Transcript Protection of the European Population from Aneugenic
Acknowledgements Previous EU-contract: ENV4-CT97-0471 Current EU-contract PEPFAC: QLK4-CT-2000-00058
James M Parry 09-05-2003 1
Protection of the European Population from Aneugenic Chemicals: the PEPFAC project
A collaboration between: • University of Wales Swansea • Free University of Brussels • GSF Neuherberg • University of Bielefeld • ENEA Rome UK Belgium Germany Germany Italy 2
RATIONALE Approximately 45% of spontaneous abortions and 0.4% of live births in the European population carry numerical chromosome aberrations i.e. they are aneuploid.
This major influence of aneuploidy upon human reproduction is paralleled by the prevalence of aneuploidy in human tumours. Thus, in both somatic and germ cells events which disturb the accurate segregation of chromosomes during cell division make a significant impact upon the health of the European population
.
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AIMS
The key factor which links the work of the PEPFAC Collaborators is the aim
to reduce the overall impact of aneuploidy
upon both reproductive and cancer rates in humans.
To this end the Collaborators have been undertaking a series of inter related activities: 1.
2.
3.
The characterisation of the
mechanisms
which ensure the
accurate
segregation of chromosomes.
The identification of the
endogenous and exogenous factors
(such as chemical exposure) which are responsible for disturbing the fidelity of chromosome segregation.
The development and validation of
methodologies
that can detect chemicals capable of inducing aneuploidy.
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Cellular targets, modifications of which may lead to aneuploidy
In Mitotic Cells
Spindle microtubules Centromeres/kinetochores Centrioles/centrosomes Microtubule-associated proteins and regulatory molecules Cytoplasmic membranes DNA
In Meiotic Cells
Process of homologous pairing (eg, formation of synaptonemal complex) Process of crossing-over 5
In vitro screening model Mechanism of aneugenic activity Models Developed Binucleate cell micronucleus assay Binucleate cell non-disjunction assay Influence of apoptosis Influence of cell cycle checkpoint genes In vivo somatic cell models In vivo germ cell models Rodent bone marrow micronucleus assay GI tract assay Lymphocyte binucleate cell assay Rodent male germ cell FISH assay Rodent in vivo/in vitro oocyte assay Rodent 2 to 8 cell embryo assay In vivo embryo model Database of Aneugenic Activity
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Chemical Insult Normal Segregation
Cytochalasin B
Non-disjunction Micronucleus Loss 1) Non-disjunction 2a) Chromosome Loss 2b) Fragment Loss 7
Role of apoptosis in determining compound related thresholds Methodology developed by Vrije Universiteit Brussel Point of inflection (threshold on dose response curve) for micronucleus (chromosome loss) induction for carbendazim is estimated at 2.47
M.
Treat with carbendazim concentrations above and below the threshold concentration.
Cells were separated into Annexin V positive and Annexin V negative, i.e. apoptotic and viable. Scored for micronuclei
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Detection of apoptotic cells by Annexin-V staining
Cell Membrane extracellular Exposure of phosphatidylserine (PS) intracellular Induction of apoptosis Incubation of cells with Annexin-V-Fluos Apoptotic cell Necrotic cell
Annexin-V conjugated to FITC (green fluorescence) binds to PS exposed to the outer leaflet of the membrane of apoptotic cells. In necrotic cells the inner leaflet of the membrane is available for binding of extrinsically applied Annexin-V, since the integrity of the membrane is lost. To discriminate between necrotic and apoptotic cells, a membrane impermeable DNA stain such as propidium iodide (PI, red fluorescence) can be added to the cells.
In this way viable (no staining), apoptotic and necrotic cells can be discriminated by fluorescence microscopy or flow cytometry.
Comparative analysis of micronuclei in viable and apoptotic human lymphocytes treated with Carbendazim Concentration μM Carbendazim 0 0.523
% o Viable fraction micro % cen + 5.0
5.3
40 33 % o Apoptotic fraction micro % cen + 12.3
8.3
30 29 0 1.5
0 2.47
4.0
5.7
5.0
4.0
25 35 20 38 10.9
7.6
11.8
11.6
25 27 40 31 0 2.65
3.0
*7.4
33 69 11.8
12.0
25 63 0 2.85
2.0
*5.5
30 73 8.6
12.6
32 76 Conclusion: Presence of micronuclei acts as a strong apoptotic signal
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In contrast monosomic and trisomic cells produced by non disjunction after Carbendazim treatment fail to produce such strong signals for apoptosis Concentration of Carbendazim μM
0 0.52
0 1.50
0 2.47
0 2.65
0 2.85
% o Viable fraction non-disjunction
24.9
24.2
24.4
29.1
36.2
38.2
27.7
40.5
24.3
38.8
Apoptotic fraction % o non-disjunction
31.7
35.6
33.6
38.7
45.4
48.9
38.5
56.7
38.0
58.9
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Mechanistic studies
• The need for an understanding of the mechanism of action of aneugenic chemicals is demonstrated by studies on Bisphenol A.
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Bisphenol A Exposure Causes Meiotic Aneuploidy in the Female Mouse
Patricia A. Hunt, Kara E. Koehler, Martha Susiarjo, Craig A. Hodges, Arlene Ilagan, Robert C. Voigt, Sally Thomas, Brian F. Thomas, and Terry J. Hassold
April 2003 13(7) 546-553
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Induction of micronuclei in human lymphoblastoid cell line MCL-5 by Bisphenol-A Concentration μg/ml % Mononucleate cells % Binucleate cells % Micronucleated binucleate cells % Kinetochore positive micronuclei % Kinetochore negative micronuclei Solvent (DMSO) control 0 63.0
66.7
36.4
33.0
0.4
0.4
38 34 62 66 5 10 15 20 30 66.5
65.8
66.6
63.5
62.7
33.1
33.8
31.5
34.8
35.9
0.75
2.05* 2.65* 3.20* 3.85* 56 59 63 44 41 37
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Induction of micronuclei in MCL-5 human cells by Bisphenol-A
4.5
4 3.5
3 2.5
2 1.5
1 0.5
0 dmso 0 5 10 Bisphenol-A ug/ml 15 20 30
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Induction of chromosome non-disjunction in human lymphoblastoid cell line MCL-5 by Bisphenol A Concentration μg/ml % Non-Disjunction Chromosome 8 Chromosome 17 Chromosome 20 Total 0.6
0.3
0.2
1.1
Solvent (DSMO) control 0 0.4
0.2
0.2
0.8
5 10 15 20 1.2
2.1
2.5
3.1
0.6
1.0
1.4
2.2
1.1
1.5
1.9
2.8
2.9* 4.6* 5.8* 8.1*
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Effect of Bisphenol-A on mitosis in Chinese HamsterV79 cells
• V79 were grown on microscope slides and exposed to graded doses of BPA for 1 cell cycle • Mitotic cells were stained with safranin O (chromosomes, red) and Brilliant blue R (spindle fibres, blue). Division abnormalities were scored.
• Similarly treated cells were investigated with antibody probes for alpha and gamma tubulins and visualised as green and red signals respectively to identify division abnormalites.
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The Induction of aberrations of mitotic cell division by Bisphenol-A in the Chinese hamster cell line V79 Concentration
g/ml % Abnormal metaphase cells % Abnormal mitotic cells % Normal mitotic cells 0 4.4
11.7
88.3
1.4
2.8
4.2
8.4
14 28 7.6
5.2
16.5* 16.3* 11.4* 22.4* 10.4
11.2
23.5* 34.5* 28.1* 54.2* 89.6
88.8
76.5
65.5
71.9
45.8
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Analysis of the induction of aberrations of the mitotic spindle and centrosome organisation in Chinese hamster V79 cells following exposure to Bisphenol-A Concentration ugml 0 14 28 Bipolar spindle 88 63* 50* Tripolar spindle 10 31* 24* Tetrapolar spindle 2 5 20* Multipolar spindle 0 1 6* -
Cells were exposed to Bishenol-A for one cell cycle, fixed and hybridised with antibody probes for -tubulin and -tubulin. The cells were then treated with secondary antibodies to illuminate -tubulin with a green fluorochrome, and -tubulin with a red fluorochrome. 100 cells per treatment were evaluated for the fidelity of microtubules in the spindle (green -tubulin) and centrosome organisation (red -tubulin).
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Cell Cycle Analysis
Alpha tubulin, gamma tubulin and Dapi stained 20
Mechanisms
• Analysis of the influence of the p53 gene product on the fidelity of chromosome separation at mitosis.
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Aberration Abnormality
Normal metaphase PSCS Premature sister chromatid separation PCD Premature centromere division TPCD Total premature centromere division ER Endoreduplication No abnormal separation or replication events Sister chromatids loose cohesion but centromeres undivided Individual chromosomes loose cohesion, including centromere All chromosomes loose cohesion, including centromeres Replication of all chromosomes without separation 22
normal Premature sister chromatid separation of some chromosomes Total premature centromere division endoreduplication 23
18 16 14 12 10 8 6 4 2 0
Chromatid separation abnormaities
(after mitotic shake-off)
PCD TPCD Type of abnormality ER Neo Scx E6
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In vivo analysis
•
In vivo
somatic endpoints are currently limited to the bone marrow • There was a need for a GI tract assay
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Swiss roll Fixation Analysis Staining Embedding Mounting Sectioning 26
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Induction of micronuclei in the GI tract of mice exposed to 1 and 1.5 mg/kg Colcemid Dose Control 1mg/kg 1.5 mg/kg Sampling Time hrs 12 18 24 36 48 12 18 24 36 48 12 18 24 36 48 Micronuclei /1000 cells 0.73
1.2
1.3
1.11
0.75
7.25
5.15
12.69
6.05
7.67
4.6
6.37
5.33
11.68
4.60
Micronuclei /cypt 0.03
0.05
0.07
0.06
0.04
0.38
0.23
0.63
0.26
0.36
0.21
0.29
0.26
0.38
0.20
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Analysis of aneuploidy in the sperm of rodents and humans Methodology developed by GSF Neuerberg
•
Labelling of chromosomes with centromere specific probes.
• •
, Probes available for most of the human chromosomes.
Probes for chromosomes 8, 13, X and Y are most frequently used for mice. Each chromosome labelled with specific fluorochrome.
• •
Typically, 10,000 sperm are analysed per male mouse and human.
•
Samples analysed manually for number of each chromosome in each sperm (typically takes 8hrs)
•
Recent development is to use laser-scaning cytometry using argon ion and helium neon laser unit.
Cells scanned directly on a slide, typically 30 minutes per 10,000 sperm.
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3-chromosome FISH-labelling for the detection of aneuploidy in sperm of mice (sperm FISH assay) 8-probe Signal CY3 X-probe Signal FITC + CY3 Y-probe Signal FITC ANEUPLOID SPERM (X-Y-8)
Phenotypes of abnormal sperm
Digitized images (software ISIS3, MetaSystems, Altlussheim, Germany) 31
Demonstration of the effectiveness of the sperm FISH methodology in the detection of induced aneuploidy Five self-poisoners taking a mean of 2.8
mg/kg/bw of Diazepam, Sperm analysed 45 days after exposure. 4 colour FISH used for chromosomes 1, 16, X and Y.
Exposure to mg/kg/bw of diazepam Sperm Analysis % Abnormal Sperm Chromosome number >23 <23 46 92 0 Mean 2.8
51069 51348 0.22
0.38* 0.09
0.14* 0.35
0.71* 0 0.02
* Significant p<0.01
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3-colour FISH analysis of sperm of mice given a single oral dose of 300 mg/kg Diazepam (probes for chromosomes X, Y and 8) Treatment Normal chromosome numbers Abnormal chromosome numbers (%)
X-8 Y-8 Hyperploid Hypoploid Diploid 0 26,115 25,907 26 (0.05) 27 (0.052) 1 (0.002) 300 25,372 25,427 41 (0.081) 27 (0.053) 9 (0.002) 33
Result of the three-colour FISH assay with epididymal sperm of mice treated with colchicine and Etoposide (VP-16 ) Number of animals Sperm scored Control 5 50087 Colchicine (3mg/kg) 5 50058 VP-16 (25 mg/kg) 5 50123 VP-16 (50 mg/kg) 5 50170 Diploid Sperm X-Y-8-8 X-X-8-8 Y-Y-8-8
Total
% Diploidies
SD
0 0 0
0
0.0
0.0
0 1 1
2
0.004
0.005
1 11 4
16
0.032
0.005** Disomic Sperm X-X-8 Y-Y-8 X-Y-8 X-8-8 Y-8-8
Total
% Disomies
SD
9 2 2 9 4
26
0.052
0.013
8 8 3 10 15
44
0.088
0.018*
15 5 3 3 11
37
0.074
0.013
#
# *p<0.05, **p<0.01, compared with the concurrent control (chi-square test) p<0.05, compared with the concurrent solvent control Mann-Whitney
U
-test.
4 19 5
28
0.056
0.005**
17 10 7 14 13
61
0.122
0.011**
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Analysis of effects of chemicals upon rodent female germ cells Basic methodology developed by Eichenlaub-Ritter’s group at the University of Bielefeld
,
Germany
.
Female mice of different ages – 12 to 26 days old are super ovulated.
•
12-14 days old
Generates pre antral follicle cultures matured for 16 to 20 hours in presence of human chlorionic gonadotrophin and recombinant epidermal growth factor .
• •
24 days old 26 days old Generates pre MI oocytes Generates MII oocytes after 12 hours in vitro culture
•
Oocytes can be exposed to test agent in vitro and effects upon meiosis analysed.
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Spindles in Living and Fixed Mouse Oocytes Spindle in Unfixed Metaphase II Mouse Oocyte Examined Non-invasively by Enhanced Polarization Microscopy Spindle Metaphase II Spindle and Chromosomes in Fixed Mouse Oocyte by Confocal Microscopy
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Analysis of spindle formation and chromosome alignment after 16h or maturation of oocytes in absence (Control) or presence of diazepam, or after exposure to diazepam for the second 8h of culture Concentration μg/ml Control Number of oocytes 81 Normal (%) Aberrant (%) 79 (97.5) Spindle 2 (2.5) Chromosomes At equator (%) 80 (98.7) Unaligned 1 (%) (1.3) Solvent 5 25 44 29 94 41 ( 26 68 93.2) (89.7) (72.3) 3 3 26 (6.8) (10.3) (27.7) 40 26 65 (90.9) (89.7) (69.1) 4 3 29 (9.1) (10.3) (30.9) 25 (2 nd ) 27 24 (88.9) 3 (11.1) 22 (81.5) 5 (18.5)
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Transfer of aneugenic damage
• Analysis of induced aneuploidy in Embryos
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App l icati on of i n terphase FISH t o aneu plo idy assessment in two -cell embr y os Non-dis juction in 1 st mitotic d i vision Chromo some los s in meiotic div i sion Non-dis junction in meiotic div i sion Chromo some los s in 1st mitotic d i vision + MN
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Why study Trichlorphon (Trichlorfon)?
§
Broad spectrum o.p. insecticide which functions by inhibition of acetylcholine esterase
§
Trichlorphon is stable in water at acid pHs, at higher pHs transformed to Dichlorvos
§
Key observation of Czeizel et al 1993, who reported a cluster of congenital abnormalities (including Downs Syndrome) in a population exposed to trichlorphon via fish.
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Induction of micronuclei in cultured human lymphoblastoid cell line AHH-1 treated with trichlorphon for 6 hrs at pH5.5
Concentration of trichlorphon μg/ml
0 5 10 20 40 80
% Binucleate cells
41.5
31.5
27.2
22.9
15.6
10.3
% Micronucleated Binucleate cells
1.85
3.55* 5.50* 6.60* 7.40* 10.20*
% Centromere positive micronuclei
56 78 75 73 49 39 * Significant p<0.05
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Induction of non-disjunction in cultured human lymphoblastoid cell line AHH-1 treated with trichlorphon for 6 hours at pH5.5
Concentration of trichlorphon μg/ml % Binucleate cells Non-disjunction in 1000 Binucleate cells Chr 2 Chr 7 Chr 18
0 1 10 20 40 80 39.00
36.49
27.23
24.47
19.23
10.05
3 9 18 22 10 14 3 9 21 25 20 6 4 10 23 26 14 14 43
Induction of disomic sperm in trichlorphon treated mice Concentration of Trichlorphon in mg/kg/bw
0 200 300 405
% sperm disomic for chromosomes X, Y and 8
0.04
0.07
0.07
0.13
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Analysis of chromosome numbers in oocytes generated from pre antral follicle culture. Exposure to Trichlorphon for 16 hours.
Concentration trichlorphon μg/ml 0 6 12 50 100 Chromosome numbers (%)in metaphase II oocytes 20 85 57 46 46 38 <20 2.3
4.6
2.9
4.0
3.8
> 20 0 1.5
1.5
1.6
1.9
No increases were observed if trichlorphon was kept for 6 hrs at neutral pH 40 13 35 50 49 57
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Conclusions
• The PEPFAC project has resulted in a package of methodologies suitable for application in the regulatory process. The package enables the assessment of aneugenic activity from cultured cells to male and female germ cells as required by international regulators and experts.
• The expertise developed is a unique facility and provides the basis for making a significant impact upon human health.
• Thank you Framework V.
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