Deciphering the origin of total estrogenic activity of complex mixtures.

Introduction: Identifying 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. Methods: The 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). Results: Seven 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. Conclusion: Finally, 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.

Interlaboratory Study to Evaluate a Testing Protocol for the Safety of Food Packaging Coatings.

According to European regulations, migration from food packaging must be safe. However, currently, there is no consensus on how to evaluate its safety, especially for non-intentionally added substances (NIAS). The intensive and laborious approach, involving identification and then quantification of all migrating substances followed by a toxicological evaluation, is not practical or feasible. In alignment with the International Life Sciences Institute (ILSI) and the European Union (EU) guidelines on packaging materials, efforts are focused on combining data from analytics, bioassays and in silico toxicology approaches for the risk assessment of packaging materials. Advancement of non-targeted screening approaches using both analytical methods and in vitro bioassays is key. A protocol was developed for the chemical and biological screening of migrants from coated metal packaging materials. This protocol includes guidance on sample preparation, migrant simulation, chemical analysis using liquid chromatography (LC-MS) and validated bioassays covering endocrine activity, genotoxicity and metabolism-related targets. An inter-laboratory study was set-up to evaluate the consistency in biological activity and analytical results generated between three independent laboratories applying the developed protocol and guidance. Coated packaging metal panels were used in this case study. In general, the inter-laboratory chemical analysis and bioassay results displayed acceptable consistency between laboratories, but technical differences led to different data interpretations (e.g., cytotoxicity, cell passages, chemical analysis). The study observations with the greatest impact on the quality of the data and ultimately resulting in discrepancies in the results are given and suggestions for improvement of the protocol are made (e.g., sample preparation, chemical analysis approaches). Finally, there was agreement on the need for an aligned protocol to be utilized by qualified laboratories for chemical and biological analyses, following best practices and guidance for packaging safety assessment of intentionally added substances (IAS) and NIAS to avoid inconsistency in data and the final interpretation.

Incorporation of Metabolic Activation in the HPTLC-SOS-Umu-C Bioassay to Detect Low Levels of Genotoxic Chemicals in Food Contact Materials.

The safety evaluation of food contact materials requires excluding mutagenicity and genotoxicity in migrates. Testing the migrates using in vitro bioassays has been proposed to address this challenge. To be fit for that purpose, bioassays must be capable of detecting very low, safety relevant concentrations of DNA-damaging substances. There is currently no bioassay compatible with such qualifications. High-performance thin-layer chromatography (HPTLC), coupled with the planar SOS Umu-C (p-Umu-C) bioassay, was suggested as a promising rapid test (~6 h) to detect the presence of low levels of mutagens/genotoxins in complex mixtures. The current study aimed at incorporating metabolic activation in this assay and testing it with a set of standard mutagens (4-nitroquinoline-N-oxide, aflatoxin B1, mitomycin C, benzo(a)pyrene, N-ethyl nitrourea, 2-nitrofluorene, 7,12-dimethylbenzanthracene, 2-aminoanthracene and methyl methanesulfonate). An effective bioactivation protocol was developed. All tested mutagens could be detected at low concentrations (0.016 to 230 ng/band, according to substances). The calculated limits of biological detection were found to be up to 1400-fold lower than those obtained with the Ames assay. These limits are lower than the values calculated to ensure a negligeable carcinogenic risk of 10-5. They are all compatible with the threshold of toxicological concern for chemicals with alerts for mutagenicity (150 ng/person). They cannot be achieved by any other currently available test procedures. The p-Umu-C bioassay may become instrumental in the genotoxicity testing of complex mixtures such as food packaging, foods, and environmental samples.

Limitations of currently available in vitro oestrogenicity bioassays for effect-based testing of whole foods as the basis for decision making.

The idea that previously unknown hazards can be readily revealed in complex mixtures such as foods is a seductive one, giving rise to the hope that data from effect-based assays of food products collected in market surveys is of suitable quality to be the basis for data-driven decision-making. To study this, we undertook a comparative study of the oestrogenicity of blinded cereal samples, both in a number of external testing laboratories and in our own facility. The results clearly showed little variance in the activities of 9 samples when using a single method, but great differences between the activities from each method. Further exploration of these findings suggest that the oestrogenic activity is likely an inherent part of the natural food matrix which the varying sample preparation methods are able to release and extract to differing degrees. These issues indicate the current poor suitability of these types of datasets to be used as the basis for consumer advice or food decision-making. Data quality must be improved before such testing is used in practice.

Use of in vitro bioassays to facilitate read-across assessment of nitrogen substituted heterocycle analogues of polycyclic aromatic hydrocarbons.

Nitrogen-containing polycyclic aromatic hydrocarbons (PANHs or azaarenes) are compounds structurally similar to PAHs (carbon substituted by a nitrogen) reported to occur at low levels in food. Although limited, literature may suggest possible higher toxicity than for PAHs. Using a battery of in vitro assays, the toxicological properties of uncharacterized PANHs of increasing ring number were compared to those of characterized structural PAH analogues. The parameters measured covered key events relevant to the AOP developed for Benzo(a)pyrene: AhR activation, mutagenicity and DNA-damage with and without metabolic activation and endocrine receptors activation/inhibition. There was a strong correlation between the chemical structure and the biological activities of the compounds. AhR activation was the most sensitive parameter with a direct correlation between potency and ring number. The most potent genotoxic chemicals were found amongst the ones with the highest number of ring, and under metabolic activation. Such an approach allowed designing sub-groups based on biological properties in addition to structural similarities. Within a sub-group, toxicological data of tested chemicals may be used to characterize hazard of biologically similar but toxicologically uncharacterized substances. This indicates that in addition to structural properties, in vitro biological data may be useful to conduct read-across.

Dicarbonyls stimulate cellular protection systems in primary rat hepatocytes and show anti-inflammatory properties.

Advanced glycation endproducts (AGEs) and their precursor dicarbonyls are generally perceived as having adverse health effects. They are also considered to be initiators and promoters of disease and aging. However, proof for a causal relationship is lacking. On the other hand, it is known that AGEs and melanoidins possess beneficial properties, such as antioxidant and metal-chelating activities. Furthermore, some AGEs may stimulate the cellular detoxification system, generally known as the phase II drug metabolizing system. We show here that several reactive dicarbonyl intermediates have the capability to stimulate the cellular phase II detoxification systems in both a reporter cell line and primary rat hepatocytes. In addition, we demonstrate that dicarbonyls can attenuate the inflammatory signaling induced by tumor necrosis factor-alpha in a reporter cell system.

Differentiating true androgen receptor inhibition from cytotoxicity-mediated reduction of reporter-gene transactivation in-vitro.

In vitro effect-based reporter assays are applied as biodetection tools designed to address nuclear receptor mediated-modulation. While such assays detect receptor modulating potential, cell viability needs to be addressed, preferably in the same well. Some assays circumvent this by co-transfecting a second constitutively-expressed marker gene or by multiplexing a cytotoxicity assay. Some assays, such as the CALUX®, lack this feature. The cytotoxic effects of unknown substances can confound in vitro assays, making the interpretation of results difficult and uncertain, particularly when assessing antagonistic activity. It's necessary to determine whether the cause of the reporter signal decrease is an antagonistic effect or a non-specific cytotoxic effect. To remedy this, we assessed the suitability of multiplexing a cell viability assay within the CALUX® transcriptional activation test for anti-androgenicity. Tests of both well-characterized anti-androgens and cytotoxic compounds demonstrated the suitability of this approach for discerning between the molecular mechanisms of action without altering the nuclear receptor assay; though some compounds were both cytotoxic and anti-androgenic. The optimized multiplexed assay was then applied to an uncharacterized set of polycyclic aromatic compounds. These results better characterized the mode of action and the classification of effects. Overall, the multiplexed protocol added value to CALUX test performance.

Integrating bioassays and analytical chemistry as an improved approach to support safety assessment of food contact materials.

Food contact materials (FCM) contain chemicals which can migrate into food and result in human exposure. Although it is mandatory to ensure that migration does not endanger human health, there is still no consensus on how to pragmatically assess the safety of FCM since traditional approaches would require extensive toxicological and analytical testing which are expensive and time consuming. Recently, the combination of bioassays, analytical chemistry and risk assessment has been promoted as a new paradigm to identify toxicologically relevant molecules and address safety issues. However, there has been debate on the actual value of bioassays in that framework. In the present work, a FCM anticipated to release the endocrine active chemical 4-nonyphenol (4NP) was used as a model. In a migration study, the leaching of 4NP was confirmed by LC-MS/MS and GC-MS. This was correlated with an increase in both estrogenic and anti-androgenic activities as measured with bioassays. A standard risk assessment indicated that according to the food intake scenario applied, the level of 4NP measured was lower, close or slightly above the acceptable daily intake. Altogether these results show that bioassays could reveal the presence of an endocrine active chemical in a real-case FCM migration study. The levels reported were relevant for safety assessment. In addition, this work also highlighted that bioactivity measured in migrate does not necessarily represent a safety issue. In conclusion, together with analytics, bioassays contribute to identify toxicologically relevant molecules leaching from FCM and enable improved safety assessment.

Inducible nitric oxide synthase-dependent stimulation of PKGI and phosphorylation of VASP in human embryonic kidney cells.

Inducible nitric oxide synthase (iNOS) production of nitric oxide (NO) has been mostly associated with so-called nitrosative stress or interaction with superoxide anion. However, recent investigations have indicated that, as for the other isoenzymes producing NO, guanylyl cyclase (GC) is a very sensitive target of iNOS activity. To further investigate this less explored signaling, the NO-cyclic guanosine 3'-5'-monophosphate (NO-cGMP)-induced vasodilator-stimulated phosphoprotein (VASP) phosphorylation on serine 239 was investigated in human embryonic kidney 293 cells (HEK cells). First, the expression and activity of alpha2 and beta1 NO-sensitive GC subunits was determined by Western blot analysis, reverse transcription-polymerase chain reaction and NO donors administration. Then, the expression of a functional cGMP-dependent protein kinase I (PKGI) was verified by addition of 8-Br-cGMP followed by determination of phosphorylation of VASP on serine 239. Finally, iNOS activation of this signaling pathway was characterized after transfection of HEK cells with human iNOS cDNA. Altogether our data show that iNOS-derived NO activates endogenous NO-sensitive GC and leads to VASP phosphorylation in HEK cells.

Glycolaldehyde-modified ß-lactoglobulin AGEs are unable to stimulate inflammatory signaling pathways in RAGE-expressing human cell lines.

SCOPE: Advanced glycation endproducts (AGEs) are suspected to stimulate inflammatory signaling pathways in target tissues via activation of the receptor for AGEs. Endotoxins are generally recognized as potential contamination of AGE preparations and stimulate biological actions that are very similar as or identical to those induced by AGEs. METHODS AND RESULTS: In our study, we used glycolaldehyde-modified ß-lactoglobulin preparations as model AGEs and employed two methods to remove endotoxin using either affinity columns or extraction with Triton X-114 (TX-114). Affinity column-purified AGEs retained their ability to stimulate inflammatory signaling as measured by mRNA expression of inflammatory cytokines in the human lung epithelial cell line Beas2b. However, glycolaldehyde-modified AGEs purified by extraction with TX-114 did not show any stimulation of mRNA expression of inflammatory cytokines. The presence of a cell stimulating endotoxin-like activity was demonstrated in the detergent phase after extraction with TX-114, thus indicating that not AGEs but a lipophilic contamination was responsible for the stimulation of inflammatory signaling. CONCLUSION: Our results demonstrate that glycolaldehyde-modified AGEs are unable to induce inflammatory signaling in receptor for AGE-expressing cells. The observed cell-activating activity can be ascribed to an endotoxin-like lipophilic contamination present in AGE preparations and affinity column purification was insufficient to remove this contamination.

N(epsilon)-carboxymethyllysine-modified proteins are unable to bind to RAGE and activate an inflammatory response.

Advanced glycation endproducts (AGEs) containing carboxymethyllysine (CML) modifications are generally thought to be ligands of the receptor for AGEs, RAGEs. It has been argued that this results in the activation of pro-inflammatory pathways and diseases. However, it has not been shown conclusively that a CML-modified protein can interact directly with RAGE. Here, we have analyzed whether beta-lactoglobulin (bLG) or human serum albumin (HSA) modified chemically to contain only CML (10-40% lysine modification) can (i) interact with RAGE in vitro and (ii) interact with and activate RAGE in lung epithelial cells. Our results show that CML-modified bLG or HSA are unable to bind to RAGE in a cell-free assay system (Biacore). Furthermore, they are unable to activate pro-inflammatory signaling in the cellular system. Thus, CML probably does not form the necessary structure(s) to interact with RAGE and activate an inflammatory signaling cascade in RAGE-expressing cells.