Differential cellular metabolite alterations in HaCaT cells caused by exposure to the aryl hydrocarbon receptor-binding polycyclic aromatic hydrocarbons chrysene, benzo[a]pyrene and dibenzo[a,l]pyrene

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the human environment. Since they are present in crude oilfractions used for the production of rubber and plastics, consumers may come into direct dermal contacts with these compounds (e.g., via tool handles) on a daily basis. Some individual PAHs are identified as genotoxic mutagens thereby prompting particular toxicological and environmental concern. Among this group, benzo[a]pyrene (BAP) constitutes a model carcinogen which is also used as reference compound for risk assessment purposes. It acts as a strong agonist of the aryl hydrocarbon receptor (AHR) and becomes metabolically activated toward mutagenic and carcinogenic intermediates by cytochrome P450-dependent monooxygenases (CYPs). While BAP has been exhaustively characterized with regard to its toxicological properties, there is much less information available for other PAHs. We treated an AHR-proficient immortal human keratinocyte cell line (i.e., HaCaT) with three selected PAHs: BAP, chrysene (CRY) and dibenzo[a,l]pyrene (DALP). Compound-mediated alterations of endogenous metabolites were investigated by an LC–MS/MS-based targeted approach. To examine AHR-dependent changes of the measured metabolites, AHR-deficient HaCaT knockdown cells (AHR-KD) were used for comparison. Our results reveal that 24 metabolites are sufficient to separate the PAH-exposed cells from untreated controls by application of a multivariate model. Alterations in the metabolomics profiles caused by each PAH show influences on the energy and lipid metabolism of the cells indicating reduced tricarboxylic acid (TCA) cycle activity and β-oxidation. Up-regulation of sphingomyelin levels after exposure to BAP and DALP point to pro-apoptotic processes caused by these two potent PAHs. Our results suggest that in vitro metabolomics can serve as tool to develop bioassays for application in hazard assessment. Keywords: Polycyclic aromatic hydrocarbons, Metabolomics, Aryl hydrocarbon receptor, Keratinocyte

identification of biomarkers for risk assessment and insight into toxicologically relevant metabolic pathways in HaCaT and MCF-7 cells

1\. Einleitung 1 1.1. Metabolomics in der Toxikologie 1 1.1.1. Methodik 1 1.1.2. Anwendungsmöglichkeiten 4 1.2. Verbrauchernahe Chemikalien 6 1.2.1. PAKs 6 1.2.2. Endokrine Disruptoren 10 2\. Zielstellung 14 3\. Ergebnisse 17 3.1. Differential cellular metabolite alterations in HaCaT cells caused by exposure to the aryl hydrocarbon receptor-binding polycyclic aromatic hydrocarbons chrysene, benzo[a]pyrene and dibenzo[a,l]pyrene 17 3.2. Combination of metabolomics with cellular assays reveals new biomarkers and mechanistic insights on xenoestrogenic exposures in MCF-7 cells 19 3.3. Identification of lipidomic biomarkers for coexposure to subtoxic doses of benzo[a]pyrene and cadmium: the toxicological cascade biomarker approach 21 4\. Diskussion 23 4.1. Differentielle Veränderungen zellulärer Metabolite in HaCaT Zellen nach Exposition mit drei karzinogenen PAKs: CRY, BAP und DALP 23 4.1.1. PAK-Einfluss auf Viabilität und CYP-Expression in HaCaT Zellen 23 4.1.2. Eingriff in biochemische Stoffwechselwege 25 4.1.3. Schlussfolgerung 27 4.2. Identifizierung von Biomarkern und mechanistische Einblicke nach Exposition mit Xenoestrogenen durch eine Kombination von Metabolomanalysen und zellulären Assays in MCF-7 Zellen 28 4.2.1. Ko-Exposition von BPA oder E2 mit dem Schwermetall Cd2+ 29 4.2.2. Mechanistische Untersuchung des estrogenen Biomarkers Pro 30 4.2.3. Schlussfolgerung 33 4.3. Identifizierung von Lipidbiomarkern für die Ko-Exposition subtoxischer Dosen von BAP und Cd2+: der toxikologische „Kaskaden-Biomarker-Ansatz“ 34 4.3.1. Ko-Exposition von BAP und Cd2+ 34 4.3.2. „Kaskaden-Biomarker-Ansatz“ 37 4.3.3. Schlussfolgerung 38 5\. Übergreifende Interpretation und Ausblick 39 6\. Zusammenfassung 43 7\. Summary 45 8\. Abkürzungsverzeichnis 47 9\. Abbildungsverzeichnis 51 10\. Literaturverzeichnis 52 11\. Publikationsliste 65 11.1. Publikationen in Peer- Review Fachzeitschriften 63 11.2. Konferenzbeiträge 64In der vorliegenden Dissertation wurde Metabolomics mit zellulären Methoden kombiniert, um die Entwicklung neuer, auf toxischen Mechanismen basierenden, in vitro Testmethoden für die Risikobewertung von verbrauchernahen Chemikalien, insbesondere von Substanzmischungen, voranzutreiben. Im ersten Teil wurden erstmals die Wirkungen drei verschiedener PAKs CRY, BAP und DALP unterschiedlicher karzinogener Potenz und AHR Bindungsaffinität auf das Metabolom in HaCaT Zellen untersucht. Unterschiede in der CYP-Expression, sowie Viabilität exponierter Zellen zeigten die differentielle AHR-abhängige Wirkweise der drei PAKs. Über einen zielgerichteten Metabolomics Ansatz wurden 24 Metabolite als Biomarker identifiziert, welche die distinkte Wirkweise der einzelnen Verbindungen der Substanzgruppe der PAKs weitergehend herausstellten. Einflüsse auf den Energiestoffwechsel und den Lipidmetabolismus wurden hierbei abgeleitet. Im zweiten Teil wurden Effekte von E2, BPA und Cd2+ auf MCF-7 Zellen untersucht. Erstmals wurde eine Gruppe von elf Biomarkern identifiziert, welche nach E2 sowie nach BPA Exposition reguliert und daher mit estrogenen Effekten assoziiert wurde. Ko- Expositionsexperimente mit E2 oder BPA und dem Schwermetall Cd2+ zeigten lediglich einen geringen modulierenden Einfluss von Cd2+ auf das veränderte Metabolitmuster. Zelluläre Testmethoden, wie Expressionsexperimente zu ER- Zielgenen, zeigten keine Effekte durch Cd2+ in der Ko-Exposition. Die Aminosäure Pro wurde als estrogener Biomarker identifiziert. Eine Korrelation des Anstiegs der Pro-Konzentrationen mit Proliferationseffekten wurde bestätigt. Weiterführende Experimente zeigten die Abhängigkeit der Pro-Level- Erhöhung von ER-alpha. Außerdem wurde ein Einfluss des Onkogens MYC auf den betreffenden Signalweg ermittelt. Im dritten Teil wurden 18 Lipidbiomarker in MCF-7 Zellen für die Ko-Exposition von BAP und Cd2+ identifiziert. Das metabolomische Profil zeigte synergistische Effekte von BAP in Kombination von Cd2+. Als Schlüsselsignalweg wurde eine durch PC-PLC Aktivierung verstärkte Aktivierung des EGFRs HER2 identifiziert, welcher mit proliferativen und karzinogenen Eigenschaften der Testsubstanzen in Verbindung gebracht wurde. Durch gezielte Inhibition von Schritten im identifizierten Signalweg wurde ein mechanistischer „Kaskaden-Biomarker-Ansatz“ erarbeitet. Dieser wurde als in vitro Testsystem für Ko-Expositionen von PAKs, wie BAP, mit Schwermetallen, wie Cd2+, postuliert. Die Anwendbarkeit dieses Ansatzes für die Erfassung additiver und synergistischer Effekte wurde gezeigt. Die Untersuchungen dieser Arbeit zeigen das hohe Potenzial von Metabolomics Methoden für den Einsatz in der Risikobewertung. Es wurden neue metabolomische Biomarker identifiziert. Außerdem wurden über ergänzende zelluläre Methoden, sowie Anwendung spezifischer Inhibitoren, Einblicke in toxische Signalwege gewonnen. Darüber hinaus wird ein neuartiger Ansatz für ein in vitro Testsystem für Mischexpositionen präsentiert.In the presented work, metabolomics was combined with cellular assays, in order to promote the development of new, mechanism based, in vitro test systems for risk assessment of consumer related chemicals, especially of substance mixtures. In the first part, effects on the metabolome of HaCaT cells triggered by the three different PAHs: CRY, BAP and DALP were investigated for the first time. The studied PAHs possess different carcinogenic potencies and affinities to bind the AHR. Differences in CYP- expression and viability of exposed cells showed the distinct effects of the studied PAHs depending on AHR activation. A targeted metabolomics approach revealed 24 metabolomic biomarkers which further pointed out the differential effects of the investigated PAHs. Metabolite level changes indicated influences on energy and lipid metabolism. In the second part, effects of E2, BPA and Cd2+ on the metabolome of MCF-7 cells were investigated. For the first time, a set of eleven estrogenic biomarkers was identified that were regulated similarly upon E2 and BPA exposure. Co-exposure experiments of E2 or BPA, with or without the heavy metal Cd2+, showed a slight modulation of the estrogenic metabolite pattern. Cellular assays, e.g. expression studies of ER target genes, revealed no effects of Cd2+ in co-exposure experiments. The amino acid Pro was identified as most prominent estrogenic biomarker. Pro increase could be correlated with proliferation in the studied MCF-7 cells. Mechanistic investigations confirmed ER-alpha dependency. Further, the underlying signaling was found to involve the oncoprotein MYC. In the third part, 18 lipid biomarkers were identified in MCF-7 cells for co-exposure of BAP and Cd2+. The metabolomic pattern showed synergistic effects of BAP in combination with Cd2+. An increased activation of EGFR HER2 caused by enhanced activation of PC-PLC activation was identified as key signaling pathway. Activation of this mechanism was associated with proliferative and carcinogenic potencies of the test chemicals. Based on specific inhibition of steps in the identified pathway and examination of the resulting metabolomic pattern changes, a mechanistic “cascade biomarker approach” was established. This was postulated as in vitro test system for co-exposure scenarios of PAHs, like BAP, with heavy metals, like Cd2+. The approach is further suitable for the detection of additive and synergistic effects. This work shows the high potential of metabolomics approaches for the application in toxicological risk assessment. New metabolomic biomarkers were identified. Further, insights into the underlying toxic signaling pathways were gained through application of cellular assays and specific inhibitors. Moreover, a new approach for an in vitro test system for mixture toxicology is presented

Assessing oral health-related quality of life among older people in home-based care - survey results of the InSEMaP study in Germany

Abstract Background Older people receiving home-based care (HBC) often face barriers to access preventive oral health care (OHC) and dental treatments. Leading to deterioration of their oral healthcare. It is further deteriorated by factors such as increasing burden of systemic diseases, medicinal side effects, limited mobility, financial constraints and lack of professional OHC at home. Older people also struggle to maintain necessary daily oral hygiene, leading to malnutrition, weight loss, and a risk of a further health degradation. This cross-sectional survey aimed to investigate the oral health-related quality of life (OHRQoL) and their associated factors in HBC recipients. Methods 5,280 older people (≥ 60 years) living in Hamburg, who were in need of care and insured with statutory health insurance DAK-Gesundheit received the questionnaire, which included the German version of the Oral Health Impact Profile (OHIP G-14) and, the EQ-5D health-related quality of life (HRQoL) measure as well as further questions regarding the extent of informal social support, subjective oral health status, oral health behaviour, subjective cognitive status, and socio-demographic variables. Results The participants (n = 1,622) had a median age of 83.2 years, with 72.0% of the sample being female. Nearly two thirds of the sample reported that their independence or abilities were significantly impaired (care level 2). Regarding oral health impacts, 40.0% of the participants reported experiencing at least one of the fourteen possible prevalent impacts of the OHIP-G14 fairly often or very often. A multivariate regression model on the severity of oral health impacts revealed, that a better HRQoL, a positive perception of one’s own dental status, fewer visits to dental practices, and no need for support in OHC were associated with better OHRQoL. Conversely, respondents with a negative perception of their oral health status, more frequent visits to a dental practice, a need for support in OHC, and subjective memory impairment showed poorer OHRQoL. Conclusions The results highlight the risk for poor oral health among older people in HBC. We conclude that there is an urgent need to prioritise oral health, especially as poor oral health can further compromise the systemic wellbeing of these already care dependent population

Identification of Lipidomic Biomarkers for Coexposure to Subtoxic Doses of Benzo[<i>a</i>]pyrene and Cadmium: The Toxicological Cascade Biomarker Approach

The search for model bioassay systems indicating activation of different toxicological signaling pathways is one of the paramount goals of modern toxicology. Especially coexposure scenarios need to be investigated with respect to synergistic and interdependent effects for the activation of toxicological signaling pathways. The present study introduces an experimental in vitro model system for nontoxic and low-dose coexposures of human mammary carcinoma MCF-7 cells against polycyclic aromatic hydrocarbons (PAHs) such as benzo­[<i>a</i>]­pyrene (BP) and heavy metals such as cadmium. For the first time, a multivariate model that identifies 18 metabolic biomarkers has been shown to be sufficient to separate BP-treated cells from coexposed or control cells. A “toxicological pathway color code model” is introduced to visualize the results. Different biomarker subsets can be associated with specific HER2 signaling steps. A tiered cascade biomarker approach is proposed that could be used to identify profiles associated with tumorigenic potency of environmental toxicants in coexposure scenarios, including possible synergistic or additive effects

Combination of Metabolomics with Cellular Assays Reveals New Biomarkers and Mechanistic Insights on Xenoestrogenic Exposures in MCF‑7 Cells

The disruptive potential of xenoestrogens like bisphenol A (BPA) lies in their 17β-estradiol (E2)-like binding to estrogen receptors (ERs) followed by concomitant modulation of ER target gene expression. Unsurprisingly, most endocrine testing systems focus on the quantification of canonical transcripts or ER-sensitive reporters. However, only little information is available about the corresponding metabolomic changes <i>in vitro</i>. This knowledge gap becomes particularly relevant in the context of potential mixture effects, for example, as a consequence of coexposure to potentially estrogenically active pollutants (e.g., Cd²⁺). Such effects are often difficult to dissect with molecular tools, especially with regard to potential physiological relevance. Metabolomic biomarkers are well-suited to address this latter aspect as they provide a comprehensive readout of whole-cell physiology. Applying a targeted metabolomics approach (FIA-MS/MS), this study looked for biomarkers indicative of xenoestrogenic exposure in MCF-7 cells. Cells were treated with E2 and BPA in the presence or absence of Cd²⁺. Statistical analysis revealed a total of 11 amino acids and phospholipids to be related to the compound’s estrogenic potency. Co-exposure to Cd²⁺ modulated the estrogenic profile. However, the corresponding changes were found to be moderate with cellular assays such as the E-screen failing to record any Cd²⁺-specific estrogenic effects. Overall, metabolomics analysis identified proline as the most prominent estrogenic biomarker. Its increase could clearly be related to estrogenic exposure and concomitant ERα-mediated induction of proliferation. Involvement of the latter was confirmed by siRNA-mediated knockdown studies as well as by receptor inhibition. Further, the underlying signaling was also found to involve the oncoprotein MYC. Taken together, this study provides insights into the underlying mechanisms of xenoestrogenic effects and exemplify the strength of the complementary use of metabolomics and cellular and molecular assays

Pathway and time-resolved benzo[a]pyrene toxicity on Hepa1c1c7 cells at toxic and subtoxic exposure

Benzo­[<i>a</i>]­pyrene (B­[<i>a</i>]­P) is an environmental contaminant mainly studied for its toxic/carcinogenic effects. For a comprehensive and pathway orientated mechanistic understanding of the effects directly triggered by a toxic (5 μM) or a subtoxic (50 nM) concentration of B­[<i>a</i>]P or indirectly by its metabolites, we conducted time series experiments for up to 24 h to study the effects in murine hepatocytes. These cells rapidly take up and actively metabolize B­[<i>a</i>]­P, which was followed by quantitative analysis of the concentration of intracellular B­[<i>a</i>]P and seven representative degradation products. Exposure with 5 μM B­[<i>a</i>]P led to a maximal intracellular concentration of 1604 pmol/5 × 10⁴ cells, leveling at 55 pmol/5 × 10⁴ cells by the end of the time course. Changes in the global proteome (>1000 protein profiles) and metabolome (163 metabolites) were assessed in combination with B­[<i>a</i>]P degradation. Abundance profiles of 236 (both concentrations), 190 (only 5 μM), and 150 (only 50 nM) proteins were found to be regulated in response to B­[<i>a</i>]P in a time-dependent manner. At the endogenous metabolite level amino acids, acylcarnitines and glycerophospholipids were particularly affected by B­[<i>a</i>]­P. The comprehensive chemical, proteome and metabolomic data enabled the identification of effects on the pathway level in a time-resolved manner. So in addition to known alterations, also protein synthesis, lipid metabolism, and membrane dysfunction were identified as B­[<i>a</i>]P specific effects

Subtoxic concentrations of benzo[a]pyrene induce metabolic changes and oxidative stress in non-activated and affect the mTOR pathway in activated Jurkat T cells

Although the activation of immune cells is the first and thereby pivotal step in the immunological cascade, the current knowledge about the details of this process is quite limited. Recent studies have shown that aromatic compounds, such as B[a]P, influence the immune system even at low concentrations. We investigated the effect of a subtoxic B[a]P concentration (50 nM) on the proteome and the metabolome of non-activated and activated Jurkat T cells. The GeLC-MS/MS analysis yielded 2624 unambiguously identified proteins. In addition to typical regulatory pathways associated with T cell activation, pathway analysis by Ingenuity IPA revealed several metabolic processes, for instance purine and pyruvate metabolism. The activation process seems to be influenced by B[a]P suggesting an important role of the mTOR pathway in the cellular adaptation. B[a]P exposure of non-activated Jurkat cells induced signaling pathways such as protein ubiquitination and NRF2 mediated oxidative stress response as well as metabolic adaptation involving pyruvate, purine and fatty acid metabolism. Thus, we validated the proteome results by determining the concentrations of 183 metabolites with FIA-MS/MS and IC-MS/MS. Furthermore, we were able to show that Jurkat cells metabolize B[a]P to B[a]P-1,6-dione. The combined evaluation of proteome and metabolome data with an integrated, genome-scale metabolic model provided novel systems biological insights into the complex relation between metabolic and proteomic processes in Jurkat T cells during activation and subtoxic chemical exposure

Interaction of Systemic Morbidity and Oral Health in Ambulatory Patients in Need of Home Care (InSEMaP) : an observational study at the sector boundary between dental and general practice care in Germany

Introduction: Older people in need of home care are at risk of declining oral health as their visits to dentists are becoming less frequent due to restricted mobility. There is increasing evidence that poor oral health and systemic diseases are closely associated, for example, in cardiological, metabolic or neurodegenerative conditions. Thus, Interaction of Systemic Morbidity and Oral Health in Ambulatory Patients in Need of Home Care (InSEMaP) is investigating the need, provision and utilisation of oral healthcare, systemic morbidity and clinical status of the oral cavity in older people. Methods and analysis: InSEMaP consists of four subprojects (SP), all involving the target population of older people in need of home care. In SP1 part a, a sample is surveyed using a self-report questionnaire. In SP1 part b, stakeholders (general practitioners, dentists, medical assistants, family and professional caregivers) are interviewed regarding barriers and facilitators using focus groups and personal interviews. In SP2, a retrospective cohort study, health insurance claims data are examined to investigate the utilisation of oral healthcare, its association with systemic morbidity and healthcare costs. In SP3, a clinical observational study will assess the oral health of participants by a dentist’s visit at home. SP4 synthesises the results of SP1, SP2 and SP3 to develop integrated clinical pathways, identifying strategies to uphold oral healthcare in older people. In assessing and evaluating the process of oral healthcare, and its associated systemic morbidity, InSEMaP aims to improve general healthcare across the sector boundary of dental and general practitioner care.PeerReviewe