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Vitamin A (VA) deficiency in very low birth weight (VLBW) infants is associated with an increased risk for disorders related to kidney and lung maturation and function. VA losses through increased urinary retinol (ROH) excretion might contribute to this deficiency risk. The mechanism accounting for ROH loss in the urine has not yet been clarified. The aim of this study was to assess the excretion of ROH, retinol-binding protein 4 (RBP4) and transthyretin (TTR) in urine from VLBW infants in comparison with that in term infants in relation to kidney function. Urine specimens were collected from 15 VLBW infants (birth weight < 1,500 g) as well as from 20 term infants during the first 2 days after birth. ROH in urine was detectable in 14 of the 15 VLBW infants at a median concentration of 234 nmol/g creatinine. In the group of term infants, 17 of the 20 excreted ROH, but at an approximately five-times lower concentration (P<0.001). Excretion of RBP4 and TTR was also much higher in VLBW infants (both P<0.001). The urinary ROH excretion in VLBW infants may be related to the impaired tubular handling of its carrier proteins RBP4 and TTR. Thus, ROH excretion might contribute to an increased risk of VA deficiency, especially in VLBW infants.
Aims: Contrast media-induced nephropathy (CIN) is associated with increased morbidity and mortality. The renal endothelin system has been associated with disease progression of various acute and chronic renal diseases. However, robust data coming from adequately powered prospective clinical studies analyzing the short and long-term impacts of the renal ET system in patients with CIN are missing so far. We thus performed a prospective study addressing this topic.
Main methods: We included 327 patients with diabetes or renal impairment undergoing coronary angiography. Blood and spot urine were collected before and 24 h after contrast media (CM) application. Patients were followed for 90 days for major clinical events like need for dialysis, unplanned rehospitalization or death.
Key findings: The concentration of ET-1 and the urinary ET-1/creatinine ratio decreased in spot urine after CM application (ET-1 concentration: 0.91 +/- 1.23pg/ml versus 0.63 +/- 1.03pg/ml, p<0.001; ET-1/creatinine ratio: 0.14 +/- 0.23 versus 0.09 +/- 0.19, p<0.001). The urinary ET-1 concentrations in patients with CIN decreased significantly more than in patients without CIN (-0.26 +/- 1.42pg/ml vs. -0.79 +/- 1.69pg/ml, p=0.041), whereas the decrease of the urinary ET-1/creatinine ratio was not significantly different (non-CIN patients: -0.05 +/- 0.30; CIN patients: -0.11 +/- 0.21, p=0.223). Urinary ET-1 concentrations as well as the urinary ET-1/creatinine ratio were not associated with clinical events (need for dialysis, rehospitalization or death) during the 90day follow-up after contrast media exposure. However, the urinary ET-1 concentration and the urinary ET-1/creatinine ratio after CM application were higher in those patients who had a decrease of GFR of at least 25% after 90days of follow-up.
Significance: In general the ET-1 system in the kidney seems to be down-regulated after contrast media application in patients with moderate CIN risk. Major long-term complications of CIN (need for dialysis, rehospitalization or death) are not associated with the renal ET system. (C) 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license.
Background The use of iodine-based contrast agents entails the risk of contrast induced nephropathy (CIN). Radiocontrast agents elicit the third most common cause of nephropathy among hospitalized patients, accounting for 11-12% of cases. CIN is connected with clinically significant consequences, including increased morbidity, prolonged hospitalization, increased risk of complications, potential need for dialysis, and increased mortality rate. The number of in hospital examinations using iodine-based contrast media has been significantly increasing over the last decade. In order to protect patients from possible complications of such examinations, new biomarkers are needed that are able to predict a risk of contrast-induced nephropathy. Urinary and plasma cyclic guanosine monophosphate (cGMP) concentrations are influenced by renal function. Urinary cGMP is primarily of renal cellular origin. Therefore, we assessed if urinary cGMP concentration may predict major adverse renal events (MARE) after contrast media exposure during coronary angiography. Methods Urine samples were prospectively collected from non-randomized consecutive patients with either diabetes or preexisting impaired kidney function receiving intra-arterial contrast medium (CM) for emergent or elective coronary angiography at the Charite Campus Mitte, University Hospital Berlin. Urinary cGMP concentration in spot urine was analyzed 24 hours after CM exposure. Patients were followed up over 90 days for occurrence of death, initiation of dialysis, doubling of plasma creatinine concentration or MARE. Results In total, 289 consecutive patients were included into the study. Urine cGMP/creatinine ratio 24 hours before CM exposure expressed as mean +/- SD was predictive for the need of dialysis (no dialysis: 89.77 +/- 92.85 mu M/mM, n = 277; need for dialysis: 140.3 +/- 82.90 mu M/mM, n = 12, p = 0.008), death (no death during follow-up: 90.60 +/- 92.50 mu M/mM, n = 280; death during follow-up: 169.88 +/- 81.52 mu M/mM, n = 9; p = 0.002), and the composite endpoint MARE (no MARE: 86.02 +/- 93.17 mu M/mM, n = 271; MARE: 146.64 +/- 74.68 mu M/mM, n = 18, p<0.001) during the follow-up of 90 days after contrast media application. cGMP/creatinine ratio stayed significantly increased at values exceeding 120 pM/mM in patients who developed MARE, required dialysis or died. Conclusions Urinary cGMP/creatinine ratio >= 120 mu M/mM before CM exposure is a promising biomarker for the need of dialysis and all-cause mortality 90 days after CM exposure in patients with preexisting renal impairment or diabetes.
Der Embryonale Stammzelltest (EST) ist ein validierter In-vitro-Embryotoxizitätstest, der zur Untersuchung embryotoxischer Wirkungen von Chemikalien eingesetzt werden kann. Während des zehntägigen Differenzierungsassays differenzieren sich die pluripotenten murinen embryonalen Stammzellen (ES-Zellen) der Linie D3 in vitro in spontan kontrahierende Herzmuskelzellen. Dabei rekapitulieren sie Prozesse der frühen Embryogenese in vivo. Ein Zytotoxizitätsassay mit D3-Zellen und ausdifferenzierten, adulten 3T3-Maus-Fibroblasten dient der Ermittlung allgemeiner zytotoxischer Effekte und unterschiedlicher Sensitivitäten beider Zelllinien. Somit basiert der EST auf den beiden wichtigsten Mechanismen pränataler Toxizität, der Störung der Differenzierung und der Zytotoxizität. Ziel dieser Arbeit war es, mit Hilfe des EST das embryotoxische Potential der vier Chemikalien Trichostatin A (TSA), Methylazoxymethanolacetat (MAMac), Natriumdodecylsulfat (SDS) und Benzoesäure (BA) abzuschätzen. Dazu wurde mikroskopisch ermittelt, bei welcher Testsubstanzkonzentration in 50 % der während der In-vitro-Differenzierung gebildeten Embryonalkörperchen die Kardiomyozytendifferenzierung inhibiert wird (ID50). Außerdem wurde die halbmaximale Hemmkonzentration des Zellwachstums auf die beiden Zelllinien bestimmt (IC50D3 bzw. IC503T3). Als Erweiterung dieses konventionellen EST wurden mittels quantitativer Real Time-PCR an den Tagen 5, 7 und 10 der Differenzierung zusätzlich Genexpressionsanalysen etablierter herzmuskelspezifischer Markergene (Mesoderm Posterior 1, Tag 5; Myosin light chain 1, Tag 7 und 10) durchgeführt. Deren Expression korreliert in den ES-Zellen mit der embryonalen Herzdifferenzierung in vivo und kann zur Ermittlung der von der Prüfsubstanz hervorgerufenen halbmaximalen Hemmung der Genexpression in den Kardiomyozyten (IC50 Exp) herangezogen werden. Um letztlich embryotoxische Effekte in vivo auf Grundlage der ermittelten In-vitro-Daten abschätzen zu können, wurden die ermittelten Parameter mittels eines für den EST empirisch abgeleiteten mathematischen Prädiktionsmodells (PM) zur Klassifizierung der Testsubstanzen als nicht, schwach oder stark embryotoxisch herangezogen. Für jede der Substanzen waren die ermittelten Halbhemmkonzentrationen in den überwiegenden Fällen vergleichbar und führten unter Verwendung des PMs im konventionellen und im molekularen EST zu deren identischer Klassifizierung. TSA wurde als „stark embryotoxisch“ klassifiziert und beeinflusste insbesondere das Differenzierungspotential der ES-Zellen. Das als „schwach embryotoxisch“ klassifizierte SDS wirkte auf die D3-Zellen stärker differenzierungsinhibierend als zytotoxisch, hemmte jedoch das Wachstum der 3T3-Zellen bereits in deutlich niedrigeren Konzentrationen. MAMac und BA wurden als „nicht embryotoxisch“ klassifiziert. Bei ihnen stand die zytotoxische Wirkung deutlich im Vordergrund. Diese Prädiktionen stimmten mit In-vivo-Befunden überein, was von der Stabilität und der Brauchbarkeit der im konventionellen und molekularen EST ermittelten Parameter zeugte. Einzige Ausnahme war das als Entwicklungsneurotoxin in vivo bekannte MAMac. Da der EST auf mesodermaler Differenzierung basiert, können spezifische Effekte auf neuronale Entwicklungsprozesse offenbar nicht vollständig erfasst werden. Substanzkonzentrationen, die sich als differenzierungsinhibierend auf die morphologische Kardiomyozytendifferenzierung erwiesen haben, führten auch zu einer messbaren Repression der herzmuskelspezifischen Genexpression. Dabei erwies sich die IC50 Exp als ebenso sensitiv wie die konventionellen Parameter und als nutzbringende Ergänzung zu diesen, da sie bereits nach 5 bzw. 7 Tagen der In-vitro-Differenzierung eine mit dem mikroskopischen Parameter übereinstimmende Einschätzung des embryotoxischen Potentials der Chemikalien in vivo ermöglichte. Genexpressionsanalysen weiterer differenzierungsspezifischer Gene können zusätzlich zur Aufklärung zu Grunde liegender Mechanismen der Embryotoxizität von Testsubstanzen dienen. Somit kann der EST durch die Vorteile der Stammzelltechnologie und der Genexpressionsanalyse als neues prädiktives Screening-Instrument zur frühzeitigen Detektion embryotoxischer Substanzeffekte in der pharmazeutischen und chemischen Industrie genutzt werden.
Die Zahl der Kolonkarzinome in den westlichen Industrieländern steigt in den letzten Jahren stetig an. Zu den Verbindungen, die mit der Zubereitung der Nahrung entstehen, mit ihr aufgenommen werden und die Kolonkanzerogenese möglicherweise begünstigen, gehört das heterozyklische aromatische PhIP, das bei der Erhitzung proteinreicher Nahrungsmittel entsteht. Neben zahlreichen Fütterungsversuchen an Nagern existieren auch Zellkulturmodelle zur Untersuchung der molekularen Mechanismen der PhIP-induzierten Kolonkanzerogenese. Die chemische Transformation von Zellen sollte durch wiederholte Exposition gegenüber dem hydroxylierten Metaboliten des Kanzerogens (N2-OH-PhIP) erzielt werden. Es wurden IEC-18-Zellen der Ratte und HCEC-Zellen des Menschen zur Untersuchung verwendet. Die Behandlung der IEC-18-Zellen führt nach 25 Behandlungszyklen mit Konzentrationen von 5 bis 20 µM nicht zur Transformation der Zellen. Die Anwesenheit von N2-OH-PhIP führt zu einer zehnfach erhöhten Induktion der GST-Aktivität, insbesondere der Untereinheiten GST-A1, -A3, -Pi und -T2, die für die effiziente Detoxifizierung des N-Acetoxy-Metaboliten vom N2-OH-PhIP verantwortlich sind. Bereits nach drei Behandlungen mit 1,5 µM N2-OH-PhIP konnte eine maligne Transformation der HCEC-Zellen erzielt werden. Die Zellen zeigten die charakteristischen Zeichen der Transformation: veränderte Wachstumseigenschaften wie klonales dreidimensionales Zellwachstum („pilling up“), Hemmung der Zell-Zell-Kontaktinhibierung, verkürzte Populationsverdopplungszeiten und tumorigene und metastasierende Eigenschaften. Außerdem exprimierten die N2-OH-PhIP-exponierten humanen Kolonzellen mit steigender Anzahl der Behandlungen größere Mengen des trunkierten APC-Proteins. Die bekannten PhIP-spezifischen Mutationen im APC-Gen resultieren in der Expression eines trunkierten Proteinproduktes und werden als frühe Ereignisse in der Kolonkanzerogenese betrachtet. Die zusammenfassende Betrachtung aller Ergebnisse zeigt, dass die IEC-18-Zelllinie zur chemischen Transformation durch N2-OH-PhIP ungeeignet ist. Dagegen wurde erstmalig eine vollständige chemische Transformation von Humandickdarmepithelzellen in vitro durch Exposition der humanen Kolonepithelzelllinie HCEC gegenüber dem Kolonkarzinogen N2-OH-PhIP erzielt.
Untersuchungen zum Transfer von Carotinoiden aus dem Plasma in die Follikelflüssigkeit der Frau
(2000)
Am Beispiel der Wiederkäuer wurde unter Zuhilfenahme von biochemischen und molekularbiologischen Methoden die Adaptation von Pflanzenfressern (Herbivoren) an pflanzliche Sekundärmetabolite wie z.B. Tannine untersucht. Tannine können in nicht an ihren Verzehr adaptierten Spezies durch ihr Proteinbindungsvermögen die Nahrungsverwertung und damit Wachstum und Gesundheit des Pflanzenfressers beeinträchtigen (antinutritive Wirkung). Einige Wiederkäuerarten wie z.B. das Reh (Capreolus capreolus) haben in ihrem Nahrungsspektrum viele stark tanninhaltige Pflanzen, leiden aber nicht unter den erwähnten postdigestiven Konsequenzen. Eine Möglichkeit, die antinutritive Wirkung von Tanninen zu neutralisieren, besteht in der Produktion tanninbindender Speichelproteine. Der Speichel verschiedener Wiederkäuerarten wurde auf das Vorhandensein tanninbindender Proteine untersucht. Diese Arten wurden so ausgewählt, dass alle drei Ernährungstypen (Konzentratselektierer, Intermediärtyp, Gras- und Rauhfutterfresser) in den Vergleich eingeschlossen werden konnten. Als Referenzspezies wurde der Konzentratselektierer Reh herangezogen. Die Speichelproteine des Rehs und die der Intermediärtypen (Rentier, Rangifer tarandus; Damhirsch, Cervus dama; Moschusochse, Ovibos moschatus) banden ungefähr doppelt so effektiv an hydrolysierbare Tannine (Tanninsäure), wie die der untersuchten Gras- und Rauhfutterfresser (Rind, Bos taurus; und Mufflon, Ovis orientalis musimon). Diese Abstufung zeigte sich auch bei der Untersuchung der Bindung an kondensierte Tannine (Quebracho). Eine Ausnahme stellte Mufflonspeichel dar, dieser band ebenso gut an Quebracho wie die Speichelproteine der anderen Ernährungstypen. Über eine Aminosäuretotalanalyse konnte festgestellt werden, dass der Speichel einiger untersuchter Wiederkäuerarten prolinreiche Proteine (PRPs) enthielt. Unter Ausnutzung ihrer Trichloressigsäure (TCA)-Löslichkeit wurden diese angereichert und genauer untersucht. Die Analyse der TCA-löslichen Speichelproteine der Konzentratselektierer (Reh, Elch) ergab einen relativen Prolingehalt von über 35 %, während beim Moschusochsen noch 29 % gemessen wurden. In Damhirsch- und Rinderspeichel wurden keine prolinreichen Proteine gefunden. Für die TCA-löslichen Speichelproteine des Rehs konnte eine hohe Tanninbindungskapazität nachgewiesen werden. Diese banden 24 - 30 x effektiver an Tannine als die TCA-löslichen Speichelproteine des Rindes. Die Tanninbindungskapazitäten der TCA-löslichen Speichelproteine von Moschusochse und Damhirsch waren ebenfalls höher als die des Rindes, aber niedriger als die des Rehs. Die Kohlenhydrat-Analyse der TCA-löslichen Speichelproteine des Rehs erbrachte, dass es sich bei ihnen um Glykoproteine handelt. Mittels Gelfiltration und zweidimensionaler Polyacrylamidgelektrophorese konnten fünf Proteingruppen mit Molekulargewichten zwischen 15 und 50 kd sowie isoelektrischen Punkten zwischen 4,0 und 8,2 detektiert werden. Von 15 dieser Proteine konnten die N-terminalen Aminosäuresequenzen ermittelt werden. Ausgehend von diesen Informationen wurden Reh-PRP spezifische mRNAs isoliert und partiell sequenziert. Die meisten dieser Fragmente hatten eine gemeinsame 18 Aminosäuren lange C-terminale Sequenz PPPEEQPEE/QSPDEE/DSPSE. Die Suche nach Übereinstimmungen der analysierten Sequenzen mit anderen Säugetier-PRPs in der Genbank ergab keine sinnvollen Ähnlichkeiten. Die Ergebnisse können zu Informationen über tanninbindende Proteine anderer Wiederkäuer führen. Die Sequenzinformationen stellen einen Ausgangspunkt bei der Analyse der evolutiven Zusammenhänge der Cerviden dar.
"Untersuchung kardioprotektiver Wirkungen des Olivenöles und seiner phenolischen Komponenten in einer Gruppe gesunder deutscher Männer" EINLEITUNG: Epidemiologische Daten belegen, dass die mediterrane Ernährung mit einer niedrigen Inzidenz an mit oxidativen Stress assoziierten kardiovaskulären Erkrankungen einhergeht. Dabei wird vor allem dem Olivenöl, als Hauptfettlieferant in der mediterranen Ernährung, eine kardioprotektive Wirkung zugesprochen. Olivenöl zeichnet sich neben dem hohen Gehalt an einfach ungesättigten Fettsäuren (MUFA) durch ein reichhaltiges Spektrum an phenolischen Verbindungen aus, deren antioxidative Wirkung bereits zahlreichen in in vitro Studien beschrieben wurde. Demnach könnte der Verzehr von phenolreichem Olivenöl auch in vivo vor oxidativen Schädigungen schützen und somit das Risiko für kardiovaskuläre Erkrankungen senken. ZIELSTELLUNG: Untersuchung der kardioprotektiven Wirkung von Olivenöl und seiner phenolischen Komponenten in einer Gruppe gesunder deutscher Männer. METHODE: Dazu wurde eine randomisierte cross-over doppelt-verblindete Interventionsstudie an 70 gesunden Männern zwischen 20 - 60 Jahren im Raum Berlin-Brandenburg durchgeführt. In jeweils drei dreiwöchigen Interventionsphasen konsumierten die Probanden täglich 25 ml natives (phenolreich), gemischtes (mittlerer Phenolgehalt) und raffiniertes (annähernd phenolfrei) Olivenöl, was sich ausschließlich im Gehalt an phenolischen Verbindungen unterschied. Das Olivenöl sollte dabei die gewöhnlich verzehrten Fette ersetzen. Die Interventionsphasen waren durch zweiwöchige Wash out-Phasen unterbrochen. Die Erhebung der Blutlipide, Biomarker der Lipidperoxidation und endogene Antioxidantien erfolgte zu Studienbeginn sowie zu Beginn und Ende jeder Verzehrsperiode.ERGEBNISSE: Bei den Blutlipiden sowie den Biomarkern der Lipidperoxidation und den endogenen Antioxidantien konnte keine signifikante Veränderung in Abhängigkeit vom Phenolgehalt der applizierten Olivenöle nachgewiesen werden. Einzig die Glutathion-Reduktase-Aktivität stieg mit zunehmendem Gehalt an phenolischen Verbindungen (pTrend = 0,041). Unabhängig von der Konzentration der Phenole im Olivenöl wurde bei den Probanden durch den Olivenölverzehr eine Senkung von Gesamtcholesterol (p = 0,007) und Triglyzeride (p = 0,013) im Serum erzielt. Diese Wirkung geht einher mit einem gestiegenen MUFA-Anteil in der Ernährung aufgrund des Olivenölkonsums (p < 0,001). SCHLUSSFOLGERUNG: Die Hypothese, dass die Phenole im Olivenöl aufgrund ihrer in in vitro und Tierstudien beschriebenen antioxidativen Wirkung dem Olivenöl neben dem einzigartigen Fettsäureprofil eine zusätzliche kardioprotektive Wirkung bescheren, konnte in der vorliegenden Studie nicht gezeigt werden. Dennoch konnte durch den Olivenölverzehr und der damit einhergehenden Erhöhung des MUFA-Anteils in der Ernährung eine vorteilhafte Beeinflussung der Blutlipide erzielt werden. Obgleich Olivenöl nicht das vorwiegend verzehrte Fett in Deutschland darstellt, zeigten die befragten Probanden eine hohe Akzeptanz. Folglich könnte die Integration von Olivenöl in die habituelle Ernährung einen Beitrag zur Senkung des kardiovaskulären Erkrankungsrisikos leisten.
Untersuchung des Recyclings Kaede-fusionierter Corticotropin-Releasing-Factor Rezeptoren Typ 1
(2009)
Aktivierte G-Protein-gekoppelte Rezeptoren (GPCR) werden schnell desensitisiert, internalisiert und anschließend entweder lysosomal degradiert oder zur Plasmamembran (PM) recycelt. Zur Resensitisierung der Zellen tragen neben recycelten auch neusynthetisierte Rezeptoren bei. Die Überlagerung beider Prozesse erschwert die Untersuchung des Rezeptorrecyclings. In dieser Arbeit sollte mit Hilfe des photokonvertierbaren Fluoreszenzproteins Kaede eine Technik entwickelt werden, mit der es möglich ist Recycling- von Neusyntheseprozessen zu trennen und das Recycling von GPCR mikroskopisch in Echtzeit zu beobachten. Als Modellproteine wurden der Vasopressin-1a-Rezeptor V1aR (recycelnder Rezeptor), der Vasopressin-2-Rezeptor V2R (degradierter Rezeptor) und der Corticotropin-Releasing Factor-Rezeptor Typ 1 (CRF1R) verwendet, wobei bei Letzterem untersucht werden sollte, ob er nach Stimulation zur PM zurücktransportiert wird. Da Kaede als fluoreszierendes Protein mit den GPCR fusioniert wird, wurde zunächst überprüft, ob es die Eigenschaften der Rezeptoren verändert und generell für Transportstudien geeignet ist. Eventuell könnte die bereits publizierte Tetramerisierung von Kaede seine Anwendung verhindern oder erschweren. Mittels Fluoreszenz-Korrelationsspektroskopie konnte gezeigt werden, dass Kaede nicht tetramerisiert, wenn es an ein Membranprotein fusioniert ist. Außerdem konnte in in vitro- und Zellkulturexperimenten belegt werden, dass die native und die photokonvertierte Form von Kaede gleichermaßen stabil sind. Darüber hinaus zeigten Kaede-fusionierte GPCR sowohl in Kolokalisationsstudien als auch in Agonistbindungs- und Rezeptoraktivierungsexperimenten die gleichen Eigenschaften wie CFP- bzw. die unfusionierte Rezeptoren. Lediglich die Expression der Kaede-fusionierten Rezeptoren war geringer. Parallel wurde anhand der bereits publizierten Kaede-Struktur versucht, die Tetramerisierung des Proteins durch den Austausch interagierender Aminosäuren zu unterbinden. Die eingeführten Mutationen bewirkten aber eine Fehlfaltung des Proteins und damit den Verlust der Fluoreszenz. Da zuvor gezeigt werden konnte, dass Kaede-fusionierte Membranproteine nicht tetramerisieren und nicht die Eigenschaften der fusionierten Proteine verändern, war monomerisiertes Kaede zur Untersuchung des Rezeptorrecyclings nicht notwendig. Im zweiten Teil der Arbeit wurde mit Hilfe von Kaede-Fusionsproteinen und mikroskopischer Testsysteme das noch unbekannte Recyclingverhalten des CRF1R untersucht. Hierfür wurden die Kaede-fusionierten Rezeptoren in eukaryotischen Zellen exprimiert und mit Agonisten internalisiert. Die internalisierten Rezeptoren wurden in Endosomen selektiv mit UV-Strahlung photokonvertiert. Anschließend wurde der Transport der photokonvertierten Form verfolgt. Sowohl beim CRF1R als auch beim V1aR wurden Signale in der PM detektiert, beim V2R hingegen nicht. Dies zeigt, dass es sich beim CRF1R um einen recycelnden Rezeptor handelt. Die als Kontrolle eingesetzten Rezeptoren verhielten sich in diesem Experiment wie erwartet: Der V1aR wurde zur PM zurücktransportiert, der V2R nicht. Diese Ergebnisse konnten mit Hilfe biochemischer und durchflusscytometrischer Experimente bestätigt werden. Die Internalisierung des CRF1R verläuft Clathrin-vermittelt in Anwesenheit von β-Arrestin. Je nach Stabilität der β Arrestin-Interaktion unterscheidet man zwei Klassen von Rezeptoren: Klasse A-Rezeptoren interagieren transient mit β Arrestin und können recyceln. Im Gegensatz dazu gehen Klasse B-Rezeptoren eine stabile Interaktion mit β Arrestin ein und werden nach Internalisierung degradiert. In mikroskopischen Untersuchungen konnte für die aktivierten CRF1R und V1aR eine Rekrutierung von β Arrestin zur PM und eine transiente Interaktion mit β Arrestin gezeigt werden (Klasse A-Rezeptoren). Für den V2R wurde dagegen eine stabile Interaktion mit β Arrestin beobachtet (Klasse B-Rezeptor). Diese Daten stützen die Ergebnisse des Kaede-basierten Recyclingversuchs und zeigen, dass der CRF1R ein recycelnder Rezeptor ist. Ferner wurde untersucht, ob der CRF1R zu den schnell oder langsam recycelnden Rezeptoren zählt. Schnell recycelnde Rezeptoren werden direkt aus frühen Endosomen, langsam recycelnde hingegen über das Trans-Golgi-Netzwerk (TGN) bzw. über Recycling-Endosomen zur PM transportiert. Als Marker für das TGN oder die Recycling-Endosomen wurde Rab11 verwendet. In Kolokalisationsstudien konnte gezeigt werden, dass der CRF1R den langsam recycelnden Rezeptoren zugeordnet werden kann. Zusammenfassend konnte in dieser Arbeit belegt werden, dass Kaede als Fusionspartner für Membranproteine genutzt werden kann um deren Transport in Echtzeit zu studieren. Damit wurde erstmals eine mikroskopische Methode etabliert, die es erlaubt recycelnde von neusynthetisierten Rezeptoren zu unterscheiden. Mit Hilfe dieser Methode war es möglich zu zeigen, dass der CRF1R ein recycelnder Rezeptor ist.
Manganese (Mn) is an essential micronutrient for development and function of the nervous system. Deficiencies in Mn transport have been implicated in the pathogenesis of Huntington's disease (HD), an autosomal dominant neurodegenerative disorder characterized by loss of medium spiny neurons of the striatum. Brain Mn levels are highest in striatum and other basal ganglia structures, the most sensitive brain regions to Mn neurotoxicity. Mouse models of HD exhibit decreased striatal Mn accumulation and HD striatal neuron models are resistant to Mn cytotoxicity. We hypothesized that the observed modulation of Mn cellular transport is associated with compensatory metabolic responses to HD pathology. Here we use an untargeted metabolomics approach by performing ultraperformance liquid chromatography-ion mobility-mass spectrometry (UPLC-IM-MS) on control and HD immortalized mouse striatal neurons to identify metabolic disruptions under three Mn exposure conditions, low (vehicle), moderate (non-cytotoxic) and high (cytotoxic). Our analysis revealed lower metabolite levels of pantothenic acid, and glutathione (GSH) in HD striatal cells relative to control cells. HD striatal cells also exhibited lower abundance and impaired induction of isobutyryl carnitine in response to increasing Mn exposure. In addition, we observed induction of metabolites in the pentose shunt pathway in HD striatal cells after high Mn exposure. These findings provide metabolic evidence of an interaction between the HD genotype and biologically relevant levels of Mn in a striatal cell model with known HD by Mn exposure interactions. The metabolic phenotypes detected support existing hypotheses that changes in energetic processes underlie the pathobiology of both HD and Mn neurotoxicity.
Manganese (Mn) is an essential micronutrient for development and function of the nervous system. Deficiencies in Mn transport have been implicated in the pathogenesis of Huntington's disease (HD), an autosomal dominant neurodegenerative disorder characterized by loss of medium spiny neurons of the striatum. Brain Mn levels are highest in striatum and other basal ganglia structures, the most sensitive brain regions to Mn neurotoxicity. Mouse models of HD exhibit decreased striatal Mn accumulation and HD striatal neuron models are resistant to Mn cytotoxicity. We hypothesized that the observed modulation of Mn cellular transport is associated with compensatory metabolic responses to HD pathology. Here we use an untargeted metabolomics approach by performing ultraperformance liquid chromatography-ion mobility-mass spectrometry (UPLC-IM-MS) on control and HD immortalized mouse striatal neurons to identify metabolic disruptions under three Mn exposure conditions, low (vehicle), moderate (non-cytotoxic) and high (cytotoxic). Our analysis revealed lower metabolite levels of pantothenic acid, and glutathione (GSH) in HD striatal cells relative to control cells. HD striatal cells also exhibited lower abundance and impaired induction of isobutyryl carnitine in response to increasing Mn exposure. In addition, we observed induction of metabolites in the pentose shunt pathway in HD striatal cells after high Mn exposure. These findings provide metabolic evidence of an interaction between the HD genotype and biologically relevant levels of Mn in a striatal cell model with known HD by Mn exposure interactions. The metabolic phenotypes detected support existing hypotheses that changes in energetic processes underlie the pathobiology of both HD and Mn neurotoxicity.
The regulation of energy homeostasis is controlled by the brain and, besides requiring high amounts of energy, it relies on functional insulin/insulin-like growth factor (IGF)-1 signalling in the central nervous system. This energy is mainly provided by mitochondria in form of ATP. Thus, there is an intricate interplay between mitochondrial function and insulin/IGF-1 action to enable functional brain signalling and, accordingly, propagate a healthy metabolism. To adapt to different nutritional conditions, the brain is able to sense the current energy status via mitochondrial and insulin signalling-dependent pathways and exerts an appropriate metabolic response. However, regional, cell type and receptor-specific consequences of this interaction occur and are linked to diverse outcomes such as altered nutrient sensing, body weight regulation or even cognitive function. Impairments of this cross-talk can lead to obesity and glucose intolerance and are linked to neurodegenerative diseases, yet they also induce a self-sustainable, dysfunctional 'metabolic triangle' characterised by insulin resistance, mitochondrial dysfunction and inflammation in the brain. The identification of causal factors deteriorating insulin action, mitochondrial function and concomitantly a signature of metabolic stress in the brain is of utter importance to offer novel mechanistic insights into development of the continuously rising prevalence of non-communicable diseases such as type 2 diabetes and neurodegeneration. This review aims to determine the effect of insulin action on brain mitochondrial function and energy metabolism. It precisely outlines the interaction and differences between insulin action, insulin-like growth factor (IGF)-1 signalling and mitochondrial function; distinguishes between causality and association; and reveals its consequences for metabolism and cognition. We hypothesise that an improvement of at least one signalling pathway can overcome the vicious cycle of a self-perpetuating metabolic dysfunction in the brain present in metabolic and neurodegenerative diseases.
Analyzing mixture toxicity requires an in-depth understanding of the mechanisms of action of its individual components. Substances with the same target organ, same toxic effect and same mode of action (MoA) are believed to cause additive effects, whereas substances with different MoAs are assumed to act independently. Here, we tested 2 triazole fungicides, propiconazole, and tebuconazole (Te), for individual and combined effects on liver toxicity-related endpoints. Both triazoles are proposed to belong to the same cumulative assessment group and are therefore thought to display similar and additive behavior. Our data show that Te is an antagonist of the constitutive androstane receptor (CAR) in rats and humans, while propiconazole is an agonist of this receptor. Both substances activate the pregnane X-receptor (PXR) and further induce mRNA expression of CYP3A4. CYP3A4 enzyme activity, however, is inhibited by propiconazole. For common targets of PXR and CAR, the activation of PXR by Te overrides CAR inhibition. In summary, propiconazole and Te affect different hepatotoxicity-relevant cellular targets and, depending on the individual endpoint analyzed, act via similar or dissimilar mechanisms. The use of molecular data based on research in human cell systems extends the picture to refine cumulative assessment group grouping and substantially contributes to the understanding of mixture effects of chemicals in biological systems.
Ultrasound evaluation of the patellar tendon cross-sectional area and its relation to maximum force
(2012)
Detection of cancer precursors contributes to cancer prevention, for example, in the case of colorectal cancer. To record more patients early, ultrasensitive methods are required for the purpose of noninvasive precursor detection in body fluids. Our aim was to develop a method for enrichment and detection of known as well as unknown driver mutations in the Adenomatous polyposis coli (APC) gene. By coupled wild-type blocking (WTB) PCR and high-resolution melting (HRM), referred to as WTB-HRM, a minimum detection limit of 0.01% mutant in excess wild-type was achieved according to as little as 1 pg mutated DNA in the assay. The technique was applied to 80 tissue samples from patients with colorectal cancer (n = 17), adenomas (n = 50), serrated lesions (n = 8), and normal mucosa (n = 5). Any kind of known and unknown APC mutations (deletions, insertions, and base exchanges) being situated inside the mutation cluster region was distinguishable from wild-type DNA. Furthermore, by WTB-HRM, nearly twice as many carcinomas and 1.5 times more precursor lesions were identified to be mutated in APC, as compared with direct sequencing. By analyzing 31 associated stool DNA specimens all but one of the APC mutations could be recovered. Transferability of the WTB-HRM method to other genes was proven using the example of KRAS mutation analysis. In summary, WTB-HRM is a new approach for ultrasensitive detection of cancer-initiating mutations. In this sense, it appears especially applicable for noninvasive detection of colon cancer precursors in body fluids with excess wild-type DNA like stool. Cancer Prev Res; 6(9); 898-907. (C) 2013 AACR.
The Saxon granulites, the type granulite locality, were deeply buried, extremely heated and then rapidly exhumed during the Variscan Orogeny; thus their evolution differs from many granulites elsewhere. The peak-metamorphic assemblages of layered felsic-mafic granulites from a 500 m deep borehole consist of garnet, kyanite, rutile, ternary feldspar and quartz in felsic granulite, and garnet, omphacite, titanite, ternary feldspar and quartz in mafic granulite. A minimum temperature of 1000-1020degreesC, calculated from reintegrated hypersolvus feldspar in felsic and mafic granulites, is consistent with the highest temperature estimates from garnet-clinopyroxene equilibria. Various equilibria in felsic and mafic granulites record a peak pressure of about 23 kbar. Diffusion zoning and local homogenisation of minerals reflect near-isothermal decompression that preceded cooling and partial hydration at medium- to low-pressure. U-Pb dating of titanite yields an age of peak metamorphism at 340.7+/-0.8 Ma (2sigma). However, chemical inheritance from precursor rutile and post-peak Pb loss are also evident, suggesting a protolith age of 499+/-2 Ma (2sigma) and partial resetting down to an age of 333+/-2 Ma (2sigma). Rb-Sr mica ages of 333.2+/-3.3 Ma (2sigma) are interpreted as dating cooling through about 620degreesC. Hence the Saxon granulites were exhumed to the upper crust during the short period of 6-11 Ma, which corresponds to average exhumation and cooling rates of 10 mm/year and 50degreesC/Ma, respectively. Such rapid exhumation is inconsistent with recent numerical models that assume foreland- directed transport of the Saxon granulites in the lower crust followed by extensional unroofing. Instead, high-pressure rocks of the Saxon Granulite Massif and the nearby Erzgebirge experienced a buoyant rise to the middle crust and subsequent juxtaposition with structurally higher units along a series of medium- to low-pressure detachment faults
A randomized, placebo-controlled, double-blind clinical study was performed to investigate the dose-dependent response of serum cholesterol after consuming an ultra-heat-treated milk containing a soy protein preparation. Eighty hypercholesterolemic subjects were assigned to one of four study groups receiving 12.5 or 25 g soy protein (active treatment) or casein (placebo) daily over a period of 4 weeks. The trial substances were provided as ready-made, ultra-heated milk preparations. Before and after the treatment, serum concentrations of total, low-density lipoprotein, and high-density lipoprotein cholesterol were determined. Unexpectedly, at the end of the study, low-density lipoprotein cholesterol concentrations were significantly increased compared with baseline in all study groups. The magnitude of this increase (17 - 19%) was similar in all active and placebo study groups. Soy protein supplements previously shown to be effective in reducing serum cholesterol had in this study no such lipid-lowering effect after ultra heat treatment.
Mutations in the gene encoding for filaggrin (FLG) are major predisposing factors for atopic dermatitis (AD). Besides genetic predisposition, immunological dysregulations considerably contribute to its pathophysiology. For example, thymic stromal lymphopoietin (TSLP) is highly expressed in lesional atopic skin and significantly contributes to the pathogenesis of AD by activating dendritic cells that then initiate downstream effects on, for example, T cells. However, little is known about the direct interplay between TSLP, filaggrin-deficient skin and other immune cells such as T lymphocytes. In the present study, FLG knockdown skin equivalents, characterised by intrinsically high TSLP levels, were exposed to activated CD4(+) T cells. T cell exposure resulted in an inflammatory phenotype of the skin equivalents. Furthermore, a distinct shift from a Th1/Th17 to a Th2/Th22 profile was observed following exposure of T cells to filaggrin-deficient skin equivalents. Interestingly, TSLP directly stimulated T cell migration exclusively in filaggrin-deficient skin equivalents even in the absence of dendritic cells, indicating a hitherto unknown role of TSLP in the pathogenesis of AD.
Treatment of caenorhabditis elegans with small selenium species enhances antioxidant defense systems
(2019)
ScopeSmall selenium (Se) species play a key role in Se metabolism and act as dietary sources of the essential trace element. However, they are redox-active and trigger pro- and antioxidant responses. As health outcomes are strongly species-dependent, species-specific characteristics of Se compounds are tested in vivo. Methods and resultsIn the model organism Caenorhabditis elegans (C. elegans), immediate and sustained effects of selenite, selenomethionine (SeMet), and Se-methylselenocysteine (MeSeCys) are studied regarding their bioavailability, incorporation into proteins, as well as modulation of the cellular redox status. While all tested Se compounds are bioavailable, only SeMet persistently accumulates and is non-specifically incorporated into proteins. However, the protection toward chemically-induced formation of reactive species is independent of the applied Se compound. Increased thioredoxin reductase (TXNRD) activity and changes in mRNA expression levels of antioxidant proteins indicate the activation of cellular defense mechanisms. However, in txnrd-1 deletion mutants, no protective effects of the Se species are observed anymore, which is also reflected by differential gene expression data. ConclusionSe species protect against chemically-induced reactive species formation. The identified immediate and sustained systemic effects of Se species give rise to speculations on possible benefits facing subsequent periods of inadequate Se intake.
OBJECTIVE-BMI and albumin are commonly accepted parameters to recognize wasting in dialysis patients and are powerful predictors of morbidity and mortality. However, both parameters reveal limitations and may not cover the entire range of patients with wasting. The visceral protein transthyretin (TTR) may be helpful in overcoming the diagnostic and prognostic gap. Therefore, the aim of this study was to assess the association of TTR with morbidity and mortality in hemodialysis patients.
RESEARCH DESIGN AND METHODS-The TTR concentration was determined in plasma samples of 1,177 hemodialysis patients with type 2 diabetes. Cox regression analyses were used to determine hazard ratios (HRs) for the risk of cardiovascular end points (CVEs) and mortality according to quartiles of TTR concentration for the total study cohort and the subgroups BMI >= 23 kg/m(2), albumin concentration >= 3.8 g/dL, and a combination of both.
RESULTS-A low TTR concentration was associated with an increased risk for CVE for the total study cohort (HR 1.65 [95% CI 1.27-2.14]), patients with BMI >= 23 kg/m(2) (1.70 [1.22-2.37]), albumin >= 3.8 g/dL (1.68 [1.17-2.42]), and the combination of both (1.69 [1.13-2.53]). Additionally, a low TTR concentration predicted mortality for the total study cohort (1.79 [1.43-2.24]) and patients with BMI >= 23 kg/m(2) (1.46 [1.09-1.95]).
CONCLUSIONS-The current study demonstrated that TTR is a useful predictor for cardiovascular outcome and mortality in diabetic hemodialysis patients. TTR was particularly useful in patients who were not identified to be at risk by BMI or albumin status.
Objective:
We address two questions relevant to infants' exposure to potentially toxic arsenolipids, namely, are the arsenolipids naturally present in fish transported intact to a mother's milk, and what is the efficiency of this transport.
Methods:
We investigated the transport of arsenolipids and other arsenic species present in fish to mother's milk by analyzing the milk of a single nursing mother at 15 sampling times over a 3-day period after she had consumed a meal of salmon. Total arsenic values were obtained by elemental mass spectrometry, and arsenic species were measured by HPLC coupled to both elemental and molecular mass spectrometry.
Results:
Total arsenic increased from background levels (0.1 mu g As kg(-1)) to a peak value of 1.72 lig As kg(-1) eight hours after the fish meal. The pattern for arsenolipids was similar to that of total arsenic, increasing from undetectable background levels (< 0.01 mu g As kg(-1)) to a peak after eight hours of 0.45 mu g As kg(-1). Most of the remaining total arsenic in the milk was accounted for by arsenobetaine. The major arsenolipids in the salmon were arsenic hydrocarbons (AsHCs; 55 % of total arsenolipids), and these compounds were also the dominant arsenolipids in the milk where they contributed over 90 % of the total arsenolipids.
Conclusions:
Our study has shown that ca 2-3 % of arsenic hydrocarbons, natural constituents of fish, can be directly transferred unchanged to the milk of a nursing mother. In view of the potential neurotoxicity of AsHCs, the effects of these compounds on the brain developmental stage of infants need to be investigated.
Methicillin resistant Staphylococcus aureus (MRSA) is one of the most important antibiotic-resistant pathogens in hospitals and the community. Recently, a new generation of MRSA, the so called livestock associated (LA) MRSA, has emerged occupying food producing animals as a new niche. LA-MRSA can be regularly isolated from economically important live-stock species including corresponding meats. The present thesis takes a methodological approach to confirm the hypothesis that LA-MRSA are transmitted along the pork, poultry and beef production chain from animals at farm to meat on consumers` table. Therefore two new concepts were developed, adapted to differing data sets.
A mathematical model of the pig slaughter process was developed which simulates the change in MRSA carcass prevalence during slaughter with special emphasis on identifying critical process steps for MRSA transmission. Based on prevalences as sole input variables the model framework is able to estimate the average value range of both the MRSA elimination and contamination rate of each of the slaughter steps. These rates are then used to set up a Monte Carlo simulation of the slaughter process chain. The model concludes that regardless of the initial extent of MRSA contamination low outcome prevalences ranging between 0.15 and 1.15 % can be achieved among carcasses at the end of slaughter. Thus, the model demonstrates that the standard procedure of pig slaughtering in principle includes process steps with the capacity to limit MRSA cross contamination. Scalding and singeing were identified as critical process steps for a significant reduction of superficial MRSA contamination.
In the course of the German national monitoring program for zoonotic agents MRSA prevalence and typing data are regularly collected covering the key steps of different food production chains. A new statistical approach has been proposed for analyzing this cross sectional set of MRSA data with regard to show potential farm to fork transmission. For this purpose, chi squared statistics was combined with the calculation of the Czekanowski similarity index to compare the distributions of strain specific characteristics between the samples from farm, carcasses after slaughter and meat at retail. The method was implemented on the turkey and veal production chains and the consistently high degrees of similarity which have been revealed between all sample pairs indicate MRSA transmission along the chain.
As the proposed methods are not specific to process chains or pathogens they offer a broad field of application and extend the spectrum of methods for bacterial transmission assessment.
Background Cardiovascular disease risk among individuals across different categories of BMI might depend on their metabolic health. It remains unclear to what extent metabolic health status changes over time and whether this affects cardiovascular disease risk. In this study, we aimed to examine the association between metabolic health and its change over time and cardiovascular disease risk across BMI categories. Findings During 2 127 391 person-years of follow-up with a median follow-up of 24 years, we documented 6306 cases of cardiovascular disease including 3304 myocardial infarction cases and 3080 strokes. Cardiovascular disease risk of women with metabolically healthy obesity was increased compared with women with metabolically healthy normal weight (HR 1.39, 95% CI 1.15-1.68), but risk was considerably higher in women with metabolically unhealthy normal weight (2.43, 2.19-2.68), overweight (2.61, 2.36-2.89) and obesity (3.15, 2.83-3.50). The majority of metabolically healthy women converted to unhealthy phenotypes (2555 [84%] of 3027 women with obesity, 22 215 [68%] of 32 882 women with normal-weight after 20 years). Women who maintained metabolically healthy obesity during follow-up were still at a higher cardiovascular disease risk compared with women with stable healthy normal weight (HR 1.57, 1.03-2.38), yet this risk was lower than for initially metabolically healthy women who converted to an unhealthy phenotype (normal-weight 1.90, 1.66-2.17 vs obesity 2.74, 2.30-3.27). Particularly incident diabetes and hypertension increased the risk among women with initial metabolic health. Interpretation Even when metabolic health is maintained during long periods of time, obesity remains a risk factor for cardiovascular disease. However, risks are highest for metabolically unhealthy women across all BMI categories. A large proportion of metabolically healthy women converted to an unhealthy phenotype over time across all BMI categories, which is associated with an increased cardiovascular disease risk. Copyright (C) 2018 Elsevier Ltd. All rights reserved.
Transfer of carotenoids, alfa-tocopherol and retinol from plasma into follicular fluid in women
(2000)
Cholesterol uptake and efflux are key metabolic processes associated with macrophage physiology and atherosclerosis. Peroxisome proliferator-activated receptor gamma (PPARgamma) and liver X receptor alpha (LXRalpha) have been linked to the regulation of these processes. It remains to be identified how activation of these receptors is connected and regulated by endogenous lipid molecules. We identified CYP27, a p450 enzyme, as a link between retinoid, PPARgamma, and LXR signaling. We show that the human CYP27 gene is under coupled regulation by retinoids and ligands of PPARs via a PPAR-retinoic acid receptor response element in its promoter. Induction of the enzyme's expression results in an increased level of 27-hydroxycholesterol and upregulation of LXR-mediated processes. Upregulated CYP27 activity also leads to LXR-independent elimination of CYP27 metabolites as an alternative means of cholesterol efflux. Moreover, human macrophage-rich atherosclerotic lesions have an increased level of retinoid-, PPARgamma-, and LXR- regulated gene expression and also enhanced CYP27 levels. Our findings suggest that nuclear receptor-regulated CYP27 expression is likely to be a key integrator of retinoic acid receptor-PPARgamma-LXR signaling, relying on natural ligands and contributing to lipid metabolism in macrophages
Small selenium (Se) species play a major role in the metabolism, excretion and dietary supply of the essential trace element selenium. Human cells provide a valuable tool for investigating currently unresolved issues on the cellular mechanisms of Se toxicity and metabolism. In this study, we developed two isotope dilution inductively coupled plasma tandem-mass spectrometry based methods and applied them to human hepatoma cells (HepG2) in order to quantitatively elucidate total cellular Se concentrations and cellular Se species transformations in relation to the cytotoxic effects of four small organic Se species. Species-and incubation time-dependent results were obtained: the two major urinary excretion metabolites trimethylselenonium (TMSe) and methyl-2-acetamido-2-deoxy-1-seleno-beta- D-galactopyranoside (SeSugar 1) were taken up by the HepG2 cells in an unmodified manner and did not considerably contribute to the Se pool. In contrast, Se-methylselenocysteine (MeSeCys) and selenomethionine (SeMet) were taken up in higher amounts, they were largely incorporated by the cells (most likely into proteins) and metabolized to other small Se species. Two new metabolites of MeSeCys, namely gamma-glutamyl-Se-methylselenocysteine and Se-methylselenoglutathione, were identified by means of HPLC-electrospray-ionization-Orbitrap-MS. They are certainly involved in the (de-) toxification modes of Se metabolism and require further investigation.
The trace elements zinc and manganese are essential for human health, especially due to their enzymatic and protein stabilizing functions. If these elements are ingested in amounts exceeding the requirements, regulatory processes for maintaining their physiological concentrations (homeostasis) can be disturbed. Those homeostatic dysregulations can cause severe health effects including the emergence of neurodegenerative disorders such as Parkinson’s disease (PD). The concentrations of essential trace elements also change during the aging process. However, the relations of cause and consequence between increased manganese and zinc uptake and its influence on the aging process and the emergence of the aging-associated PD are still rarely understood. This doctoral thesis therefore aimed to investigate the influence of a nutritive zinc and/or manganese oversupply on the metal homeostasis during the aging process. For that, the model organism Caenorhabditis elegans (C. elegans) was applied. This nematode suits well as an aging and PD model due to properties such as its short life cycle and its completely sequenced, genetically amenable genome. Different protocols for the propagation of zinc- and/or manganese-supplemented young, middle-aged and aged C. elegans were established. Therefore, wildtypes, as well as genetically modified worm strains modeling inheritable forms of parkinsonism were applied. To identify homeostatic and neurological alterations, the nematodes were investigated with different methods including the analysis of total metal contents via inductively-coupled plasma tandem mass spectrometry, a specific probe-based method for quantifying labile zinc, survival assays, gene expression analysis as well as fluorescence microscopy for the identification and quantification of dopaminergic neurodegeneration.. During aging, the levels of iron, as well as zinc and manganese increased.. Furthermore, the simultaneous oversupply with zinc and manganese increased the total zinc and manganese contents to a higher extend than the single metal supplementation. In this relation the C. elegans metallothionein 1 (MTL-1) was identified as an important regulator of metal homeostasis. The total zinc content and the concentration of labile zinc were age-dependently, but differently regulated. This elucidates the importance of distinguishing these parameters as two independent biomarkers for the zinc status. Not the metal oversupply, but aging increased the levels of dopaminergic neurodegeneration. Additionally, nearly all these results yielded differences in the aging-dependent regulation of trace element homeostasis between wildtypes and PD models. This confirms that an increased zinc and manganese intake can influence the aging process as well as parkinsonism by altering homeostasis although the underlying mechanisms need to be clarified in further studies.