Selection of cytotoxicity markers for the screening of new chemical entities in a pharmaceutical context: A preliminary study using a multiplexing approach

Gerets, Helga; Hanon, Étienne; Cornet, Miranda; Dhalluin, Stéphane; Depelchin, Olympe; Canning, Michael; Atienzar, Franck

doi:10.1016/j.tiv.2008.11.012

Cited by 67 publications

(47 citation statements)

References 26 publications

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“…The average assay window for ELEC was 60.5 and the average Z= value was 0.76, indicating that this system is adequate to be used for hit identification in a medium-throughput format (26). Eighty-five percent (101/119) of the primary hits were confirmed to be true hits by means of EC 50 determinations (Table 5), with some of them (n ϭ 19) showing efficacy at a concentration of Ͻ1 M. In parallel to the determination of antileishmanial activity, we quantified the level of cellular cytotoxicity of the hits using an established dose response assay in a human hepatocyte cell line (27,28,41) to calculate the concentration of the compound that killed 50% of the cells (CC 50 ). Taking the ratio of the cytotoxicity to the antileishmanial activity, we calculated the in vitro therapeutic index (IVTI ϭ CC 50 /EC 50 ) as an estimate of potential drug efficacy.…”

Section: Resultsmentioning

confidence: 99%

“…We used a cellbased assay as an alternative to animal testing to determine the toxicity of the selected compounds (27,28). The cytotoxicity was evaluated in HepG2 cells (human hepatocellular carcinoma; ATCC HB-8065) maintained in minimum essential medium (MEM) (Gibco) supplemented with 5% heat-inactivated FBS, 1 mM sodium pyruvate (Gibco), and 1ϫ MEM amino acid solution (Sigma).…”

Section: Assessment Of Cell Toxicity (50% Cytotoxicity Concentration mentioning

confidence: 99%

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Development of anEx VivoLymph Node Explant Model for Identification of Novel Molecules Active against Leishmania major

Peniche

Osorio

Renslo

et al. 2014

Antimicrob Agents Chemother

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e Leishmaniasis is a vector-borne zoonotic infection affecting people in tropical and subtropical regions of the world. Current treatments for cutaneous leishmaniasis are difficult to administer, toxic, expensive, and limited in effectiveness and availability. Here we describe the development and application of a medium-throughput screening approach to identify new drug candidates for cutaneous leishmaniasis using an ex vivo lymph node explant culture (ELEC) derived from the draining lymph nodes of Leishmania major-infected mice. The ELEC supported intracellular amastigote proliferation and contained lymph node cell populations (and their secreted products) that enabled the testing of compounds within a system that mimicked the immunopathological environment of the infected host, which is known to profoundly influence parasite replication, killing, and drug efficacy. The activity of known antileishmanial drugs in the ELEC system was similar to the activity measured in peritoneal macrophages infected in vitro with L. major. Using the ELEC system, we screened a collection of 334 compounds, some of which we had demonstrated previously to be active against L. donovani, and identified 119 hits, 85% of which were confirmed to be active by determination of the 50% effective concentration (EC 50 ). We found 24 compounds (7%) that had an in vitro therapeutic index (IVTI; 50% cytotoxic/effective concentration [CC 50 ]/EC 50 ) > 100; 19 of the compounds had an EC 50 below 1 M. According to PubChem searchs, 17 of those compounds had not previously been reported to be active against Leishmania. We expect that this novel method will help to accelerate discovery of new drug candidates for treatment of cutaneous leishmaniasis.

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Section: Resultsmentioning

confidence: 99%

Section: Assessment Of Cell Toxicity (50% Cytotoxicity Concentration mentioning

confidence: 99%

Development of anEx VivoLymph Node Explant Model for Identification of Novel Molecules Active against Leishmania major

Peniche

Osorio

Renslo

et al. 2014

Antimicrob Agents Chemother

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“…We also measured caspase-3/7 activity at 24 hr after treatment with the compounds as an index of apoptosis. Although caspase-3/7 activity has been measured after short-term treatment with compounds in some reports (Gerets et al, 2009), we employed caspase-3/7 activity after 24 hr treatment with compounds to detect apoptosis induced by mitochondrial dysfunction through various mechanisms including not only direct and rapid suppression of the mitochondrial respiration but also other mechanisms. The results of the multiple assays in this report are summarized in Table 1.…”

Section: Discussionmentioning

confidence: 99%

Usefulness of <i>in vitro</i> combination assays of mitochondrial dysfunction and apoptosis for the estimation of potential risk of idiosyncratic drug induced liver injury

et al. 2016

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-Drug-induced liver injury (DILI) is one of the serious and frequent drug-related adverse events. This adverse event is a main reason for regulatory action pertaining to drugs, including restrictions in clinical indications and withdrawal from clinical trials or the marketplace. Idiosyncratic DILI especially has become a major clinical concern because of its unpredictable nature, frequent hospitalization, need for liver transplantation and high mortality. The estimation of the potential for compounds to induce idiosyncratic DILI is very difficult in non-clinical studies because the precise mechanism of idiosyncratic DILI is still unknown. Recently, many in vitro assays which indicate a possibility of the prediction of the idiosyncratic DILI have been reported. Among these, some in vitro assays focus on the effects of compounds on mitochondrial function and the apoptotic effects of compounds on human hepatocytes. In this study, we measured oxygen consumption rate (OCR) and caspase-3/7 activity as an endpoint of mitochondrial dysfunction and apoptosis, respectively, with human hepatocytes after treatment with compounds causing idiosyncratic DILI (troglitazone, leflunomide, ranitidine and diclofenac). Troglitazone and leflunomide decreased the OCR but did not affect caspase-3/7 activity. Ranitidine increased caspase-3/7 activity but did not affect the OCR. Diclofenac decreased the OCR and increased caspase-3/7 activity. Acetaminophen and ethanol, which are also hepatotoxicants but do not induce idiosyncratic DILI, did not affect the OCR or caspase-3/7 activity. These results indicate that a combination assay of mitochondrial dysfunction and apoptosis is useful for the estimation of potential risk of compounds to induce idiosyncratic DILI.

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“…Assuming that toxic effects seen in a whole organism are due to prior failure of basic cellular function, cytotoxicity studies offer a good source of information about the mechanism of toxicity (8), especially if a battery of tests is used (9,11,12,21).…”

Section: Introductionmentioning

confidence: 99%

Cytotoxic effects of the synthetic oestrogens and androgens on Balb/c 3T3 and HepG2 cells

Minta

Radko

Stypuła-Trębas

et al. 2014

Bulletin of the Veterinary Institute in Pulawy

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The aim of the study was to test and compare the cytotoxic potential of two synthetic oestrogens: diethylstilboestrol (DES) and ethinyloestradiol (EE2) and two androgens: testosterone propionate (TP) and trenbolone (TREN) on two cell lines. The fibroblast cell line Balb/c 3T3 and the hepatoma cell line HepG2 were selected. To get more insight into the mode of toxic action, four methods were used, which evaluated different biochemical endpoints: mitochondrial activity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay), lysosomal activity (neutral red uptake assay), total protein content, and lactate dehydrogenase release. Cytotoxicity was assessed after 24, 48, and 72 h exposure to eight concentrations ranging from 0.78 to 100 µg/mL. Concentration-and time-dependent effects were observed. Depending on the line and assay used, half maximal effective concentration after 72 h (EC50-72h) values ranged as follows: DES 1-13.7 µg/mL (Balb/c 3T3) and 3.7-5.2 µg/mL (HepG2); EE2 2.1-14.3 µg/mL (Balb/c 3T3) and 1.8-7.8 µg/mL (HepG2); TP-14.9-17.5 µg/mL (Balb/c 3T3), and 63.9-100 µg/mL (HepG2); and TREN 11.3-31.4 µg/mL (Balb/c 3T3) and 12.5-59.4 µg/mL (HepG2). The results revealed that oestrogens were more toxic than androgens and the most affected endpoint was mitochondrial activity. In contrast to oestrogens, for which EC50-72h values were similar in both lines and by all assays used, Balb/c 3T3 cells were more sensitive than HepG2 cells to TP.

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Selection of cytotoxicity markers for the screening of new chemical entities in a pharmaceutical context: A preliminary study using a multiplexing approach

Cited by 67 publications

References 26 publications

Development of anEx VivoLymph Node Explant Model for Identification of Novel Molecules Active against Leishmania major

Development of anEx VivoLymph Node Explant Model for Identification of Novel Molecules Active against Leishmania major

Usefulness of <i>in vitro</i> combination assays of mitochondrial dysfunction and apoptosis for the estimation of potential risk of idiosyncratic drug induced liver injury

Cytotoxic effects of the synthetic oestrogens and androgens on Balb/c 3T3 and HepG2 cells

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