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«Time- and Concentration-dependent response of a liver cell line to Benzo(a)pyrene Time- and Concentration-dependent response of a liver cell line to ...»

-- [ Page 1 ] --

Danielle Jannuzzi Madureira

DISS. ETH NO. 20716

Time- and Concentration-dependent

response of a liver cell line to

Benzo(a)pyrene

Time- and Concentration-dependent response of a liver cell line to Benzo(a)pyrene

Danielle Jannuzzi Madureira

DISS. ETH NO. 20716

Time- and Concentration-dependent response of

a liver cell line to Benzo(a)pyrene exposure

A dissertation submitted to

ETH ZURICH

for the degree of

Doctor of Sciences Presented by Danielle Jannuzzi Madureira Master in Genetics and Molecular Biology, State University of Campinas born on November 12, 1981 Citizen of Brazil / Portugal Accepted on the recommendation of Prof. Dr. Kristin Schirmer, examiner Prof. Dr. Martin Ackermann, co-examiner Dr. Juliette Legler, co-examiner Prof. Dr. Rik I. Eggen, co-examiner Table of Content Table of Content

Abstract

Zusammenfassung

1. General Introduction

1.1 ORGANIC CHEMICALS IN THE ENVIRONMENT

1.1.1 Polycyclic Aromatic Hydrocarbons

1.1.2 Benzo(a)pyrene (BaP)

1.2 THE ARYL HYDROCARBON RECEPTOR SIGNAL TRANSDUCTION PATHWAY.................. 5 1.2.1 The Aryl Hydrocarbon receptor (AhR) as transcription factor

1.2.2 BaP as a ligand of AhR

1.3 TOXICOGENOMICS

1.3.1 Transcriptomics

1.3.2 Phenotypic anchoring (Adverse outcome pathway)

1.3.3 Cellular models

1.4 SCOPE OF THE THESIS

1.5 REFERENCES

2.Differential time-resolved molecular response of a mouse liver cell line on low and high benzo(a)pyrene exposure

ABSTRACT

2.1 INTRODUCTION

2.2 MATERIAL AND METHODS

2.3 RESULTS

2.4 DISCUSSION

2.5 REFERENCES

2.6 SUPPORT INFORMATION

3. Characterization of tiPARP in BaP-exposed Hepa1c1c7 wild-type and tiPARP silenced cells

ABSTRACT

3.1 INTRODUCTION

3.2 MATERIAL AND METHODS

3.3 RESULTS

III Table of Content

3.4 DISCUSSION

3.5 REFERENCES

4. Quantification in a time- and concentration-dependent manner of BaP uptake and DNA adduct formation

ABSTRACT

4.1 INTRODUCTION

4.2 MATERIAL AND METHODS

4.3 RESULTS

4.4 DISCUSSION

4.6 REFERENCES

5. Conclusion & Outlook

Appendix

Acknowledgements

Curriculum vitae

–  –  –

Abstract Benzo(a)pyrene (BaP) is a ubiquitous pollutant derived by incomplete combustion of organic matter. BaP is classified by the International Agency of Cancer (IARC) as carcinogenic to humans and is controlled in drinking water in the United States and Europe.

BaP needs to be metabolized to become carcinogenic. Its metabolism is induced by the interaction between BaP and the aryl hydrocarbon receptor (AhR) and subsequent gene regulation. Despite this general knowledge, details about BaP entry into cells, distribution and internal concentrations able to elicit a cellular response are largely unexplored and mechanisms of actions incompletely understood. This PhD thesis therefore aims to investigate in a time and concentration dependent manner the molecular response of a murine hepatoma cell line, Hepa1c1c7, exposed to BaP, combining high throughput techniques, bioinformatics, phenotype characterization and chemical analysis.

To elucidate the temporal response of the murine liver cell line, Hepa1c1c7, a transcriptome data set was first established. Despite the 100-fold difference in BaP exposure concentration, i.e. 50 nM of BaP, where no cytotoxic effect was observed, and 5 µM of BaP, being a common concentration used in toxicology studies, the pattern of transcript regulation was shown to be comparable in cells for up to 4 h of exposure. A total of 20 genes was regulated by the low BaP concentration with a large fraction of them also being regulated by the high BaP exposure condition. However, while the transcriptome returned to control levels for the low concentration of BaP between 4 and 12 h of exposure, the transcriptome response diverged for the high concentration of BaP, with more than 1000 genes identified as significantly regulated. Selected descriptors of (sub)cellular phenotype were related to the regulation of selected groups of genes. Cells exposed to the low (50 nM) BaP showed a slight reduction in cell doubling time but no impact on cell viability or signs of formation of ROS.

In contrast, cells exposed to the high (5 µM) BaP concentration greatly reduced proliferation and were prone to cell death. Both cyp1a1 mRNA transcripts and Cyp1a catalytic activity were induced by BaP, implying BaP metabolism and production of toxic and/or carcinogenic metabolites. Therefore, the slight decrease in proliferation observed for the low concentration of BaP is an indication that cells allow for damage repair in order to return to normal cell cycling. However, on exposure to the high BaP concentration, this defence is overruled and cells undergo cell death. Based on the regulation of the transcriptome, the 4 h time point is highlighted as an important window in the cells’ fate.

VAbstract





Based on the transcriptome analysis an early regulated gene for both BaP concentrations used, namely TCDD-inducible poly(ADP-ribose) polymerase (tiPARP), was chosen to be further investigated. The tiPARP gene was silenced in Hepa1c1c7 cells and different end points were tested in wild type and tiPARP silenced cells, exposed and nonexposed to BaP. The hypothesis underlying this study was that tiPARP’s main function was to protect cells against damage induced by dioxin-like compounds, such as BaP. However, no change in cell viability upon exposure to BaP was seen when tiPARP was silenced by siRNA.

One potential explanation was that tiPARP protein was not down-regulated sufficiently.

However, attempts to detect the endogenous tiPARP protein levels using western blot and MudPIT were unsuccessful.

Transcriptome analysis indicated rapid uptake of BaP into cells along with transcription of genes related to BaP metabolism, cell cycle arrest and/or repair. At the same time, no impact on cell viability occurred and cell doubling time was only slightly increased for the low BaP concentration. In contrast, sever impact on cell viability and an almost complete block of cell proliferation was noted at the high BaP concentration. In order to investigate if these differences could be explained by cell-internal BaP amounts, BaP cellular concentrations were quantified using liquid scintillation counting (LSC) and high performance liquid chromatography (HPLC). BaP was indeed taken up by cells fast (within the first 4 h), although most of the BaP stayed in the culture medium, apparently associated to the culture medium components. The number of BaP molecules within one cell reached a maximum after 2 and 4 h and ranged between 1.2 to 1.9 106 and 1.2 to 1.7 109 for 50 nM and 5 µM, respectively. Biotransformation became apparent between 4 and 8 h of BaP exposure and most of the added BaP ( 90 %) was bio-transformed within 24 h for both concentrations.

Rates of BaP uptake and biotransformation were calculated, showing that uptake is faster than biotransformation (0.17 and0.15 h-1 for 50 nM and 0.12 and 0.1 h-1 for 5 µM BaP, respectively) for both concentrations. DNA adduct quantification showed the presence of adducts as early as 8 h of BaP exposure. Both DNA adduct isomers, t(+)Benzo(a)pyrene diolepoxide (BPDE) dGuo and t(-)BPDEdGuo, were detected in Hepa1c1c7 cells exposed to 50 nM and 5 µM BaP, with the first being more abundant. DNA repair was seen at the 24 h time point in the cells exposed to 50 nM BaP while repair mechanisms seemed to be overwhelmed at the 5 µM BaP exposure and DNA adducts continued to accumulate. These results are in agreement with the transcriptome analysis, where genes related to nucleotide excision repair (NER) were regulated at the high (5µM) BaP concentration and later (12 and VI

Abstract

24 h) time points. NER pathway is responsible to remove bulky DNA adducts as caused by BPDE.

In conclusion, this work demonstrates that studying the cell response at different levels in a time and concentration dependent manner helps to better understand the mechanisms allowing cells to survive or having cells succumb to chemical exposure. Such knowledge and the rate constants calculated in this thesis can contribute to building computational models with the goal to predict cellular responses to BaP in the Hepa1c1c7 cells or other cells, as well as to other stressors with similar mechanisms of toxic action.

VIIZusammenfassung

Zusammenfassung Benzo(a)pyren (BaP) ist ein allgegenwärtiger Schadstoff, welcher aus der unvollständigen Verbrennung von organischem Material entsteht. Von der „International Agency of Cancer“ (IARC) wurde BaP als krebserregend für den Menschen eingestuft und Trinkwasser in den USA und in Europa wird auf sein Vorkommen kontrolliert. Um krebserregend zu wirken, muss BaP metabolisiert werden. Dieser Prozess wird durch die Bindung von BaP an den Aryl-Hydrocarbon-Rezeptor (AhR) und anschliessende Genregulation durch BaP selbst induziert. Trotz dieses allgemeinen Wissens sind Details über Eintritt in die Zelle, Verteilung und interne Konzentrationen, welche molekulare Reaktionen in den Zellen auslösen können, weitgehend unerforscht, und die zugrunde liegenden Mechanismen schlecht verstanden. Das Ziel dieser Doktorarbeit ist daher, die molekulare Antwort von Mausleberzellen, speziell der Hepatomzelllinie Hepa1c1c7, in Abhängigkeit von Zeit und BaP-Konzentrationen zu untersuchen. Dazu wurden High-Throughput-Verfahren, Bioinformatik sowie phänotypische und chemische Analysen kombiniert.

Um die zeitliche Antwort der Leberzellen auf BaP zu eruieren, wurde zunächst ein Transkriptomdatensatz etabliert. Es zeigte sich, dass das Muster der Transkriptregulation nach 2 und 4 Stunden BaP-Exposition für beide eingesetzten Konzentrationen, d. h. 50 nM (kein zytotoxischer Effekt) und 5 µM (häufig eingesetzte Konzentration in vorhergehenden Studien), vergleichbar waren. Bis zu 20 Gene wurden durch die niedrige BaP-Konzentration reguliert, wobei eine Reihe dieser Gene auch durch die hohe BaP-Konzentration reguliert wurde. Während jedoch das Transkriptom für die niedrige BaP-Konzentration zwischen 4 und 12 Stunden Exposition auf Kontrollniveau zurückreguliert wurde, löste die hohe BaPKonzentration über die Zeit mit 1165 signifikant regulierten Genen eine umfangreiche Antwort des Transkriptoms aus. Ausgewählte Deskriptoren des (sub)zellulären Phänotyps wurden mit der Regulation von Gruppen von Genen in Verbindung gebracht. Zellen, die gegenüber der niedrigen (50 nM) BaP-Konzentration exponiert wurden zeigten einen leichten Anstieg der Verdopplungszeit aber keinen Einfluss auf die Zellvitalität oder die Bildung reaktiver Sauerstoffspezies. Dagegen war die Zellproliferation in Zellen, die gegenüber der hohen (5 µM) BaP-Konzentration exponiert wurden, sehr stark reduziert und die Zellen waren anfällig für Zelltod. Sowohl cyp1a1-mRNA als auch katalytische Aktivität von Cyp1a wurden durch BaP induziert, was anzeigt, dass BaP metabolisiert wurde und dabei toxische und/oder kanzerogene Metabolite gebildet wurden. Der leichte Anstieg der Verdopplungszeit, welche für die niedrige BaP Konzentration beobachtet wurde, ist somit ein Anzeichen dafür, dass die VIII

Zusammenfassung

Zellen DNA-Schäden reparieren um danach wieder in den normalen Zellzyklus einzutreten.

Bei Exposition gegenüber der hohen BaP-Konzentration reichen dagegen die Reparaturmechanismen nicht aus und die Zellen sterben ab. Aufgrund der Regulation des Transkriptoms wurde deutlich, dass 4 Stunden Exposition gegenüber dem Stressor ein sehr wichtiger Zeitpunkt für das Schicksal der Zellen ist.

Eines der Gene, welches bei den Transkriptomstudien bereits nach 2 Stunden durch beide eingesetzten BaP-Konzentrationen reguliert wurde, ist die TCDD-inducible poly(ADPribose)polymerase (tiPARP). Um dessen Funktion näher zu charakterisieren, wurden Hepa1c1c7 Zellen mit tiPARP silencing (si)RNA transfiziert und verschiedene Effekte auf die Zellvitalität in Wildtyp- und behandelten Zellen in der An- oder Abwesenheit von BaP untersucht. Die Hypothese war, dass die primäre Funktion von tiPARP der Schutz gegen Dioxin-ähnliche Verbindungen, wie BaP, ist. Allerdings konnten keine Unterschiede in der Zellvitalität festgestellt werden wenn tiparp-mRNA durch die siRNA stark reduziert vorlag.

Eine mögliche Erklärung dafür war, dass das tiPARP Protein nur ungenügend reduziert werden konnte. Um dieser Erklärung nachzugehen wurde versucht, das Protein in den Zellen mittels Western Blot-Analyse und MudPIT (Multidimensional Protein Identification Technology) zu detektieren, was allerdings erfolglos blieb.

Die schnelle Reaktion des Transkriptoms auf BaP Exposition (2 und 4 Stunden) zeigte eine schnelle Aufnahme von BaP in die Zellen an. Mit dem Ziel, die Wirkung von BaP auf Zellen mit der zellulären Aufnahme und zellinternen Konzentrationen zu verknüpfen, wurde die zeit- und konzentrationsabhängige Aufnahme per „liquid scintillation counter“ (LSC) und „high performance liquid chromatography“ (HPLC) analysiert. Es konnte gezeigt werden, dass BaP schnell aufgenommen wird (in den ersten 4 Stunden), auch wenn das meiste BaP im Kulturmedium verblieb. Die Biotransformation wurde zwischen 4 Stunden und 8 Stunden BaP Einwirkung messbar und der Grossteil des BaP (90%) wurde innerhalb von 24 Stunden bei beiden eingesetzten BaP-Konzentrationen metabolisiert. DNA-Adduktquantifizierung zeigte das Vorhandensein von Addukten nach 8 Stunden BaP-Exposition. DNA-Reparatur war nach 24 Stunden in den mit 50 nM BaP inkubierten Zellen nachweisbar, wohingegen Reparaturmechanismen bei 5 µM BaP nicht Schritt halten konnten und es zu einer Zunahme von DNA-Addukten über die Zeit kam.

Abschliessend zeigt diese Studie, dass die Untersuchung der Zellantwort auf verschiedenen Ebenen in Abhängigkeit von Zeit und Konzentration sehr hilfreich für ein besseres Verständnis der Mechanismen ist, die einer Zelle erlauben, die Exposition gegenüber BaP unschädlich zu machen oder ihr zu unterliegen Dieses Wissen kann auch dazu beitragen,

–  –  –



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