Tandem screening of toxic compounds on GFP-labeled bacteria and cancer cells in microtiter plates (original) (raw)
Related papers
Tandem screening of toxic compunds on GFP-labeled bacteria and cancer cells in microtiter plates
Biochemical and Biophysical Research Communications
A 96-well fluorescence-based assay has been developed for the rapid screening of potential cytotoxic and bacteriocidal compounds. The assay is based on detection of green fluorescent protein (GFP) in HeLa human carcinoma cells as well as gram negative (Escherichia coli) and gram positive bacteria (Mycobacterium avium). Addition of a toxic compound to the GFP marked cells resulted in the loss of the GFP fluorescence which was readily detected by fluorometry. Thirty-nine distinct naphthoquinone derivatives were screened and several of these compounds were found to be toxic to all cell types. Apart from differences in overall toxicity, two general types of toxic compounds were detected, those that exhibited toxicity to two or all three of the cell types and those that were primarily toxic to the HeLa cells. Our results demonstrate that the parallel screening of both eukaryotic and prokaryotic cells is not only feasible and reproducible but also cost effective.
Microplate-based microbial assay for risk assessment and (eco)toxic fingerprinting of chemicals
Analytica Chimica Acta, 2003
We have developed a multi-species microbial assay, MARA, for assessing the (eco)toxic risks of chemical compounds and for the determination of their toxic fingerprints. The main advantages with MARA are (1) the simultaneous testing on several microbial strains; (2) the concept of toxic fingerprinting; (3) the simple and inexpensive handling and reading of the test. The toxic activity is measured in parallel on 11 different micro-organisms lyophilised in a microplate. A concentration gradient of the chemical to be tested is added and growth is indicated through the reduction of tetrazolium red (TTC). The microplates are read by a common flatbed scanner or a microplate spectrophotometer. The array of the 11 different inhibition values constitute a toxic fingerprint, characteristic for each type of chemical compound, and it is shown that the assay can distinguish between 12 standard chemicals. Both the reproducibility (CV ≈ 20%) and the sensitivity are similar to other toxicity tests based on micro-organisms.
Journal of Applied Toxicology, 2012
Twenty-two of Galderma's proprietary compounds were tested in the GADD45a-GFP 'GreenScreen HC' assay (GS), the SOS-ChromoTest and the Mini-Ames to evaluate GSs performance for early genotoxicity screening purposes. Forty more characterized compounds were also tested, including antibiotics: metronidazole, clindamycin, tetracycline, lymecycline and neomycin; and catecholamines: resorcinol mequinol, hydroquinone, one aneugen carbendazim, one corticoid dexamethasone, one peroxisome proliferator-activated receptor rosiglitazone, one pesticide carbaryl and two further proprietary molecules with in vitro genotoxicity data. With proprietary molecules, this study concluded that the GS renders the SOS-ChromoTest obsolete for in vitro screening. The GS confirmed all results of the Mini-Ames test (100% concordance). Compared with the micronucleus test, the GS showed a concordance of 82%. With known compounds, the GS ranked the potency of positive results for catecholamines in accordance with other genotoxicity tests and showed very reproducible results. It confirmed positive results for carbendazim, for tetracycline antibiotics and for carbaryl. The GS produced negative results for metronidazole, a nitroreduction-specific bacterial mutagen, for dexamethasone (a non-genotoxic apoptosis inducer), for rosiglitazone (a GADD45g promoter inducer) and for clindamycin and neomycin (inhibitors of macromolecular synthesis in bacteria). As such, the GS appears to be a reproducible, robust, specific and sensitive test for genotoxicity screening.
Journal of Biomolecular Screening, 2008
There is a pressing need to develop rapid yet accurate screening assays for the identification of genotoxic liability and for early hazard assessment in drug discovery. The GADD45a-GFP human cell-based genotoxicity assay (GreenScreen HC) has been reformatted to test 12 compounds per 96-well microplate in a higher throughput, automated screening mode and the protocol applied to the analysis of 1266 diverse, pharmacologically active compounds. Testing from a fixed starting concentration of 100 μM and over 3 serial dilutions, the hit rates for genotoxicity (7.3%) and cytotoxicity (33%) endpoints of the assay have been determined in a much wider chemical space than previously reported. The degree of interference from color, autofluorescence, and low solubility has also been assessed. The assay results have been compared to an in silico approach to genotoxicity assessment using Derek for Windows software. Where carcinogenicity data were available, GreenScreen HC demonstrated a higher specificity than in silico methods while identifying genotoxic species that were not highlighted for genotoxic liability in structure-activity relationship software. Higher throughput screening from a fixed, low concentration reduces sensitivity to less potent genotoxins, but the maintenance of the previously reported high specificity is essential in early hazard assessment where misclassification can lead to the needless rejection of potentially useful compounds in drug development. (Journal of Biomolecular Screening 2009:16-30)
Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2000
Yeast strains transformed with high copy number plasmids carrying the gene encoding a green fluorescent protein Ž . optimised for yeast yEGFP3 under the control of the RAD54 or RNR2 promoter were used to investigate the activity of potentially DNA-damaging substances. The assays were performed on 96-well microtitre plates in the presence of different concentrations of the test substances. The synthesis of GFP protein was measured through the fluorescence signal and cell growth was monitored by absorption. Here, we demonstrate that this system can be used as a biosensor to assess the genotoxic potential of drugs and other chemical substances. The use of microtitre plates will enable full automation of the system and allows the inclusion of internal reference standards in each assay. q
Whole Cell Sensing Systems Ii, 2010
Genotoxicity test systems that are based on bacteria display an important role in the detection and assessment of DNA damaging chemicals. They belong to the basic line of test systems due to their easy realization, rapidness, broad applicability, high sensitivity and good reproducibility. Since the development of the Salmonella microsomal mutagenicity assay by Ames and coworkers in the early 1970s, significant development in bacterial genotoxicity assays was achieved and is still a subject matter of research. The basic principle of the mutagenicity assay is a reversion of a growth inhibited bacterial strain, e.g., due to auxotrophy, back to a fast growing phenotype (regain of prototrophy). Deeper knowledge of the mutation events allows a mechanistic understanding of the induced DNA-damage by the utilization of base specific tester strains. Collections of such specific tester strains were extended by genetic engineering. Beside the reversion assays, test systems utilizing the bacterial SOS-response were invented. These methods are based on the fusion of various SOS-responsive promoters with a broad variety of reporter genes facilitating numerous methods of signal detection.
Green Fluorescent Protein as a Biosensor for Toxic Compounds
Reviews in Fluorescence, 2006
In this brief review, we present recent results in the development of fluorescencebased assays for the detection of compounds with cytotoxic, anticancer and antimicrobial properties. As other reviews have explored various aspects related to these topics, this review will focus on the use of the Green Fluorescent Protein (GFP) for the detection of potentially toxic and/or therapeutic compounds. Since high-throughput screening of chemical compounds can be both expensive and laborious, development of low cost and efficient cell-based assays to determine biological activity should greatly enhance the early screening process. In our recent studies, we have developed a couple of GFP-based assays for the rapid screening of compounds with cytotoxic and bacteriocidal properties. As will be described in more detail in subsequent sections, a new 96-well assay has recently been developed that allows for the simultaneous screening of test compounds on gram positive and negative bacteria as well as mammalian (human cancer) cells. Our results demonstrate that both mammalian cells and bacteria can be analyzed in tandem to rapidly determine which compounds are specifically toxic to one of these cell types. The parallel screening of both eukaryotic and prokaryotic cells was found to be feasible, reproducible, and cost effective.
Mutagenesis, 1996
In vitro assays for mutagenicity are an important feature of pre-clinical testing and form part of the current regulatory testing conducted early in drug development They can also play a part in compound selection since mutagenic compounds can be eliminated from a range of potential candidates. Bacterial tests are particularly useful in this area because they generate results quickly, though their use may be limited because they can require up to 4 g of material. A scaled-down version of the Ames test has been developed which requires only ~20 mg of material. Initial experiences with this assay using a range of known mutagens and novel compounds have shown that the Miniscreen has similar sensitivity to the Ames test The major exception is for those mutagens preferentially detected with strains TA1537 and TA1535, which, because of their low spontaneous counts, are not employed in the Miniscreen.