The use of 3D cultures of MCF-10A and MCF-12A cells by high content screening for effect-based analysis of non-genotoxic carcinogens.

The human breast epithelial cell lines MCF-10A and MCF-12A form well-differentiated acinus-like structures when grown in three-dimensional matrigel culture over a period of 20 days. In the present study, both cell lines were tested for their suitability to serve as an effect-based in vitro test system for non-genotoxic carcinogens. A software solution for automated Acinus Detection And Morphological Evaluation (ADAME) was developed to automatically acquire acinus images and to determine morphological parameters such as acinus size, lumen size, and acinus roundness. A number of test compounds were tested for their capacity to affect acinus formation and cellular differentiation. Human epidermal growth factor stimulated acinus growth for both cell lines whereas all-trans retinoic acid inhibited acinus growth. The strong estrogen 17ß-estradiol had no effect on acinus formation of estrogen receptor (ER)-negative MCF-10A cells, but yielded larger MCF-12A (ER-positive) acini. Thus, the parallel use of both cell lines allows the identification of estrogenic properties of a given test compound.

The urinary metabolites of DINCH® have an impact on the activities of the human nuclear receptors ERα, ERß, AR, PPARα and PPARγ.

DINCH® (di-isononyl cyclohexane-1,2-dicarboxylate) is a non-phthalate plasticizer that has been developed to replace phthalate plasticizers such as DEHP (di-2-ethylhexyl phthalate) or DINP (di-isononyl phthalate). DINCH® is metabolized to its corresponding monoester and subsequently to oxidized monoester derivatives. These are conjugated to glucuronic acid and subject to urinary excretion. In contrast to DINCH®, there are almost no toxicological data available regarding its primary and secondary metabolites. The present study aimed at the characterization of potential endocrine properties of DINCH® and five DINCH® metabolites by using reporter gene assays to monitor the activity of the human nuclear receptors ERα, ERß, AR, PPARα and PPARγ in vitro. DINCH® itself did not have any effect on the activity of these receptors whereas DINCH® metabolites were shown to activate all these receptors. In the case of AR, DINCH® metabolites predominantly enhanced dihydrotestosterone-stimulated AR activity. In the H295R steroidogenesis assay, neither DINCH® nor any of its metabolites affected estradiol or testosterone synthesis. In conclusion, primary and secondary DINCH® metabolites exert different effects at the molecular level compared to DINCH® itself. All these in vitro effects of DINCH® metabolites, however, were only observed at high concentrations such as 10 µM or above which is about three orders of magnitude above reported DINCH® metabolite concentrations in human urine. Thus, the in vitro data do not support the notion that DINCH® or any of the investigated metabolites may exert considerable endocrine effects in vivo at relevant human exposure levels.

Agonistic and antagonistic effects of phthalates and their urinary metabolites on the steroid hormone receptors ERα, ERß, and AR.

Phthalate plasticizers have been reported to exert adverse effects via activation of the estrogen receptors ERα and ERß and inhibition of the androgen receptor AR as molecular initiating events. After oral uptake, phthalates are metabolized to their corresponding monoesters and subsequently to oxidized phthalate monoester derivatives, which are in turn conjugated to glucuronic acid and finally excreted with the urine. In contrast to the parent phthalates, toxicological data regarding their primary and secondary metabolites are rare. The present study aimed at the characterization of potential endocrine effects of 15 phthalates and 19 phthalate metabolites by using reporter gene assays to monitor human ERα, ERß, and AR activity. In these in vitro assays, the phthalates either stimulated or inhibited ERα and ERß activity and inhibited AR activity, whereas the phthalate metabolites had no impact on the activity of these human hormone receptors. In contrast, the metabolites of di-(2-ethylhexyl) phthalate (DEHP) stimulated transactivation of the human peroxisome proliferator-activated receptors PPARα and PPARγ in analogous reporter gene assays, although DEHP itself did not activate these nuclear receptors. Therefore, primary and secondary phthalate metabolites appear to exert different effects at the molecular level compared to the parent compounds.

Morphological and molecular characterization of the human breast epithelial cell line M13SV1 and its tumorigenic derivatives M13SV1-R2-2 and M13SV1-R2-N1.

BACKGROUND: The estrogen receptor-positive M13SV1 breast epithelial cell line was proposed to be a suitable in vitro model for breast cancer research since two derivatives with graduated tumorigenicity-M13SV1-R2-2 and M13SV1-R2-N1-are available for this cell line. In the present study, these three cell lines were comparatively examined for their morphological and their biochemical properties on the molecular level. METHODS: A transcriptomic approach (gene array analysis) was chosen to unravel differences in gene expression among the three cell lines. Network analysis was conducted to identify deregulated signaling pathways. Cellular viability was determined by impedance measurements as well as by neutral red uptake assay. Apoptosis was determined by using a caspase assay. For morphological characterization, cells were grown in three-dimensional cell culture, and cellular differentiation and spheroid formation was followed by immunofluorescence staining by using confocal laser scanning microscopy. RESULTS: The gene array results indicated that there were only marginal differences in gene expression among the three cell lines. Network analysis predicted the R2-N1 derivative (1) to display enhanced apoptosis and (2) to have a higher migration capability compared to its parent cell line M13SV1. Enhanced apoptosis was confirmed by elevated caspase activity, and increased migration was observed in 3D culture when cells migrated out of the globular spheroids. In 3D cell culture, all three cell lines similarly formed spheroids within three days, but there was no acini formation until day 21 which is indicated by a growth arrest around day 15, cellular polarization, and the formation of hollow lumen inside the spheroids. These characteristics, however, are crucial to study, e.g., the differentiation process of breast epithelial cells in vitro. CONCLUSION: Due to the molecular and morphological features, the M13SV1 cell line and its tumorigenic derivatives seem to be less suitable as in vitro models than other cell lines such as the MCF-10A cell line which displays proper acini formation in 3D culture.

Receptor Polymorphism and Genomic Structure Interact to Shape Bitter Taste Perception.

The ability to taste bitterness evolved to safeguard most animals, including humans, against potentially toxic substances, thereby leading to food rejection. Nonetheless, bitter perception is subject to individual variations due to the presence of genetic functional polymorphisms in bitter taste receptor (TAS2R) genes, such as the long-known association between genetic polymorphisms in TAS2R38 and bitter taste perception of phenylthiocarbamide. Yet, due to overlaps in specificities across receptors, such associations with a single TAS2R locus are uncommon. Therefore, to investigate more complex associations, we examined taste responses to six structurally diverse compounds (absinthin, amarogentin, cascarillin, grosheimin, quassin, and quinine) in a sample of the Caucasian population. By sequencing all bitter receptor loci, inferring long-range haplotypes, mapping their effects on phenotype variation, and characterizing functionally causal allelic variants, we deciphered at the molecular level how a subjects' genotype for the whole-family of TAS2R genes shapes variation in bitter taste perception. Within each haplotype block implicated in phenotypic variation, we provided evidence for at least one locus harboring functional polymorphic alleles, e.g. one locus for sensitivity to amarogentin, one of the most bitter natural compounds known, and two loci for sensitivity to grosheimin, one of the bitter compounds of artichoke. Our analyses revealed also, besides simple associations, complex associations of bitterness sensitivity across TAS2R loci. Indeed, even if several putative loci harbored both high- and low-sensitivity alleles, phenotypic variation depended on linkage between these alleles. When sensitive alleles for bitter compounds were maintained in the same linkage phase, genetically driven perceptual differences were obvious, e.g. for grosheimin. On the contrary, when sensitive alleles were in opposite phase, only weak genotype-phenotype associations were seen, e.g. for absinthin, the bitter principle of the beverage absinth. These findings illustrate the extent to which genetic influences on taste are complex, yet arise from both receptor activation patterns and linkage structure among receptor genes.