A high-throughput cell-based toxicity analysis of drug metabolites using flow cytometry

Buenz, Eric J.

doi:10.1007/s10565-007-0226-1

Cited by 11 publications

(9 citation statements)

References 16 publications

(23 reference statements)

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“…Trophozoites of E. histolytica strain HM1:IMSS and G. lamblia strain WB were axenically maintained in TYI-S-33 medium supplemented with penicillin (100 U/ml) and streptomycin (100 g/ml) (38). Jurkat T lymphocytes (clone E6-1) were maintained in RPMI 1640 medium with HEPES, supplemented with penicillin (100 U/ml), streptomycin (100 g/ml), and 10% heat-inactivated fetal bovine serum, and cultured according to ATCC specifications (ATCC, Manassas, VA) (39). E. histolytica and G. lamblia trophozoites and Jurkat cells were counted using a particle counter (Beckman Coulter, Fullerton, CA).…”

Section: Methodsmentioning

confidence: 99%

“…We found that SNX-2112, SMDC-304198, SMDC-304201, SMDC-304203, SMDC-304205, and SMDC-304209 were active against both E. histolytica and G. lamblia, with EC 50 s better than those of metronidazole (Table 1). SMDC-304200 failed to show potency, possibly due to the presence of a (39). No cytotoxicity was seen at inhibitor concentrations of Ͼ100 M for 48 h ( Table 1).…”

Section: Effect Of Hsp90 Inhibitors Against E Histolytica G Lamblimentioning

confidence: 99%

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Hsp90 Inhibitors as New Leads To Target Parasitic Diarrheal Diseases

Debnath

Shahinas

Bryant

et al. 2014

Antimicrob Agents Chemother

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Entamoeba histolytica and Giardia lamblia are anaerobic protozoan parasites that cause amebiasis and giardiasis, two of the most common diarrheal diseases worldwide. Current therapy relies on metronidazole, but resistance has been reported and the drug has significant adverse effects. Therefore, it is critical to search for effective, better-tolerated antiamebic and antigiardial drugs. We synthesized several examples of a recently reported class of Hsp90 inhibitors and evaluated these compounds as potential leads for antiparasitic chemotherapy. Several of these inhibitors showed strong in vitro activity against both E. histolytica and G. lamblia trophozoites. The inhibitors were rescreened to discriminate between amebicidal and giardicidal activity and general cytotoxicity toward a mammalian cell line. No mammalian cytotoxicity was found at >100 M for 48 h for any of the inhibitors. To understand the mechanism of action, a competitive binding assay was performed using the fluorescent ATP analogue bis-ANS (4,4=-dianilino-1,1=-binaphthyl-5,5=-disulfonic acid dipotassium salt) and recombinant E. histolytica Hsp90 preincubated in both the presence and absence of Hsp90 inhibitors. There was significant reduction in fluorescence compared to the level in the control, suggesting that E. histolytica Hsp90 is a selective target. The in vivo efficacy and safety of one Hsp90 inhibitor in a mouse model of amebic colitis and giardiasis was demonstrated by significant inhibition of parasite growth at a single oral dose of 5 mg/kg of body weight/day for 7 days and 10 mg/kg/day for 3 days. Considering the results for in vitro activity and in vivo efficacy, Hsp90 inhibitors represent a promising therapeutic option for amebiasis and giardiasis.

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Section: Methodsmentioning

confidence: 99%

Section: Effect Of Hsp90 Inhibitors Against E Histolytica G Lamblimentioning

confidence: 99%

Hsp90 Inhibitors as New Leads To Target Parasitic Diarrheal Diseases

Debnath

Shahinas

Bryant

et al. 2014

Antimicrob Agents Chemother

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“…19,20 Cell lines of hepatic origin, such as HepG2 cells, have been previously adapted to HTS formats 21,22 and utilized to assess hepatotoxicity. 19,20,23 However, HepG2 cells lack the full expression of hepatocyte proteins, such as phase I and phase II metabolizing enzymes and transporters, and thus may not correlate to in vivo hepatotoxicity.…”

Section: Introductionmentioning

confidence: 99%

Assessment of Compound Hepatotoxicity Using Human Plateable Cryopreserved Hepatocytes in a 1536-Well-Plate Format

Moeller¹,

Shukla

Xia

2012

ASSAY and Drug Development Technologies

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Hepatotoxicity is a major concern for both drug development and toxicological evaluation of environmental chemicals. The assessment of compound-induced hepatotoxicity has traditionally relied on in vivo testing; however, it is being replaced by human in vitro models due to an emphasis on the reduction of animal testing and species-specific differences. Since most cell lines and hybridomas lack the full complement of enzymes at physiological levels found in the liver, primary hepatocytes are the gold standard to study liver toxicities in vitro due to the retention of most of their in vivo activities. Here, we optimized a cell viability assay using plateable cryopreserved human hepatocytes in a 1536-well-plate format. The assay was validated by deriving inhibitory concentration at 50% values for 12 known compounds, including tamoxifen, staurosporine, and phenylmercuric acetate, with regard to hepatotoxicity and general cytotoxicity using multiple hepatocyte donors. The assay performed well, and the cytotoxicity of these compounds was confirmed in comparison to HepG2 cells. This is the first study to report the reliability of using plateable cryopreserved human hepatocytes for cytotoxicity studies in a 1536-well-plate format. These results suggest that plateable cryopreserved human hepatocytes can be scaled up for screening a large compound library and may be amenable to other hepatocytic assays such as metabolic or drug safety studies.

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“…However, selecting the appropriate plants for examination of pharmacologic properties can be challenging (Schnepple et al, 2006). Fortunately, ethnobotany can provide clues as to the medicinal properties of plants (Buenz, 2006). However, because of the generational loss of traditional knowledge (Cox, 2000;Lee et al, 2001), and the loss of plant resources (Buenz, 2005), ethnobotanical investigations are becoming more difficult to conduct as time passes.…”

Section: Introductionmentioning

confidence: 99%

“…For example, our group has used historic herbal texts (Buenz, 2006(Buenz, , 2007bBuenz et al, 2006Buenz et al, , 2005Buenz et al, , 2004a to re-discover promising ethnobotanical compounds.…”

Section: Introductionmentioning

confidence: 99%

Cytotoxic Properties of Diospyros Seychellarum Extract

Buenz

Bauer²,

Motley

et al. 2007

J. Toxicol. Sci.

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-During a recent botanical expedition in the Seychelles archipelago we identified healers using Diospyros seychellarum as a tonic. Since this plant lacks any medicinal record in the current literature, we assessed the cytotoxic potential of D. seychellarum. Using Jurkat cells as a model system we show, by flow cytometry, that treatment with the leaf extract results in mitochondrial depolarization and subsequent loss of cellular membrane integrity. Additionally, by transmission electron microscopy, we show that treatment with the extract results in chromatin condensation, mitochondrial swelling, and loss of nuclear membrane integrity. Through these morphological and biochemical observations we concluded that the extract of Diospyros seychellarum is able to induce apoptosis. While it is difficult to extrapolate a potential pharmacologic function based on the ethnomedical use as a tonic, the ability of this extract to induce apoptosis warrants further investigation of the medicinal properties of this plant.

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A high-throughput cell-based toxicity analysis of drug metabolites using flow cytometry

Cited by 11 publications

References 16 publications

Hsp90 Inhibitors as New Leads To Target Parasitic Diarrheal Diseases

Hsp90 Inhibitors as New Leads To Target Parasitic Diarrheal Diseases

Assessment of Compound Hepatotoxicity Using Human Plateable Cryopreserved Hepatocytes in a 1536-Well-Plate Format

Cytotoxic Properties of Diospyros Seychellarum Extract

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