity have shown that many environmental compounds have the potential to cause neurodegeneration, through a variety of pathways similar to those described in neurodegenerative diseases. Since the population is exposed to a huge number of potentially neurotoxic compounds, there is an important need for rapid and efficient procedures for hazard evaluation. Xenobiotics elicit a cascade of reactions that, most of the time, involve numerous interactions between the different brain cell types. A reliable in vitro model for the detection of environmental toxins potentially at risk for neurodegenerative diseases should therefore allow maximal cell-cell interactions and multiparametric endpoints determination. The combined use of in vitro models and new analytical approaches using "omics" technologies should help to map toxicity pathways, and advance our understanding of the possible role of xenobiotics in the etiology of neurodegenerative diseases.

Evaluation in vitro de la réactivité des particules fines et ultrafines : rapport scientifique final

Une des percées les plus importantes dans la recherche sur les nanoparticules (ambiantes et manufacturées) a été la reconnaissance de leur potentiel à générer un stress oxydatif au niveau cellulaire. Dans cette optique, la mesure du potentiel oxydant intrinsèque des particules pourrait présenter une première étape dans l'évaluation des dangers. Ce projet méthodologique avait pour but de caractériser le potentiel oxydant de différentes nanoparticules « modèles » (ambiantes et manufacturées) au moyen de trois tests acellulaires (Test DTT, Test DCFH, Test oxymétrique) et d'utiliser ces résultats pour proposer une méthode de « référence ». D'autre part, nous avons appliqué la méthode sélectionnée à deux cas (exposition d'ouvriers à des particules de combustion et évaluation du danger de différentes nanoparticules manufacturées) afin de déterminer quels sont les paramètres qui influencent la mesure. Les résultats obtenus indiquent que la préparation des suspensions joue un rôle dans la mesure de ce potentiel oxydant. La réactivité dépend de la concentration du surfactant et de la durée de sonication. D'autre part, l'ordre de réactivité est dépendant de la métrique utilisée (masse ou surface) pour exprimer les résultats. Parmi les trois tests considérés, le test DTT pourrait être le plus utile pour effectuer une évaluation initiale du danger potentiel de nanoparticules ambiantes ou manufacturées. Ce test pourrait être intégré dans une stratégie d'évaluation de la toxicité des nanoparticules. Le test DTT correspond bien un test intégratif. Pour des situations de travail dans lesquelles les particules de combustion sont majoritaires, les paramètres physico-chimiques qui corrèlent de manière significative avec la réactivité DTT sont la surface des particules, les concentrations de carbone organique, la somme des concentrations de quatre quinones et les concentrations de fer et cuivre. Un nombre plus faible de corrélations est observé dans des ateliers mécaniques, suggérant que d'autres composés non mesurés interviennent également dans cette réactivité. Concernant les nanoparticules carbonées manufacturées, les fonctions chimiques de surface corrélées avec la réactivité DTT sont des fonctions acides et des fonctions inconnues pouvant dismuter. D'autre part, la solubilité et la possibilité de former des complexes avec le DTT sur la surface des NP manufacturées influencent le résultat de réactivité.

Workgroup report: incorporating in vitro alternative methods for developmental neurotoxicity into international hazard and risk assessment strategies.

This is the report of the first workshop on Incorporating In Vitro Alternative Methods for Developmental Neurotoxicity (DNT) Testing into International Hazard and Risk Assessment Strategies, held in Ispra, Italy, on 19-21 April 2005. The workshop was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) and jointly organized by ECVAM, the European Chemical Industry Council, and the Johns Hopkins University Center for Alternatives to Animal Testing. The primary aim of the workshop was to identify and catalog potential methods that could be used to assess how data from in vitro alternative methods could help to predict and identify DNT hazards. Working groups focused on two different aspects: a) details on the science available in the field of DNT, including discussions on the models available to capture the critical DNT mechanisms and processes, and b) policy and strategy aspects to assess the integration of alternative methods in a regulatory framework. This report summarizes these discussions and details the recommendations and priorities for future work.

Pharmacokinetic and pharmacodynamic analysis of efavirenz dose reduction using an in vitro-in vivo extrapolation model.

The pharmacokinetics (PK) of efavirenz (EFV) is characterized by marked interpatient variability that correlates with its pharmacodynamics (PD). In vitro-in vivo extrapolation (IVIVE) is a "bottom-up" approach that combines drug data with system information to predict PK and PD. The aim of this study was to simulate EFV PK and PD after dose reductions. At the standard dose, the simulated probability was 80% for viral suppression and 28% for central nervous system (CNS) toxicity. After a dose reduction to 400 mg, the probabilities of viral suppression were reduced to 69, 75, and 82%, and those of CNS toxicity were 21, 24, and 29% for the 516 GG, 516 GT, and 516 TT genotypes, respectively. With reduction of the dose to 200 mg, the probabilities of viral suppression decreased to 54, 62, and 72% and those of CNS toxicity decreased to 13, 18, and 20% for the 516 GG, 516 GT, and 516 TT genotypes, respectively. These findings indicate how dose reductions might be applied in patients with favorable genetic characteristics.

Drug-eluting beads loaded with antiangiogenic agents for chemoembolization: in vitro sunitinib loading and release and in vivo pharmacokinetics in an animal model.

PURPOSE: The combination of embolic beads with a multitargeted tyrosine kinase inhibitor that inhibits tumor vessel growth is suggested as an alternative and improvement to the current standard doxorubicin-eluting beads for use in transarterial chemoembolization. This study demonstrates the in vitro loading and release kinetics of sunitinib using commercially available embolization microspheres and evaluates the in vitro biologic efficacy on cell cultures and the resulting in vivo pharmacokinetics profiles in an animal model. MATERIALS AND METHODS: DC Bead microspheres, 70-150 µm and 100-300 µm (Biocompatibles Ltd., Farnham, United Kingdom), were loaded by immersion in sunitinib solution. Drug release was measured in saline in a USP-approved flow-through apparatus and quantified by spectrophotometry. Activity after release was confirmed in cell culture. For pharmacokinetics and in vivo toxicity evaluation, New Zealand white rabbits received sunitinib either by intraarterial injection of 100-300 µm sized beads or per os. Plasma and liver tissue drug concentrations were assessed by liquid chromatography-tandem mass spectroscopy. RESULTS: Sunitinib loading on beads was close to complete and homogeneous. A total release of 80% in saline was measured, with similar fast-release profiles for both sphere sizes. After embolization, drug plasma levels remained below the therapeutic threshold (< 50 ng/mL), but high concentrations at 6 hours (14.9 µg/g) and 24 hours (3.4 µg/g) were found in the liver tissue. CONCLUSIONS: DC Bead microspheres of two sizes were efficiently loaded with sunitinib and displayed a fast and almost complete release in saline. High liver drug concentrations and low systemic levels indicated the potential of sunitinib-eluting beads for use in embolization.

Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro.

Doxorubicin (DOX) is a potent available antitumor agent; however, its clinical use is limited because of its cardiotoxicity. Cell death is a key component in DOX-induced cardiotoxicity, but its mechanisms are elusive. Here, we explore the role of superoxide, nitric oxide (NO), and peroxynitrite in DOX-induced cell death using both in vivo and in vitro models of cardiotoxicity. Western blot analysis, real-time PCR, immunohistochemistry, flow cytometry, fluorescent microscopy, and biochemical assays were used to determine the markers of apoptosis/necrosis and sources of NO and superoxide and their production. Left ventricular function was measured by a pressure-volume system. We demonstrated increases in myocardial apoptosis (caspase-3 cleavage/activity, cytochrome c release, and TUNEL), inducible NO synthase (iNOS) expression, mitochondrial superoxide generation, 3-nitrotyrosine (NT) formation, matrix metalloproteinase (MMP)-2/MMP-9 gene expression, poly(ADP-ribose) polymerase activation [without major changes in NAD(P)H oxidase isoform 1, NAD(P)H oxidase isoform 2, p22(phox), p40(phox), p47(phox), p67(phox), xanthine oxidase, endothelial NOS, and neuronal NOS expression] and decreases in myocardial contractility, catalase, and glutathione peroxidase activities 5 days after DOX treatment to mice. All these effects of DOX were markedly attenuated by peroxynitrite scavengers. Doxorubicin dose dependently increased mitochondrial superoxide and NT generation and apoptosis/necrosis in cardiac-derived H9c2 cells. DOX- or peroxynitrite-induced apoptosis/necrosis positively correlated with intracellular NT formation and could be abolished by peroxynitrite scavengers. DOX-induced cell death and NT formation were also attenuated by selective iNOS inhibitors or in iNOS knockout mice. Various NO donors when coadministered with DOX but not alone dramatically enhanced DOX-induced cell death with concomitant increased NT formation. DOX-induced cell death was also attenuated by cell-permeable SOD but not by cell-permeable catalase, the xanthine oxidase inhibitor allopurinol, or the NADPH oxidase inhibitors apocynine or diphenylene iodonium. Thus, peroxynitrite is a major trigger of DOX-induced cell death both in vivo and in vivo, and the modulation of the pathways leading to its generation or its effective neutralization can be of significant therapeutic benefit.

In Vitro Activity of Gentamicin-Loaded Bioabsorbable Beads against Different Microorganisms

Abstract: Osteomyelitis is responsible for high treatment costs, long hospital stays, and results in substantial morbidity. Treatment with surgical debridement and antibiotic-impregnated Polymethylmetacrylate (PMMA) beads is the standard of care, providing high local but low serum antibiotic concentrations, thereby avoiding systemic toxicity. However, for several reasons, the beads require surgical removal. Alternative antibiotic delivery systems should improve the treatment of bone infection, actively encourage bone healing and require no additional surgery for removal. We investigated the activity of gentamicin-loaded bioabsorbable beads against different microorganisms (Staphylococcus epidermidis, S. aureus, Escherichia coli, Enterococcus faecalis, Candida albicans) commonly causing surgical site bone infection, by microcalorimetry. Calcium sulphate beads containing gentamicin were incubated in microcalorimetry ampoules containing different concentrations of the corresponding microorganism. Growth medium with each germ and unloaded beads was used as positive control, growth medium with loaded beads alone as negative control. Bacterial growth-related heat production at 37 °C was measured for 24 h. Cultures without gentamicin-loaded beads produced heat-flow peaks corresponding to the exponential growth of the corresponding microorganisms in nutrient-rich medium. In contrast, cultures with gentamicin-loaded beads completely suppressed heat production during 24 h, demonstrating their antibiotic activity. Gentamicin-loaded beads effectively inhibited growth of susceptible microorganisms, under the described in vitro conditions.

Comparative sensitivity of tumor and non-tumor cell lines as reliable approach for in vitro cytotoxicity screening of lysine-based surfactants with potential pharmaceutical applications

Surfactants are used as additives in topical pharmaceuticals and drug delivery systems. The biocompatibility of amino acid-based surfactants makes them highly suitable for use in these fields, but tests are needed to evaluate their potential toxicity. Here we addressed the sensitivity of tumor (HeLa, MCF-7) and non-tumor (3T3, 3T6, HaCaT, NCTC 2544) cell lines to the toxic effects of lysine-based surfactants by means of two in vitro endpoints (MTT and NRU). This comparative assay may serve as a reliable approach for predictive toxicity screening of chemicals prior to pharmaceutical applications. After 24-h of cell exposure to surfactants, differing toxic responses were observed. NCTC 2544 and 3T6 cell lines were the most sensitive, while both tumor cells and 3T3 fibroblasts were more resistant to the cytotoxic effects of surfactants. IC50-values revealed that cytotoxicity was detected earlier by MTT assay than by NRU assay, regardless of the compound or cell line. The overall results showed that surfactants with organic counterions were less cytotoxic than those with inorganic counterions. Our findings highlight the relevance of the correct choice and combination of cell lines and bioassays in toxicity studies for a safe and reliable screen of chemicals with potential interest in pharmaceutical industry.

Impaired uptake of glutathione by hepatic mitochondria from chronic ethanol-fed rats. Tracer kinetic studies in vitro and in vivo and susceptibility to oxidant stress.

Isolated hepatocytes incubated with [35S]-methionine were examined for the time-dependent accumulation of [35S]-glutathione (GSH) in cytosol and mitochondria, the latter confirmed by density gradient purification. In GSH-depleted and -repleted hepatocytes, the increase of specific activity of mitochondrial GSH lagged behind cytosol, reaching nearly the same specific activity by 1-2 h. However, in hepatocytes from ethanol-fed rats, the rate of increase of total GSH specific radioactivity in mitochondria was markedly suppressed. In in vivo steady-state experiments, the mass transport of GSH from cytosol to mitochondria and vice versa was 18 nmol/min per g liver, indicating that the half-life of mitochondrial GSH was approximately 18 min in controls. The fractional transport rate of GSH from cytosol to mitochondria, but not mitochondria to cytosol, was significantly reduced in the livers of ethanol-fed rats. Thus, ethanol-fed rats exhibit a decreased mitochondrial GSH pool size due to an impaired entry of cytosol GSH into mitochondria. Hepatocytes from ethanol-fed rats exhibited a greater susceptibility to the oxidant stress-induced cell death from tert-butylhydroperoxide. Incubation with glutathione monoethyl ester normalized the mitochondrial GSH and protected against the increased susceptibility to t-butylhydroperoxide, which was directly related to the lowered mitochondrial GSH pool size in ethanol-fed cells.