Assessment of chromosomal genotoxicity of steroidal hormones related to drug development [Elektronische Ressource] / vorgelegt von Susanne Barbara Dorn

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• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2007
• Nombre de lectures: 17
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Assessment of chromosomal genotoxicity of
steroidal hormones related to drug development









Inaugural-Dissertation





zur
Erlangung des Doktorgrades der
Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf




vorgelegt von
Susanne Barbara Dorn
aus Rottweil




Oktober 2007 The present dissertation was performed according to the European Graduate College “Molecular
Mechanisms in Food Toxicology”, Heinrich-Heine-Universität Düsseldorf, at the Institut für
Arbeitsphysiologie an der Universität Dortmund.





















Gedruckt mit der Genehmigung der
Mathematisch-Naturwissenschaftlichen Fakultät der
Heinrich-Heine-Universität Düsseldorf

Referent: Prof. Dr. Dr. Hermann M. Bolt
1. Koreferent: Prof. Dr. Frank Wunderlich
2. Koreferent: Prof. Dr. Hartmut Greven


Tag der mündlichen Prüfung: 20.12.2007































Parts of the present thesis have been published in:



Dorn,S.B., Bolt,H.M., Thevis,M., Diel,P., and Degen,G.H. (2007) Induction of micronuclei in V79 cells by
the anabolic doping steroids tetrahydrogestrinone and trenbolone. Arch. Toxicol. Epub ahead of print:
[DOI 10.1007/s00204-007-0241-2].

Dorn,S.B., Degen G.H., Bolt,H.M., van der Louw,J., van Acker,F.A.A., van den Dobbelsteen,D.J., and
Lommerse,J.P.M. (2007) Some molecular descriptors for non-specific chromosomal genotoxicity based
on hydrophobic interactions. Arch. Toxicol. Epub ahead of print: [DOI 10.1007/s00204-007-0256-8].

Dorn,S.B., Degen,G.H., Müller,T., Bonacker,D., Joosten,H.F., van der Louw,J., van Acker,F.A.A., and
Bolt,H.M. (2007) Proposed criteria for specific and non-specific chromosomal genotoxicity based on
hydrophobic interactions. Mutat. Res. 628: 67-75. Contents
Table of contents
Table of contents ............................................................................................................................... I
Abbreviations....................................................................................................................................V
1 Introduction .............................................................................................................................. 8
1.1 Genotoxicity testing in drug development.......................................................................... 8
1.2 Androgenic steroids of pharmacological interest ............................................................. 12
1.2.1 Androgens as drugs .................................................................................................... 12
1.2.2 Compounds examined in the present study ................................................................ 14
1.3 Relationship of molecular properties and chromosomal genotoxicity .............................. 16
1.4 Aim of the present thesis ................................................................................................. 18
2 Materials and methods .......................................................................................................... 19
2.1 Materials.......................................................................................................................... 19
2.1.1 Chemicals and biochemicals....................................................................................... 19
2.1.2 Hormonally active steroids .......................................................................................... 20
2.1.3 Kits.............................................................................................................................. 20
2.1.4 Consumables .............................................................................................................. 21
2.1.5 Instruments ................................................................................................................. 21
2.1.6 Cell line. 22
2.1.7 Solutions and buffers .................................................................................................. 23
2.1.7.1. Ready-for-use solutions....................................................................................... 23
2.1.7.2. Cell culture........................................................................................................... 23
2.1.7.3. Cytotoxicity assays .............................................................................................. 23
2.1.7.4. Micronucleus assay ............................................................................................. 25
2.1.7.5. Cell cycle analysis ............................................................................................... 26
2.1.7.6. Apoptosis detection 27
2.1.7.7. Detection of reactive oxygen species .................................................................. 28
2.1.7.8. Test compounds .................................................................................................. 28
2.1.8 Software...................................................................................................................... 28
2.2 Methods........................................................................................................................... 29
2.2.1 Cell culture .................................................................................................................. 29
2.2.1.1. Culture and subculture......................................................................................... 29
I Contents
2.2.1.2. Cryopreservation ................................................................................................. 30
2.2.2 Substance exposure ................................................................................................... 30
2.2.3 Cytotoxicity tests ......................................................................................................... 30
2.2.3.1. Neutral Red uptake assay ................................................................................... 30
TM 2.2.3.2. CellTiter Blue assay.......................................................................................... 32
2.2.3.3. Measurement of protein content .......................................................................... 33
2.2.4 Micronucleus test ........................................................................................................ 34
2.2.4.1. Standard assay.................................................................................................... 34
2.2.4.2. CREST analysis .................................................................................................. 37
2.2.5 Cell cycle analysis....................................................................................................... 39
2.2.6 Detection of apoptosis................................................................................................. 40
2.2.6.1. Annexin-V/PI assay ............................................................................................. 41
2.2.6.2. Caspase-3/7 assay.............................................................................................. 43
2.2.7 Detection of reactive oxygen species.......................................................................... 45
2.2.8 Statistical evaluation of the test results ....................................................................... 47
2.2.9 In silico methodologies................................................................................................ 47
2.2.9.1. Lipophilicity - genotoxicity relationship................................................................. 47
2.2.9.2. Physicochemical parameters............................................................................... 50
2.2.9.3. Relationship of microtubule assembly and lipophilicity ........................................ 52
3 Results .................................................................................................................................... 54
3.1 Genotoxicity..................................................................................................................... 54
3.1.1 Standard micronucleus assay ..................................................................................... 54
3.1.2 CREST (kinetochore) analysis of micronuclei ............................................................. 57
3.2 Genotoxicity - lipophilicity relationship ............................................................................. 59
3.2.1 Characterization of data sets....................................................................................... 59
3.2.2 Correlation of genotoxicity and lipophilicity.................................................................. 60
3.2.3 Evaluation of the genotoxicity - lipophilicity relationship.............................................. 64
3.3 Physicochemical parameters.............................