Planar chromatography-bioassays for the parallel and sensitive detection of androgenicity, anti-androgenicity and cytotoxicity

Riegraf, Carolin; Bell, Anna Maria; Ohlig, Marina; Reifferscheid, Georg; Buchinger, Sebastian

doi:10.1016/j.chroma.2022.463582

Cited by 8 publications

(2 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This need may be at least partially fulfilled by effect-based methods, which provide a complementary approach to analytical techniques by detecting the biological activity of a sample or of its individual components, rather than analyzing its exact composition [1,[6][7][8][9][10][11]. In most cases, these methods are based on the use of whole-cell bioreporters, genetically engineered to detect EDCs by providing a selective and dose-dependent quantitative signal in their presence.…”

Section: Introductionmentioning

confidence: 99%

Enhanced Detection of Estrogen-like Compounds by Genetically Engineered Yeast Sensor Strains

Abu-Rmailah,

Moscovici,

Riegraf

et al. 2024

Biosensors

Self Cite

View full text Add to dashboard Cite

The release of endocrine-disrupting compounds (EDCs) to the environment poses a health hazard to both humans and wildlife. EDCs can activate or inhibit endogenous endocrine functions by binding hormone receptors, leading to potentially adverse effects. Conventional analytical methods can detect EDCs at a high sensitivity and precision, but are blind to the biological activity of the detected compounds. To overcome this limitation, yeast-based bioassays have previously been developed as a pre-screening method, providing an effect-based overview of hormonal-disruptive activity within the sample prior to the application of analytical methods. These yeast biosensors express human endocrine-specific receptors, co-transfected with the relevant response element fused to the specific fluorescent protein reporter gene. We describe several molecular manipulations of the sensor/reporter circuit in a Saccharomyces cerevisiae bioreporter strain that have yielded an enhanced detection of estrogenic-like compounds. Improved responses were displayed both in liquid culture (96-well plate format) as well as in conjunction with sample separation using high-performance thin-layer chromatography (HPTLC). The latter approach allows for an assessment of the biological effect of individual sample components without the need for their chemical identification at the screening stage.

show abstract

Section: Introductionmentioning

confidence: 99%

Enhanced Detection of Estrogen-like Compounds by Genetically Engineered Yeast Sensor Strains

Abu-Rmailah,

Moscovici,

Riegraf

et al. 2024

Biosensors

Self Cite

View full text Add to dashboard Cite

show abstract

“…In this technique, mixtures are first separated by HPTLC, and then active substances are revealed with a specific bioassay conducted directly on the plate. Its feasibility has been well documented for several activities such as estrogen receptor activation ( 11 ) as well as for androgen receptor activation and inhibition ( 12 , 13 ). Furthermore, bioactive bands can be recovered from autobiograms followed by mass spectrometry analysis, facilitating the elucidation of the substances responsible for activity ( 14 ).…”

Section: Introductionmentioning

confidence: 99%

Deciphering the origin of total estrogenic activity of complex mixtures

et al. 2023

View full text Add to dashboard Cite

IntroductionIdentifying compounds with endocrine properties in food is getting increasingly important. Current chemical analysis methodology is mainly focused on the identification of known substances without bringing insight for biological activity. Recently, the application of bioassays has been promoted for their potential to detect unknown bioactive substances and to provide information on possible interactions between molecules. From the toxicological perspective, measuring endocrine activity cannot inform on endocrine disruption and/or health risks without sufficient knowledge on the nature of the responsible factors.MethodsThe present study addresses a promising approach using High Performance Thin-Layer Chromatography (HPTLC) coupled to bioassays were analyzed using the Liquid Chromatography Mass-Spectrometry (LC-MS). The estrogen receptor activation was assessed using the transcription activation Estrogen Receptor Alpha Chemical Activated LUciferase gene eXpression assay (ERα- CALUX) and the HPTLC coupled to the Estrogen Screen Yeast assay (p-YES).ResultsSeven isoflavones were identified in the soy isolates. Estrogen receptor activation was assessed for both, the identified isoflavones and the soy isolates with ERα-CALUX test. Correlation between the soy isolates extracts and the identified isoflavones was shown. Moreover, p-YES revealed the presence of an estrogenic bioactive zone. Analysis of the bioactive zone through LCHRMS highlighted signals corresponding to several isoflavones already detected in the isolates as well as two additional ones. For all detected isoflavones, an estrogenic activity dose-response was established in both bioassays.ConclusionFinally, genistein, daidzein, and naringenin were found as the most active substances. A concordance analysis integrating the analytical and bioassay data indicated that genistein and daidzein were the drivers of the estrogenic activity of these soy protein isolates. Altogether, these data suggest that the integration of HPTLC-bioassay together with chemical analysis is a powerful approach to characterize the endocrine activity of complex mixtures.

show abstract

Extrapolation of cytotoxic masked effects in planar in vitro assays

Rosenberger,

Bell,

Reifferscheid

et al. 2024

Anal Bioanal Chem

View full text Add to dashboard Cite

The masking of specific effects in in vitro assays by cytotoxicity is a commonly known phenomenon. This may result in a partial or complete loss of effect signals. For common in vitro assays, approaches for identifying and quantifying cytotoxic masking are partly available. However, a quantification of cytotoxicity-affected signals is not possible. As an alternative, planar bioassays that combine high-performance thin layer chromatography with in vitro assays, such as the planar yeast estrogen screen (p-YES), might allow for a quantification of cytotoxically affected signals. Affected signals form a typical ring structure with a supressed or completely lacking centre that results in a double peak chromatogram. This study investigates whether these double peaks can be used for fitting a peak function to extrapolate the theoretical, unaffected signals. The precision of the modelling was evaluated for four individual peak functions, using 42 ideal, undistorted peaks from estrogenic model compounds in the p-YES. Modelled ED50-values from bisphenol A (BPA) experiments with cytotoxically disturbed signals were 13 times higher than for the apparent data without compensation for cytotoxicity (320 ± 63 ng versus 24 ± 17 ng). This finding has a high relevance for the modelling of mixture effects according to concentration addition that requires unaffected, complete dose–response relationships. Finally, we applied the approach to results of a p-YES assay on leachate samples of an elastomer material used in water engineering. In summary, the fitting approach enables the quantitative evaluation of cytotoxically affected signals in planar in vitro assays and also has applications for other fields of chemical analysis like distorted chromatography signals. Graphical abstract

show abstract

Planar chromatography-bioassays for the parallel and sensitive detection of androgenicity, anti-androgenicity and cytotoxicity

Cited by 8 publications

References 55 publications

Enhanced Detection of Estrogen-like Compounds by Genetically Engineered Yeast Sensor Strains

Enhanced Detection of Estrogen-like Compounds by Genetically Engineered Yeast Sensor Strains

Deciphering the origin of total estrogenic activity of complex mixtures

Extrapolation of cytotoxic masked effects in planar in vitro assays

Contact Info

Product

Resources

About