Pre-validation of a reporter gene assay for oxidative stress for the rapid screening of nanobiomaterials

Engineered nanomaterials have been found to induce oxidative stress. Cellular oxidative stress, in turn, can result in the induction of antioxidant and detoxification enzymes which are controlled by the nuclear erythroid 2-related factor 2 (NRF2) transcription factor. Here, we present the results of a pre-validation study which was conducted within the frame of BIORIMA (“biomaterial risk management”) an EU-funded research and innovation project. For this we used an NRF2 specific chemically activated luciferase expression reporter gene assay derived from the human U2OS osteosarcoma cell line to screen for the induction of the NRF2 mediated gene expression following exposure to biomedically relevant nanobiomaterials. Specifically, we investigated Fe3O4-PEG-PLGA nanomaterials while Ag and TiO2 “benchmark” nanomaterials from the Joint Research Center were used as reference materials. The viability of the cells was determined by using the Alamar blue assay. We performed an interlaboratory study involving seven different laboratories to assess the applicability of the NRF2 reporter gene assay for the screening of nanobiomaterials. The latter work was preceded by online tutorials to ensure that the procedures were harmonized across the different participating laboratories. Fe3O4-PEG-PLGA nanomaterials were found to induce very limited NRF2 mediated gene expression, whereas exposure to Ag nanomaterials induced NRF2 mediated gene expression. TiO2 nanomaterials did not induce NRF2 mediated gene expression. The variability in the results obtained by the participating laboratories was small with mean intra-laboratory standard deviation of 0.16 and mean inter laboratory standard deviation of 0.28 across all NRF2 reporter gene assay results. We conclude that the NRF2 reporter gene assay is a suitable assay for the screening of nanobiomaterial-induced oxidative stress responses

Novel insights in the molecular mechanisms of action of retinoids and their potential repercussions on breast cancer cell proliferation

In chapter 1 of this thesis, background information on the molecular actions and effects elicited by retinoids in the context of nuclear receptor (NR) signaling and breast cancer treatment was given. In addition, the state of the art and the knowledge gap still remaining in this area was highlighted. The principal objective of the thesis was defined which was to investigate different molecular events possibly elicited after retinoid treatment to better understand the observed effects on breast cancer cell proliferation Finally, the general outline of the present thesis was provided. Chapter 2 assessed whether the ligand binding domain (LBD) of the Retinoic Acid Receptor (RAR) can be used to study RAR-coregulator binding events as an alternative to the use of the full length (FL) RAR. Thus, this work aimed to characterize and compare ligand-driven coregulator interactions of both the RARα-LBD and the RARα-FL. We studied the coregulator motif interactions of RARα-FL and RARα-LBD upon incubation with the RAR agonist all-trans retinoic acid (AtRA) and the RAR antagonist Ro415253 using the Microarray Assay for Real-time Coregulator-Nuclear Receptor Interaction (MARCoNI). The results obtained showed that the coregulator binding profiles are highly similar for the RARα-FL and the RARα-LBD regarding both qualitative aspects like the type of coregulators that bind or dissociate upon (ant)agonist action and quantitative parameters such as EC50s and the maximum responses (modulation indexes at saturating ligand concentration). Moreover, based on the coregulator binding signatures, the RAR agonist AtRA could be clearly discriminated from the RAR antagonist Ro415253 using both the RARα-FL and the RARα-LBD. Once established that coregulator binding patterns could be adequately characterised using LBDs, Chapter 3 assessed the coregulator binding profiles of the LBDs belonging to the RAR alpha (RARα), beta (RARβ) and gamma (RARγ) focusing on possible differential outcomes between the different RARs. To this end, this study used the MARCoNI coregulator binding assay to characterize coregulator motif binding patterns of the distinct RAR subtypes upon incubation with the general agonist all-trans-Retinoic Acid (AtRA); the subtype-selective agonists Am80 (RARα), CD2314 (RARβ) and BMS961 (RARγ); and the antagonist Ro415253. Chapter 3 showed multiple ligand-dependent RAR-coregulator interactions for all RARs, including binding events not described before. It also confirmed a greater ligand-independent functioning of RARβ based on both higher basal and lower ligand-induced coregulator binding. In addition, despite the generally high similarity, various coregulator motifs showed subtype-selective binding, providing a basis for a potential role of coregulator binding in the receptor subtype-specific responses. Finally, Chapter 3 also proved that subtype-selective agonists and agonistic/antagonistic actions of ligands for all the RAR variants can be easily discriminated by the MARCoNI coregulator binding assay. After characterizing a possible role of coregulator binding in RAR subtype-specific responses, in chapter 4 and chapter 5 of this thesis, the crosstalk between retinoid and estrogen signaling in the estrogen receptor positive (ER+) breast cancer MCF7/BUS cell line was investigated. To this end, in chapter 4, the effects of AtRA on the basal and E2-induced estrogen receptor alpha (ERα) signaling were characterised using the MCF7/BUS and the U2OS-ERα-Luc cell lines. The effects of AtRA on different ERα-related events such as ERα-mediated cell proliferation and gene expression, ERα-coregulator binding and ERα subcellular localization were evaluated. While AtRA-driven inhibition of E2-induced signalling was found in the proliferation and gene expression studies, no significant effects on the E2-directed coregulator binding and subcellular distribution of ERα were observed. Strikingly, chapter 4 showed that in the absence of E2, basal ERα-mediated gene expression, ERα-coregulator binding (in lysate samples) and ERα nuclear localization were increased when exposing the cells to micromolar concentrations of AtRA. However, further coregulator binding assays making use of isolated ERα-LBDs suggested that direct binding of AtRA to ERα does not take place so that other molecular mechanisms must be involved in this phenomenon, which must be studied in the future. In Chapter 5 the studies performed in chapter 4 using AtRA were extended to the RAR subtype-selective ligands. Thus, it was investigated whether the distinct effects of RAR subtype-selective agonists on breast cancer cell proliferation could partially be driven by differential effects on the ERα pathway. To test that hypothesis, evaluation of the effects driven by the general agonist AtRA and the subtype-selective agonists Am80 (RARα), CD2314 (RARβ) and BMS961 (RARγ) on basal ERα-related events was carried out using the MCF7/BUS and the U2OS-ERα-Luc cell lines. The results obtained indicated that proliferation, ERα-mediated gene expression, ERα-coregulator binding and ERα subcellular localization can be affected by some of the retinoids in a compound-specific manner. Nonetheless, the responses did not reveal a clear distinction between the different effective (anti-proliferative) AtRA and Am80 and the more ineffective retinoids CD2314 and BMS961. In addition, the coregulator binding studies with the isolated ERα-LBD showed that none of the compounds modulated ERα by direct binding (ligand-receptor) suggesting other pathways as underlying causal factors of the distinct effects of RAR subtype-selective ligands on ERα signaling. From all retinoids tested, the general RAR-agonist AtRA and the RARβ agonist CD2314 displayed the most remarkable effects on ERα signaling. Upon AtRA and CD2314 treatment at micromolar concentrations up-regulation of ER-mediated transcription was observed. However these retinoids differed when comparing other ERα-related molecular events. Thus, while AtRA increased ERα nuclear localization and coregulator binding, CD2314 exerted no significant effects on these molecular endpoints. To obtain further insight in the crosstalk between retinoid and estrogen signaling in breast cancer, in chapter 6 of the present thesis, the effects of the general RAR agonist AtRA on the cell proliferation and the related gene expression profiles upon treatment with E2 were studied in the ER+ breast cancer cell line T47D. For this purpose, different assays determining the effects on cell proliferation and the transcriptomic pattern (RNA-seq) were used. In addition, the effects of AtRA on the specific ERα- and ERβ-mediated gene expression were assessed by means of reporter gene assays with the T47D-ERα-Luc and U2OS-ERβ-Luc cell lines. The results obtained from the proliferation and reporter gene studies pointed at AtRA as a repressor of the E2-induced breast cancer growth and as inhibitor of the E2-driven expression mediated by ERα and ERβ. To further understand the underlying modes of action of RAR-ER crosstalk, in a next step chapter 6 acquired further insights on the possible interplay between estrogen and retinoid signalling by means of a transcriptomic analysis. A high proportion of estrogen-responsive genes were found to be modulated by a single treatment with AtRA. In addition, RNA-seq data showed that AtRA can exert a variable modulation of the E2-induced transcriptomic responses. In other words, AtRA both counteracted and enhanced gene expression responses induced by E2. Further analysis showed that the estrogen-responsive genes whose E2-driven expression was counteracted upon co-exposure to AtRA were more often related to biological processes involved in breast cancer cell proliferation, differentiation or apoptosis. Altogether, the results of chapter 6 supported the existence of genomic crosstalk between estrogen and retinoid signaling eliciting distinct responses depending on the gene considered. However, co-treatment with AtRA more frequently presented inhibiting/counteracting effects on E2-induced genes involved in breast cancer growth. In Chapter 7, the studies assessing the effects of the general agonist AtRA on the proliferation and related transcriptomic patterns in the T47D breast cancer cell line were extended to the RAR subtype-selective agonists. The effects of the different retinoids on RAR or other NR pathways of importance in breast cancer cell proliferation (e.g. ER pathway) were assessed by analyzing gene expression patterns. This study showed that the general AtRA and the RARα agonist Am80 highly hampered T47D breast cancer cell proliferation while the RARβ agonist CD2314 and the RARγ agonist BMS961 showed to be ineffective anticancer agents which even slightly increased proliferative rates. The transcriptomic profiles clearly differentiated between the effective and ineffective retinoids showing multiple differences in gene expression. Chapter 7 also suggested RARα as the main RAR subtype involved in the effects of retinoids on gene expression as shown by the higher and relatively similar effects observed on the transcriptomic signature elicited by AtRA and Am80. Differences between the anti-proliferative retinoids (AtRA and Am80) and the ineffective retinoids (CD2314 and BMS961) mainly occurred in terms of magnitude of expression. Thus, relevant biological processes such as proliferation, differentiation and apoptosis and pathways such as the RAR, the ER, the VDR and the GR pathways were differentially modulated by the effective and ineffective retinoids. Altogether, the main conclusion drawn in this chapter was that small differences in effects on multiple pathways regulating breast cancer cell proliferation may sum up leading to the distinct responses elicited by the different retinoids in T47D breast cancer cells. Finally, in Chapter 8 we first provided an overview on the principal results generated throughout the present thesis. Next, we went in detail with a discussion of all the findings of the thesis. In this analysis, comparison with previous literature, implications of the results obtained and follow-up studies to be performed in the future, were incorporated.</p

The Gut Microbial Metabolite Pyrogallol Is a More Potent Inducer of Nrf2-Associated Gene Expression Than Its Parent Compound Green Tea (-)-Epigallocatechin Gallate

(-)-Epigallocatechin gallate (EGCG) has been associated with multiple beneficial effects. However, EGCG is known to be degraded by the gut microbiota. The present study investigated the hypothesis that microbial metabolism would create major catechol-moiety-containing microbial metabolites with different ability from EGCG to induce nuclear factor-erythroid 2-related factor 2 (Nrf2)-mediated gene expression. A reporter gene bioassay, label-free quantitative proteomics and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) were combined to investigate the regulation of Nrf2-related gene expression after exposure of U2OS reporter gene or Hepa1c1c7 cells in vitro to EGCG or to its major microbial catechol-moiety-containing metabolites: (-)-epigallocatechin (EGC), gallic acid (GA) and pyrogallol (PG). Results show that PG was a more potent inducer of Nrf2-mediated gene expression than EGCG, with a 5% benchmark dose (BMD5) of 0.35 µM as compared to 2.45 µM for EGCG in the reporter gene assay. EGC and GA were unable to induce Nrf2-mediated gene expression up to the highest concentration tested (75 µM). Bioinformatical analysis of the proteomics data indicated that Nrf2 induction by PG relates to glutathione metabolism, drug and/or xenobiotics metabolism and the pentose phosphate pathway. Taken together, our findings demonstrate that the microbial metabolite PG is a more potent inducer of Nrf2-associated gene expression than its parent compound EGCG

Contrasting dose response relationships of neuroactive antidepressants on the behavior of C. elegans

Antidepressant prescriptions are on a rise worldwide and this increases the concerns for the impacts of these pharmaceuticals on nontarget organisms. Antidepressants are neuroactive compounds that can affect organism’s behavior. Behavior is a sensitive endpoint that may also propagate effects at a population level. Another interesting aspect of antidepressants is that they have shown to induce non-monotonic dose-response (NMDR) curves. While such NMDR relationships may have clear implications for the environmental risk, the resolution of current studies is often too coarse to be able to detect relevant NMDR. Therefore, the current study was performed into the behavioral effects (activity, feeding and chemotaxis) in Caenorhabditis elegans as the model organism of the selective serotonin reuptake inhibitors fluoxetine and sertraline and the acetylcholinesterase inhibiting pesticide chlorpyrifos, using a wide range of concentrations (ng/l to mg/l). In order to statistically examine the non-monotonicity, nonlinear regression models were applied to the results. The results showed a triphasic dose-response relationship for activity and chemotaxis after exposure to fluoxetine, but not to sertraline or chlorpyrifos. Effects of fluoxetine already occurred at low concentrations in the range of ng/l while sertraline only showed effects at concentrations in the μg/l range, similar to chlorpyrifos. The different responses between fluoxetine and sertraline, both SSRIs, indicate that response patterns may not always be extrapolated from chemicals with the same primary mode of action. The effects of fluoxetine at low concentrations, in a non-monotonic manner, confirm the relevance of examining such responses at low concentrations

In Vitro Methodologies to Study the Role of Advanced Glycation End Products (AGEs) in Neurodegeneration

Advanced glycation end products (AGEs) can be present in food or be endogenously produced in biological systems. Their formation has been associated with chronic neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, and amyotrophic lateral sclerosis. The implication of AGEs in neurodegeneration is related to their ability to bind to AGE-specific receptors and the ability of their precursors to induce the so-called “dicarbonyl stress”, resulting in cross-linking and protein damage. However, the mode of action underlying their role in neurodegeneration remains unclear. While some research has been carried out in observational clinical studies, further in vitro studies may help elucidate these underlying modes of action. This review presents and discusses in vitro methodologies used in research on the potential role of AGEs in neuroinflammation and neurodegeneration. The overview reveals the main concepts linking AGEs to neurodegeneration, the current findings, and the available and advisable in vitro models to study their role. Moreover, the major questions regarding the role of AGEs in neurodegenerative diseases and the challenges and discrepancies in the research field are discussed

The Influence of Intracellular Glutathione Levels on the Induction of Nrf2-Mediated Gene Expression by α-Dicarbonyl Precursors of Advanced Glycation End Products

α-Dicarbonyl compounds, particularly methylglyoxal (MGO), glyoxal (GO), and 3-deoxyglucosone (3-DG), are highly reactive precursors for the formation of advanced glycation end products (AGEs). They are formed in vivo and during food processing. This study aimed to investigate the role of intracellular glutathione (GSH) levels in the induction of Nrf2-mediated gene expression by α-dicarbonyl compounds. The reactions between α-dicarbonyl compounds (MGO, GO, and 3-DG) and GSH were studied by LC-MS in a cell-free system. It was shown that these three α-dicarbonyl compounds react instantaneously with GSH, with the GSH-mediated scavenging decreasing in the order MGO > GO > 3DG. Furthermore, in a cell-based reporter gene assay MGO, GO, and 3-DG were able to induce Nrf2-mediated gene expression in a dose-dependent manner. Modulation of intracellular GSH levels showed that the cytotoxicity and induction of the Nrf2-mediated pathway by MGO, GO and 3-DG was significantly enhanced by depletion of GSH, while a decrease in Nrf2-activation by MGO and GO but not 3-DG was observed upon an increase of the cellular GSH levels. Our results reveal subtle differences in the role of GSH in protection against the three typical α-dicarbonyl compounds and in their induction of Nrf2-mediated gene expression, and point at a dual biological effect of the α-dicarbonyl compounds, being reactive toxic electrophiles and-as a consequence-able to induce Nrf2-mediated protective gene expression, with MGO being most reactive

Differences in kinetics and dynamics of endogenous versus exogenous advanced glycation end products (AGEs) and their precursors

Advanced glycation end products (AGEs) and their precursors, referred to as glycation products, are a heterogenous group of compounds being associated with adverse health effects. They are formed endogenously and in exogenous sources including food. This review investigates the roles of endogenously versus exogenously formed glycation products in the potential induction of adverse health effects, focusing on differences in toxicokinetics and toxicodynamics, which appeared to differ depending on the molecular mass of the glycation product. Based on the available data, exogenous low molecular mass (LMM) glycation products seem to be bioavailable and to contribute to dicarbonyl stress and protein cross-linking resulting in formation of endogenous AGEs. Bioavailability of exogenous high molecular mass (HMM) glycation products appears limited, while these bind to the AGE receptor (RAGE), initiating adverse health effects. Together, this suggests that RAGE-binding in relevant tissues will more likely result from endogenously formed glycation products. Effects on gut microbiota induced by glycation products is proposed as a third mode of action. Overall, studies which better discriminate between LMM and HMM glycation products and between endogenous and exogenous formation are needed to further elucidate the contributions of these different types and sources of glycation products to the ultimate biological effects

Estrogen receptor alpha (ERα)–mediated coregulator binding and gene expression discriminates the toxic ERα agonist diethylstilbestrol (DES) from the endogenous ERα agonist 17β-estradiol (E2)

Diethylstilbestrol (DES) is a synthetic estrogen and proven human teratogen and carcinogen reported to act via the estrogen receptor α (ERα). Since the endogenous ERα ligand 17β-estradiol (E2) does not show these adverse effects to a similar extent, we hypothesized that DES’ interaction with the ERα differs from that of E2. The current study aimed to investigate possible differences between DES and E2 using in vitro assays that detect ERα-mediated effects, including ERα-mediated reporter gene expression, ERα-mediated breast cancer cell (T47D) proliferation and ERα-coregulator interactions and gene expression in T47D cells. Results obtained indicate that DES and E2 activate ERα-mediated reporter gene transcription and T47D cell proliferation in a similar way. However, significant differences between DES- and E2-induced binding of the ERα to 15 coregulator motifs and in transcriptomic signatures obtained in the T47D cells were observed. It is concluded that differences observed in binding of the ERα with several co-repressor motifs, in downregulation of genes involved in histone deacetylation and DNA methylation and in upregulation of CYP26A1 and CYP26B1 contribute to the differential effects reported for DES and E2.</p

The effects of all-trans retinoic acid on estrogen receptor signaling in the estrogen-sensitive MCF/BUS subline

Estrogen receptor alpha (ERα) and retinoic acid receptors (RARs) play important and opposite roles in breast cancer growth. While exposure to ERα agonists such as 17β-estradiol (E2) is related to proliferation, RAR agonists such as all-trans retinoic acid (AtRA) induce anti-proliferative effects. Although crosstalk between these pathways has been proposed, the molecular mechanisms underlying this interplay are still not completely unraveled. The aim of this study was to evaluate the effects of AtRA on ERα-mediated signaling in the ERα positive cell lines MCF7/BUS and U2OS-ERα-Luc to investigate some of the possible underlying modes of action. To do so, this study assessed the effects of AtRA on different ERα-related events such as ERα-mediated cell proliferation and gene expression, ERα-coregulator binding and ERα subcellular localization. AtRA-mediated antagonism of E2-induced signaling was observed in the proliferation and gene expression studies. However, AtRA showed no remarkable effects on the E2-driven coregulator binding and subcellular distribution of ERα. Interestingly, in the absence of E2, ERα-mediated gene expression, ERα-coregulator binding and ERα subcellular mobilization were increased upon exposure to micromolar concentrations of AtRA found to inhibit cell proliferation after long-term exposure. Nevertheless, experiments using purified ERα showed that direct binding of AtRA to ERα does not occur. Altogether, our results using MCF7/BUS and U2OS-ERα-Luc cells suggest that AtRA, without being a direct ligand of ERα, can indirectly interfere on basal ERα-coregulator binding and basal ERα subcellular localization in addition to the previously described crosstalk mechanisms such as competition of ERs and RARs for DNA binding sites

Characterization of the differential coregulator binding signatures of the Retinoic Acid Receptor subtypes upon (ant)agonist action

Retinoic Acid Receptor alpha (RARα/NR1B1), Retinoic Acid Receptor beta (RARβ/NR1B2) and Retinoic Acid Receptor gamma (RARγ/NR1B3) are transcription factors regulating gene expression in response to retinoids. Within the RAR genomic pathways, binding of RARs to coregulators is a key intermediate regulatory phase. However, ligand-dependent interactions between the wide variety of coregulators that may be present in a cell and the different RAR subtypes are largely unknown. The aim of this study is to characterize the coregulator binding profiles of RARs in the presence of the pan-agonist all-trans-Retinoic Acid (AtRA); the subtype-selective agonists Am80 (RARα), CD2314 (RARβ) and BMS961 (RARγ); and the antagonist Ro415253. To this end, we used a microarray assay for coregulator-nuclear receptor interactions to assess RAR binding to 154 motifs belonging to > 60 coregulators. The results revealed a high number of ligand-dependent RAR-coregulator interactions among all RAR variants, including many binding events not yet described in literature. Next, this work confirmed a greater ligand-independent activity of RARβ compared to the other RAR subtypes based on both higher basal and lower ligand-driven coregulator binding. Further, several coregulator motifs showed selective binding to a specific RAR subtype. Next, this work showed that subtype-selective agonists can be successfully discriminated by using coregulator binding assays. Finally this study demonstrated the possible applications of a coregulator binding assay as a tool to discriminate between agonistic/antagonistic actions of ligands. The RAR-coregulator interactions found will be of use to direct further studies to better understand the mechanisms driving the eventual actions of retinoids.</p