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

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155 pages
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.
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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........................................................................................... 65
3.3.1 Correlation of genotoxicity and other physicochemical parameters............................. 65
3.3.2 Evaluation of the relationship of genotoxicity and physicochemical parameters ......... 69
3.4 Relationship of microtubule assembly and lipophilicity .................................................... 72
3.5 Mechanistical studies ...................................................................................................... 74
3.5.1 Cytotoxicity.................................................................................................................. 74
II Contents
3.5.2 Cell cycle analysis....................................................................................................... 76
3.5.3 Apoptosis induction ..................................................................................................... 78
3.5.4 Production of reactive oxygen species........................................................................ 80
4 Discussion.............................................................................................................................. 82
4.1 General aspect: genotoxins and thresholds of effects..................................................... 82
4.1.1 Genotoxic mechanisms leading to a threshold............................................................ 82
4.1.2 The threshold concept for genotoxic compounds in cancer risk assessment.............. 84
4.2 Methodological points of discussion ................................................................................ 88
4.2.1 Determination of cytotoxicity ....................................................................................... 88
4.2.2 Genotoxicity testing..................................................................................................... 89
4.2.3 Methodological problems ............................................................................................ 90
4.3 Mechanistic considerations regarding genotoxicity.......................................................... 91
4.3.1 Cytotoxicity and genotoxicity 91
4.3.2 Cell cycle progression and genotoxicity ...................................................................... 94
4.3.3 Apoptosis and genotoxicity.......................................................................................... 96
4.3.4 Reactive oxygen species and genotoxicity.................................................................. 98
4.3.5 Lipophilicity and genotoxicity..................................................................................... 100
4.4 Compounds with specific modes of action..................................................................... 101
4.4.1 Compounds studied with known specific modes of action......................................... 101
4.4.2 Compounds characterized in the present study ........................................................ 102
4.5 The concept of specific versus non-specific genotoxins ................................................ 103
4.6 Final conclusions for drug development ........................................................................ 106
5 Summary............................................................................................................................... 109
6 References............................................................................................................................ 111
7 Appendix............................................................................................................................... 123
7.1 Additional information.................................................................................................... 123
7.1.1 Physicochemical parameters .................................................................................... 123
7.1.1.1. Detailed description of some molecular properties ............................................ 123
7.1.2 Microtubule assembly turbidity assay........................................................................ 124
7.1.2.1. Procedure (experiments performed at the IMB, Jena)....................................... 124
7.2 Results in detail ............................................................................................................. 125
7.2.1 MN results................................................................................................................. 125
III Contents
7.2.2 Physicochemical parameters .................................................................................... 126
7.2.3 Cytotoxicity................................................................................................................ 129
7.2.4 Cell cycle analysis..................................................................................................... 131
7.2.5 Detection of apoptosis............................................................................................... 134
7.2.5.1. Annexin-V/PI assay ........................................................................................... 134
7.2.6 Detection of reactive oxygen species........................................................................ 134
7.2.7 Microtubule assembly turbidity assay 136
7.2.7.1. Results .............................................................................................................. 136
7.2.8 Cytotoxicity and genotoxicity – a comparison............................................................ 139
Acknowledgements ...................................................................................................................... 148
Curriculum Vitae............ 150
IV Abbreviations
Abbreviations
8-oxoG 8-Oxoguanin
acc Number of hydrogen bond acceptor atoms
A. dest. aqua destillata (distilled water)
ANDRO androstenedione
AP apurinic/apyrimidinic
AR androgen receptor
ARE androgen responsive element
BCA bicinchoninic acid
BSA bovine serum albumin
Carboxy -HDCFDA 5-(and-6)-carboxy-2 ′,7 ′-dichlorodihydrofluorescein diacetate 2
CAT Chromosomal aberration test
clog D Lipophilicity of ionizable molecules at pH=7.4
(c)log P Lipophilicity of neutralized molecules
CREST Calcinosis, Raynaud phenomenon, Esophageal dismotility,
Sclerodactyly and Telangiectasia (disease leading to
centromere antibodies)
TMCTB CellTiter Blue
DAPI 4`6`diamidino-2-phenylindole
dipole Molecular dipole moment
DMSO dimethylsulphoxide
DNA deoxyribonucleic acid
don Number of hydrogen bond donor atoms
EDTA ethylenediaminetetraacetic acid
ETHI ethisterone
em. emission
ex. excitation
Equ. Equation
FACS fluorescence activated cell scanner
FCS fetal calf serum (for cell culture)
Fig. Figure
FITC fluorescein-5-isothiocyanate
FSH Follicle Stimulating Hormone
V Abbreviations
GFA genetic function approximation
h hour
HBSS Hank's Balanced Salt Solution
IC inhibitory concentration with cell viability reduction of 20% 20
IC inhibitory concentration with cell viability reduction of 50% 50
ICH International Conference on Harmonisation of Technical
Requirements for Registration of Pharmaceuticals for Hu-
man Use
Ig immunoglobulin
im intramuscular
LH Luteinizing Hormone
log P Lipophilicity
log S Solubility
MAD madol
min minute(s)
MMS methylmethane sulfonate
MN micronucleus/micronuclei
MT 7 α-methyltestosterone
mw Molecular Weight
NA 19-norandrostenedione
NE 19-norethisterone
NOEC/NOEL no-observed-effect-concentration/ -level
NR Neutral Red
NT 19-nortestosterone
OECD Organisation for Economic Co-operation and Development
PBS phosphate- buffered saline
PM 17 α-propylmesterolone
polsurf Polar surface area
PS phosphatidylserine
QSAR Quantitative Structure Activity Relationship
robo Number of rotatable bonds
ROS reactive oxygen species
rpm rounds per minute
VI Abbreviations
s seconde(s)
SD standard deviation
SHBG sex hormone-binding globulin
surface Total molecular surface
T testosterone
Tab. Table
TB trenbolone
THG tetrahydrogestrinone
Tris tris-(hydroxymethyl)-aminomethane
V79 cells Chinese hamster cell line (for cell culture)
v/v volume/volume
VCR vincristine
vol Molecular Volume
vs. versus
w/o without
w/v weight/volume
WoE weight of evidence
Z-DEVD-R110 rhodamine 110, bis-(N-CBZ-L-aspartyl-L-glutamyl-L-valyl-L-
aspartic acid amide)


VII

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