Coupling High-Performance Thin-Layer Chromatography with Bacterial Genotoxicity Bioreporters

Shakibai, Dror; Riegraf, Carolin; Moscovici, Liat; Reifferscheid, Georg; Buchinger, Sebastian; Belkin, Shimshon

doi:10.1021/acs.est.9b00921

Cited by 18 publications

(7 citation statements)

References 35 publications

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“…Alternative approaches have been proposed combining chemical and bioassays solutions as high performance thin-layer chromatography (HPTLC). Most probably a breakthrough improvement of LOBDs would require a very different test system design such as the coupling of bioassay with high performance thin-layer chromatography [ 49 , 50 , 51 ]. Finally, when considering the fact that the Ames MPF™ has not only practical advantages, but also provides significantly lower LEC values and LOBDs, its use might be preferred over the standard pre-incubation Petri-dish agar-based Ames test.…”

Section: Discussionmentioning

confidence: 99%

Direct Comparison of the Lowest Effect Concentrations of Mutagenic Reference Substances in Two Ames Test Formats

Rainer

Pinter

Prielinger

et al. 2021

Toxics

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The Ames assay is the standard assay for identifying DNA-reactive genotoxic substances. Multiple formats are available and the correct choice of an assay protocol is essential for achieving optimal performance, including fit for purpose detection limits and required screening capacity. In the present study, a comparison of those parameters between two commonly used formats, the standard pre-incubation Ames test and the liquid-based Ames MPF™, was performed. For that purpose, twenty-one substances with various modes of action were chosen and tested for their lowest effect concentrations (LEC) with both tests. In addition, two sources of rat liver homogenate S9 fraction, Aroclor 1254-induced and phenobarbital/β-naphthoflavone induced, were compared in the Ames MPF™. Overall, the standard pre-incubation Ames and the Ames MPF™ assay showed high concordance (>90%) for mutagenic vs. non-mutagenic compound classification. The LEC values of the Ames MPF™ format were lower for 17 of the 21 of the selected test substances. The S9 source had no impact on the test results. This leads to the conclusion that the liquid-based Ames MPF™ assay format provides screening advantages when low concentrations are relevant, such as in the testing of complex mixtures.

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Section: Discussionmentioning

confidence: 99%

Direct Comparison of the Lowest Effect Concentrations of Mutagenic Reference Substances in Two Ames Test Formats

Rainer

Pinter

Prielinger

et al. 2021

Toxics

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“…32−34 One possible EDA approach is the combination of high-performance thinlayer chromatography (HPTLC), allowing a chromatographic separation of the sample into fractions, with a subsequent in vitro bioassay performed on the surface of the HPTLC plate for the assessment of possible adverse biological effects of its components. 35,36 The detection and quantification of triazine herbicides by a Hill reaction inhibition using isolated chloroplasts was already shown in combination with thinlayer chromatography in previous studies. 37,38 The aim of the current study was to provide a proof of principle for the applicability of the PSII-inhibition algal test on HPTLC.…”

Section: Introductionmentioning

confidence: 89%

“…As described above, classical analytical nontarget approaches may not be able to detect biologically active substances, and conversely, a direct identification of the compounds responsible for the detected biological activity by means of bioassays is not possible . To combine the advantages of both approaches, i.e., chemical analysis and bioassays, an effect-directed analysis (EDA) can be pursued. − One possible EDA approach is the combination of high-performance thin-layer chromatography (HPTLC), allowing a chromatographic separation of the sample into fractions, with a subsequent in vitro bioassay performed on the surface of the HPTLC plate for the assessment of possible adverse biological effects of its components. , The detection and quantification of triazine herbicides by a Hill reaction inhibition using isolated chloroplasts was already shown in combination with thin-layer chromatography in previous studies. , …”

Section: Introductionmentioning

confidence: 99%

Detection and Quantification of Photosystem II Inhibitors Using the Freshwater Alga Desmodesmus subspicatus in Combination with High-Performance Thin-Layer Chromatography

Riegraf

Reifferscheid

Becker

et al. 2019

Environ. Sci. Technol.

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We present a novel tool for detecting and monitoring photosystem II (PSII) inhibitors, using the freshwater alga Desmodesmus subspicatus, in environmental samples fractionated by high-performance thin-layer chromatography (HPTLC). After chromatographic separation of a sample on a HPTLC plate, the algal suspension is sprayed homogeneously on the plate, and PSII-inhibition by specific sample components is detected based on changes in fluorescence yield, viewed by a maxi Imaging-Pulse-Amplitude-Modulation fluorometer. Dose-dependent responses to the PSII-inhibitor herbicides atrazine and diuron, frequently detected in water bodies, are demonstrated without and with chromatographic separation. The limits of quantification for atrazine and diuron with chromatographic separation were 1.94 ng and 99 pg, respectively, allowing the detection of environmentally relevant concentrations of these herbicides. The developed method was also employed to analyze sample extracts collected during a passive sampling campaign in surface waters. The obtained data correlated well with results from LC-MS/MS chemical analysis but also revealed unknown PSII-inhibiting activities. The proposed methodology represents a rapid and sensitive screening tool for the simultaneous effect-based detection of PSII-inhibitors in environmental samples.

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“…A notable disadvantage of this approach is the inability to distinguish between different components exerting similar biological effects [14]. To overcome this difficulty, a number of reports describing the direct coupling of effect-based methods to high-performance thin-layer chromatography (HPTLC) have been published recently [13,15,16] The combination of chemical separation by high-performance thin-layer chromatography (HPTLC) and the effect-based assay by yeast-based sensor strains allowed the separation of environmental samples and the discovery of individual sample components exhibiting hormonal activity. These active compounds can then be removed from the HPTLC plate and identified via traditional analytical methods (e.g., LC/MS, GC/MS) [17,18].…”

Section: Introductionmentioning

confidence: 99%

Yeast-Based Fluorescent Sensors for the Simultaneous Detection of Estrogenic and Androgenic Compounds, Coupled with High-Performance Thin Layer Chromatography

et al. 2020

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The persistence of endocrine disrupting compounds (EDCs) throughout wastewater treatment processes poses a significant health threat to humans and to the environment. The analysis of EDCs in wastewater remains a challenge for several reasons, including (a) the multitude of bioactive but partially unknown compounds, (b) the complexity of the wastewater matrix, and (c) the required analytical sensitivity. By coupling biological assays with high-performance thin-layer chromatography (HPTLC), different samples can be screened simultaneously, highlighting their active components; these may then be identified by chemical analysis. To allow the multiparallel detection of diverse endocrine disruption activities, we have constructed Saccharomyces cerevisiae-based bioreporter strains, responding to compounds with either estrogenic or androgenic activity, by the expression of green (EGFP), red (mRuby), or blue (mTagBFP2) fluorescent proteins. We demonstrate the analytical potential inherent in combining chromatographic compound separation with a direct fluorescent signal detection of EDC activities. The applicability of the system is further demonstrated by separating influent samples of wastewater treatment plants, and simultaneously quantifying estrogenic and androgenic activities of their components. The combination of a chemical separation technique with an optical yeast-based bioassay presents a potentially valuable addition to our arsenal of environmental pollution monitoring tools.

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Coupling High-Performance Thin-Layer Chromatography with Bacterial Genotoxicity Bioreporters

Cited by 18 publications

References 35 publications

Direct Comparison of the Lowest Effect Concentrations of Mutagenic Reference Substances in Two Ames Test Formats

Direct Comparison of the Lowest Effect Concentrations of Mutagenic Reference Substances in Two Ames Test Formats

Detection and Quantification of Photosystem II Inhibitors Using the Freshwater Alga Desmodesmus subspicatus in Combination with High-Performance Thin-Layer Chromatography

Yeast-Based Fluorescent Sensors for the Simultaneous Detection of Estrogenic and Androgenic Compounds, Coupled with High-Performance Thin Layer Chromatography

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