From hazard detection to risk assessment – a journey in Genetic Toxicology
James M Parry, Centre for Molecular Genetics and Toxicology,
University of Wales Swansea,
Swansea, SA2 8PP, UK.
The following presentation is an updated version of the EEMS
Frits Sobels Prize Lecture in Aberdeen 2003

Introduction
During the course of this presentation I will be covering the key features of my research career with particular emphasis upon the people with whom I collaborated in both research and teaching.
In my view, these interactions turn research from an often boring activity into something that aids progress, generates excitement and sometimes even generates something useful for future generations.
I have been particularly fortunate in that the major portion of my career has involved the training of graduate students and observing the development of their careers has brought me very great pleasure.
I will highlight throughout this presentation the roles in various projects of particular graduate students and post-doctoral scientists.
I

Background
My early research experience (Ph.D. and Post-Doc) was in Liverpool, Belfast and Oxford and involved the study of recombination and DNA repair in the yeast Saccharomyces cerevisiae.
Working with Brian Cox in Oxford we isolated the first comprehensive package of over 100 DNA repair mutants which we mapped to more than 20 genes!
At that time few people believed that there could be more than a few genes involved in DNA repair. When we presented our data at a Genetical Society meeting in Glasgow we were treated with some disbelief and amusement
When I moved to Swansea in 1967, I decided to focus my work on characterising the kinetics of repair of radiation-induced DNA damage together with my first Ph.D. student Ray Waters and on radiation-induced recombination with my students Richard Tippins, Emrys Evans, Peter Davies and Steve Kelly. Richard and Steve are shown in the Plate 1 discussing their research on radiation-induced damage during mitotic and meiotic cell division

Professor Steve Kelly and Dr Richard
Tippins discussing the results of their yeast studies over a glass of wine.

•
Early in the 1970’s the members of my laboratory started to work with chemical mutagens and directed our research into the rapidly developing area of Environmental Mutagenesis.
• At that stage in my career, I was sufficiently confident in my own ability to believe that I could construct yeast strains capable of detecting all those genetic endpoints that can be modified by chemicals and radiations.
•
Also my research interests and those of my wife Elizabeth started to coalesce and developed a common theme.
•
Elizabeth had undertaken a D.Phil. in Oxford which involved the study of the stability of triploid and aneuploid yeasts and she had developed a high level of expertise in constructing new yeast strains.
Her work acted as a stimulus for me to attempt to engineer yeast strains which would be suitable for studying the mechanisms of aneuploidy induction in yeast.
•
This work led to collaboration with Fritz Zimmermann (from
Darmstadt) and competition with Silvio Albertini (currently at
Hoffmann La Roche)who had related research interests.

Aneuploidy Induction and its biological significance
• During the 1970’s I developed a passion to detect and understand the mechanisms of action of chemicals which induce numerical chromosome changes – i.e. aneugens.
• An event of particular interest was my 1 st meeting with Frits Sobels in Zurich hearing about his Drosophila models to detect radiationinduced aneuploidy.
• I talked to him about some of the progress in the development of yeast models to detect chemical and physical induced mitotic and meiotic aneuploidy.
• I tried to convince Frits that we could develop yeast models which would be more effective than Drosophila models. Personally, I never could face getting to work without breakfast just to be able to separate virgin flies.
• Needless to say I didn’t convince Frits but he did encourage me to join the early EU research programmes and invited me to join the
Editorial Board of Mutation Research.

Professor Frits Sobels and Professor Diana
Anderson enjoying the social programme at the European Environmental Mutagen
Society Meeting in Berlin .

•
•
By the early 80’s we were coming to the conclusion that there was a need to undertake a thorough validation of yeast test systems for the detection of aneuploidy and other genetic endpoints such as gene conversion and point mutation
We thus decided to participate in a series of both UK and International
Collaborations involving the comparisons of the specificity and sensitivity of assay systems for the detection of chemical carcinogens.
•
•
An important validation project was the so-called “42 compound study” initiated by the UK Health and Safety Executive and led by John Ashby,
Peter Brooks, Ian Purchase and Bryn Bridges.
Our Swansea contributions to these projects involved the development and application of a range of yeast systems we had developed. These studies included the Ph.D. studies of Phil Wilcox, Stelianos Piperakis and Terry Brookes.
Methods were developed to measure:-
•
1.
The induction of mitotic crossing-over
2.
The induction of mitotic gene conversion
3.
The induction of point mutation
4.
The induction of aneuploidy

Dr Phil Wilcox, a Vice-president of the pharmaceutical company GlaxoSK, a former graduate student who worked on yeast genetics

A collection of the presidents of
UKEMS, John Ashby, Michael Green
Barry Elliott and the author

My conclusions from the collaborative studies were that:
•
•
•
•
•
•
Many of the available assays were unsuitable for routine screening.
It is a very bad idea to become emotionally committed to a particular test system.
Chemical carcinogens function by a range of mechanisms and thus their detection and assessment requires the use of a range of test systems.
Might be valuable to have some guiding concepts for our research and we selected the following quotations:
“Out of the nettle danger, we pluck this flower, safety”
William Shakespeare
“If it is not fun don’t do it"
Jim Parry

Problems with yeast test systems
• The Mitotic Test Systems fail to detect reference aneugens such as colcemid.
• The Meiotic Test Systems had a major defect – sporulating yeasts fail to take up large chemical molecules. They are thus only of value in the study of radiations.
Where should we go now?

Development and application of new test systems based upon the use of cultured mammalian cells.
Key events:
• Ray Waters had returned from Oak Ridge with expertise in the use of cultured mammalian cells.
•
I had met Ilse-Dore Adler (GSF Neuherberg) at the EEMS meeting in Dublin.
• We decided that the group needed cytogenetics expertise so we appointed Natalie Danford to develop this expertise. Isle-Dore kindly agreed to provide initial training for Natalie.
• We set up a collaboration with Brian Dean at Shell who had expertise in the culturing of rat and Chinese hamster cell lines.
• Elizabeth had returned to laboratory research and wished to develop methods to study the mechanisms of aneuploidy induction.

Professor Ray and Mrs Eileen Waters during their period at the Oak Ridge
National Laboratory

Dr Ilse-Dore Adler during a visit to my laboratory in Swansea

Interacting Developments :-
• Development by my wife Elizabeth of morphological methods to study mitotic aberrations.
• Development by Natalie of method to accurately score cells with chromosome gains.
• Development of chromosome specific probes and metabolically active genetically engineered cell lines
(collaboration with J. Doemer and Rolle Wolf) and their application by Tracey Warr, Sian Ellard, Anna
Lafi, Kevin Adams, John Stefford, Russ Bourner and
Chiara Corso allowed us to study induced clastogenic and aneugenic changes in cultured mammalian cells.
But, we still needed a system which would give us a higher throughput but still maintain quality

Example of a Chinese hamster cell at the metaphase of cell division stained to illustrate both the chromosomes and the spindle structure

There were a number of important steps and personalities that had a major influence of the way I was thinking about where our laboratory would progress

•
•
•
•
•
I was aware of the 1950s work of John Evans on radiation-induced micronuclei in plants.
Michael Fenech was on sabbatical at Sussex University. At small
Workshop organised by Colin Arlett at CTL, Alderley Park, we heard about Michael’s development of the binucleate cell assay.
A series of meetings had been initiated by Baldev Vig of the University of
Reno which brought together people interested in chromosome segregation.
At these meetings we learnt about Bill Ernshaw’s (currently in
Edinburgh) work on kinetochore proteins. Elizabeth and I suggested that antibodies to these kinetochore proteins had the potential to detect whole chromosomes in micronuclei. This concept was validated by studies of Francesca Degrassi (Rome) and Paul Perry (Edinburgh).
At various meetings we heard about the development of centromere probes for specific chromosomes which could be used to identify specific chromosomes in interphase cells. Such probes would enable the measurement of non-disjunction in binucleate cells produced by treatment with the actin inhibitor, cytochalasin B.

A conversation with Michael Fenech during a conference in Crete

Baldev Vig and Andreas Kappas at an aneuploidy meeting in Crete

Integration of methods
•
These methods were integrated in our laboratory by Anthony Lynch, Tracey Warr,
Ann Doherty, Thierry Hermione, Justine
Williamson, Frank Haddad, Sian Ellard and
Mahmood Kayani for the study of a wide range of genotoxic chemicals.
• More recently, Emma Quick, George Johnson and Paul Fowler have been using these methods to study mechanisms of action of aneugenic chemicals and to investigate dose-response relationships of genotoxins.

Dr Anthony Lynch (now of GlaxoSK) receiving the UKEMS Young Scientist
Prize from Professor Colin Garner (York)

Dr Tracey Warr analysing her data on the mechanisms of action of aneugens

Thierry Hermione, Gareth Jenkins and Frank Haddard after receiving the
1995 UKEMS and Mutagenesis prizes for their graduate work in
Swansea

Dr Sian Ellard about to leave
Swansea for a new career in
University of Exeter Medical School

Sian Ellard and Ann Doherty (currently of
AstraZeneca) enjoying the delights of the
EEMS meeting in Barcelona

Swansea laboratory cytogenetic staff in
2003
Emma Quick, Shareen Doak, Margaret
Clatworthy, Sally James and Anna
Seager

Chemical
Insult
Normal Segregation
Cytochalasin B
Non-disjunction
Micronucleus Loss
1) Non-disjunction 2a) Chromosome Loss 2b) Fragment Loss

Mn

Chr Loss

Chr 17
Chr 1
Chr 7

The application of the micronucleus assay to analyse the potential genotoxicity of the environmental chemical bisphenol A. Our work was stimulated by the publication of
Hunt et al., 2003 which suggested that bisphenol A was a potent inducer of aneuploidy in mouse germ cells.

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

The structure of BP-A strongly resembles that of the potent oestrogen DES which has known carcinogenic effect.

11.0
16.5
27.5
33.0
66.0
132
Concentration

M
Analysis of the induction of micronuclei by
Bisphenol A in cultured AHH-1 cells
 g/ml
%
Binucleate
Cells
%
Viability
Micronuclei per 1000 binucleate cells
0 solvent DMSO 63 100 2.7
5.5
1.25
62 98 2.9
2.5
3.75
6.25
7.5
15.0
30.0
60
59
61
52
29
16
96
94
97
83
46
25
5.0
7.0
9.2
8.1
10.7
27.1

20
30
10
15
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
63.0
36.4
0.4
38 62 Solvent
(DMSO) control
0
5
66.7
66.5
33.0
33.1
0.4
0.75
34
-
66
-
65.8
66.6
63.5
62.7
33.8
31.5
34.8
35.9
2.05*
2.65*
3.20*
3.85*
63
-
56
59
37
-
44
41

Induction of chromosome non-disjunction in human lymphoblastoid cell line MCL-5 by
Bisphenol A
Concentration
μg/ml
% NonDisjunction
Chromosom e 8
Chromosom e 17
Chromosom e 20
Total
0.6
0.3
0.2
1.1
Solvent (DSMO) control
0
5
10
15
20
2.1
2.5
0.4
1.2
3.1
1.0
1.4
0.2
0.6
2.2
1.5
1.9
0.2
1.1
2.8
0.8
2.9*
4.6*
5.8*
8.1*

Example of a dividing cell stained to demonstrate the structure of the mitotic spindle in untreated cells

Cell treated with bisphenol A showing the presence of a cell with a tripolar spindle

Alpha tubulin, gamma tubulin and Dapi stained

• BPA is capable of inducing aneuploidy in both somatic and germ cells
• The aneugenic activity of BPA shows thresholds of activity below which no induction occurs
• The aneugenic activity occurs at dose substantially higher than those which produce hormonal effects

Collaborative Aneuploidy Projects
Our developing interest in aneuploidy and the encouragement by the EU to develop collaborative research led to interactions which have been the most pleasurable and scientifically profitable part of my research over the past 15 years.
•
•
•
•
Currently this research is represented by the PEPFAC
Project which involves collaborations between Swansea and:-
Micheline Kirsch-Volders
Ilse-Dore Adler
Ursula Eichenlaub-Ritter
Francesca Pacchierotti
- Brussels
- Neuberberg
- Bielefeld
- Rome

The PEPFAC team in Brussels

The PEPFAC team in Rome

Some of the major developments of the PEPFAC Project
Development of the ability to investigate the induction of aneuploidy and mechanisms of action of aneugens in systems ranging:
From Cultured cells (detection and mechanisms of action )
Rodent bone marrow, haemopoetic system, GI tract
Male germ cells, rodent and human
To Female germ cells and embryos

Environmental Genetic Toxicology
Back in the 1980’s David Tweats and I decided to ask the questions about whether genotoxins were present in the marine environment.
A major consideration our decision to undertake environmental studies was David’s passion for fishing and my desire to find an excuse to go to the beach.

David Tweats and David Gatehouse
(both then at GlaxoSK) working in our Swansea Laboratory in 1975

My beach assistants Jane and
Matthew (my children) making mussel collecting trips

The Swansea Beach which we monitored to ensure the absence of genotoxins

Environmental Genetic Toxicology
Over the next 15 years our group undertook studies into the presence and significance of mutagens in the marine environment based primarily upon the sampling of shellfish (mussels) and a number of free swimming fish from polluted and pristine environments.
The group included Munira Kadhim, Brett Lyons, Jim Harvey, Colette Jones,
Kristen Elwin, Claudia Browning, Bill and Linda Barnes from the USA.
We demonstrated the presence of complex mixtures of mutagens in various polluted environments and seasonal variations in genotoxin levels by measuring the induction (using 32 P-post labelling) of DNA lesions and their repair.
We characterised the metabolic potential of a number of marine species and their ability to metabolise compounds such as benzo(a)pyrene
We developed the use of a clawed toad (Xenopus) model for study of mutagens in freshwater samples.
The most complete story in our studies was that of the Sea Empress.

My wife Dr Elizabeth Parry and Dr
Munira Kadhim (Medical Research
Council, Harwell) at the ICEM in 2000
Awaji Island Japan

Bill and Linda Barnes (visiting
American Post-Docs) preparing to collect mussels to evaluate for the presence of mutagens

Dr Jim Harvey (currently of
GlaxoSK) preparing to collect shellfish to measure DNA adducts

Dr Brett Lyons monitoring the quality of the environment around oil rigs

•
•
•
•
•
Environmental Genetic Toxicology
The saga of the Sea Empress
15 – 21 February 1996
Released 72,000 tons crude and 360 tons of heavy fuel oil.
Onto the Pembrokeshire coast of Wales
Area contained 2 marine nature reserves.
We monitored the area for a period of 12 months from the day the oil reached the beaches.

• Crude oil contains DNA reactive compounds (both direct and indirect)
• These compounds cause genetic damage
• Environments recover from genetic damage

A m odel for freshwater studies – clawed toad
Induction of micronuclei in larvae of Xenopus laevis
Benzo(a)pyrene – DNA reactive
Concentration
 g/ml
0
0.1
0.2
0.5
0.1
0.2
0.5
1.0
Micronuclei in 2000 erythrocytes
7
12
15
28
35
49
31
25
Kinetochore positive micronuclei %
22
23
19
20
19
23
21
24

• Clear evidence for the induction of kinetochore negative micronuclei
• Conclusion clastogenic activity
• B(a)P metabolised to a genotoxin in the clawed toad

The Clawed Toad Model
Induction of micronuclei in larvae of Xenopus laevis
Benomyl – an aneugen
Concentration
 g/ml
0
1
5
10
50
100
500
Micronuclei in 2000 erythrocytes
Kinetochore positive micronuclei %
47
56
83
72
9
15
28
65
72
81
79
29
59
58

• Clear evidence for the induction of kinetochore positive micronuclei.
• Conclusion, benomyl has aneugenic activity

Clawed Toad Model
Induction of micronuclei in larvae of Xenopus laevis
Trichlorphon -aneugen
Concentration
 g/ml
Micronuclei in
2000
Erythrocytes
Kinetochore positive micronuclei %
0
0.1
0.5
1.0
5.0
10.0
50.0
11
9
23
59
87
153
216
25
29
51
67
72
84
81

• Clear evidence for the induction of kinetochore positive micronuclei
• Conclusion, trichlorphon has aneugenic activity

Development and application of the restriction site mutation assay
During laboratory discussions we concluded that there was a need for an assay which could detect rare mutations in any gene of mutagen exposed species.
These discussions lead to the development and application of the
Restriction site mutation (RSM) methodology which theoretically allows us to detect mutations in restriction enzyme recognition sites of any gene for which the sequence is known and in any species
The work on this methodology has been the basis of the Ph.D. and post doctoral work of Gareth Jenkins, Hong-lin Song, Bryan
Myers, Rosa Sueiro and Nobuo Takahashi.

Outline of the principles of the
RSM assay

A.
CC
GG
GGCC
CCGG
Digest
G
A
CC
C
T
GG
GGCC
CCGG
G
A
CC
C
T
GG
GG
CC
Restriction fragments
G
A
CC
C
T
GG
PCR amplify
B.

Isolate target gene
Amplify external standard dilution series alongside
Digest DNA with RE
PCR amplify with proof reading
Taq
Digest PCR product with RE
Electrophorese
Quantitate mutation frequency
Re-PCR and sequence
Mid PCR digest to remove wild type

ENU treated spleens Controls
Ava II fragments
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

5
259 bp
Human p53 gene restriction sites
6
188 bp
7
79 bp
8
262 bp
9
196 bp
75 bp

2
2
All mutations Amino acid altering mutations
247 248 249 250
AAC CGG AGG CCC
247 248 249 250
AAC CGG AGG CCC g T A T a a A t t g
T a
T T AC A T
T a A

T
A
T
T
T
T
G T
A
A C A
T
A
CCGG GGCC
GGCC CCGG
248 249
A
A
A
A
A
A
T
GGCC
248
A
T
A
A A
A
A T
GGCC CCGG
T
T
T
T
248 249
Hydrogen Peroxide Oesophageal Gastric

Dr Bryan Myers a member of our
RSM development group

Mrs Diane Elwell and Dr Gareth
Jenkins following a successful RSM experiment

Application of Restriction Site
Mutagenicity in the Clawed Toad Model
Larval Beta-globin gene exon 1 B(a)P treatment –
Dose
0.05 mg/ml
0.1
0.05
Recovery time
6 hr
6 hr
6 hr
Enzyme
Dra 1
Dra 1
Rsa 1
0.1
6 hr Rsa 1
Greater than 10x increase in mutation observed
Mutation frequency
4.5*10
–4
1.7*10 -3
1.8*10 -3
5.4*10 -3

Analysis of chromosome changes in the rat model
•
Collaboration with developed George Moen’s group at
Bilthoven, particularly Joyce de Stoppelar and Barbara
Hoebee who developed probes for the analysis of rat chromosomes.
This involved the application of Comparative Genomic
Hybridisation (CGH) by Dr Chiara Corso (currently in
Berlin) to investigate chromosome changes in rat gastric tumours.

The Bilthoven and Swansea groups meeting to discuss the genetics of the rat

Chiara Corso who introduced the
CGH technique to the Swansea laboratory

Principles of Comparative genomic hybridisation

The CGH methodology is highly effective in identifying deletions and gains (whole or partial) of specific chromosomes in solid tumours of humans and rodents.

Analysis of the mitochondrial genome
• Like many old (mature) yeast geneticists I had an interest in mitochondrial mutations (petites). I met Ethel Moustacchi as a post-doc in Oxford in 1966 and encouraged Ray Waters to join
Ethel for his post-doc to work on DNA repair capacity of yeast mitochondria.
• Our previous work was undertaken in collaboration with a post–doc Paul Lewis, two clinicians Prue Baxter and Paul
Griffiths and our current researchers are Misses Sue Fradley and
Sarah Prior.
•
The work has involved the analysis of mitochondrial changes in colorectal and oral tumours.

Our mitochondrial researchers Sarah
Prior and Sue Fradley

The Human mitochondrial genome

•
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•
Mitochondrial Studies
Warthins tumour of salivary gland characterised by excessive numbers of mitochondria. Smokers have 8x increased risk of developing the tumour.
The questions we wanted to address were whether the excess of mitochondria was due to a proliferation of normal or abnormal mitochondria and whether the proliferation plays a role in the aetiology of the tumour.
Analysis of mitochondrial deletions using 2 colour FISH for normal and deleted mitochondrial DNA in both tumour tissue and control tissue.
The frequency of the deleted mitochondrial DNA was also measured by quantitative PCR.

Mitochondrial Studies
Probes red fluorescent probe binds to all mtDNA molecules green fluorescent probe binds only to mtDNA that contains deleted regions
2 colour probing normal mt DNA fluorescent yellow deleted mtDNA fluroescent only red

Fluorescence-labelled mitochondria

Mitochondrial Studies
Fluorescence analysis
In oncocytes from tumour tissue approximately 10% of mitochondria carry the deletion.
Quantitative PCR
Demonstrated that the deleted mtDNA was present in control tissue at a level of approximately 0.1%
In tumour tissue the deletion was present at a mean of
2.54%.

Mitochondrial Studies - Non tumour tissue
Methodology
•
•
Comparison of frequency of mitochondrial DNA.
Deletion in non-tumour parotid gland tissue of smokers and non smokers.
Also analysed the relative frequency of point mutations in the parotid gland tissue using single strand conformational polymorphism (SSCP) analysis.

WT DNA DNA containing a point mutation
Denature double-stranded DNA
Run products through a polyacrylamide gel at 4

C
WT Mutant

Mitochondrial Studies - Non tumour tissue
Results
Deletions mtDNA deletions were detected in
•
•
• all samples ranging from 0.028 to 0.08% no correlation with smoking habit strong correlation with age
•
•
•
Point Mutations
No point mutations detectable by SSCP found in tissue of nonsmokers.
21.7% of smokers carry point mutations detectable by SSCP.
These point mutations were transitions characteristic of oxidative damage.
Work now being extended to oral squamous cell carcinomas.

•
Genetic instability
Most cancers are clonal and also karyotypically heterogeneous.
•
• Relative contribution of point mutation and chromosome I instability?
How to establish the relative timing of these events?
•
• How to establish the causative events at each stage of progression?
Combine together the analysis of point and chromosomal mutation.
•
Characteristic gene expression and protein changes associated with changes.

Application of our methods to study the aetiology of cancers
Frequency of Aneuploidy in Tumour Progression
Studies of Shareen Doak, Lisa Williams, Claire Morgan, Gareth Jenkins, Jeanette
Croft and Liz Parry.
• Barrett’s Oesophagus
• Condition where the normal squamous oesophagus is replaced by a columnar epithelium.
epithelium of the distal
•
•
•
• Progression condition via to malignancy can occur from this metaplastic
Low grade dysplasia
High grade dysplasia
Adenocarcinoma

Plate 43
Barrett’s Oesophagus
The red columnar epithelium representing Barrett’s Oesophagus can be clearly distinguished from the normal, pink squamous cell mucosa of the oesophagus.
Normal Squamous Cell
Mucosa
Barrett’s Columnar Cell
Epithelium

Analysis of tumour progression in Barrett’s Oesophagus
•
Methods
Interphase cell analysis of touch preparations from biopsies or brush samples over complete progression
• Two-colour fluorescence in situ hybridisation (FISH) using direct fluorescent labelled probes for chromosome and/or gene specific sites.
• Chromosome or gene copy numbers counted in approximately 500 cells/patient.
• Markers: cen1, cen4, cen9 and p16(pp21), Rb(13q14,1),
P53(17P13.1), cenX and cenY

Chromosomal analysis of tissue samples from the upper GI tract

Plate 45
FISH images displaying p53 (red probe) deletions in a cluster of cells within an oesophageal adenocarcinoma. The green signal represents the centromeric region of chromosome 8. Coupled maps of the images indicate the cells demonstrating p53 loss.

Research Related Activities
• Writing of “Mutagenicity Testing” with Stan Venitt.
• Founding Editor with Stan as Associate Editor of “Mutagenesis” in
1986.
• Member of the UK Advisory Committee on the Mutagenicity of
Chemicals. Chairman 1990-2000. Production of “Guidance on
Mutagenicity Testing”
• UK Government Committees and Working Parties on
Mutagenicity of Pesticides, Hormones and Food Chemicals.
•
Various OECD Meetings on Testing Methods.
• Vice-President and President of EEMS

Dr Stan Venitt at ease in his office at the Institute of Cancer Research

Our textbook showing the world how to undertake mutagenicity studies

The first issue of the journal
Mutagenesis

The 2000 Mutagenesis Editorial
Board Meeting in Japan

The UK Committee on Mutagenicity
Guidelines 2000

Developments in Toxicogenetics
• Like many laboratories worldwide the development in microarrays have provided us with the opportunity to investigate the influence of some of the genetic changes we have observed on gene expression of various cell and tissue types.
• This has involved the work of Gareth Jenkins and Shareen Doak on oral cancers, Sue Fradley on bowel cancers and Paul Fowler upon the significance of high toxicity clastogens.

Paul Fowler and Gareth Jenkins taking a break from their gene expression studies.

•
Assessment of genotoxicity of new and existing chemicals relies on the measurement of mutagenic ability in a number of different test systems and over a variety of exposure concentrations
•
Where a mutational effect is observed at high levels of cell survival it is relatively straightforward to predict that the chemical may have significant biological effect in vivo dependant on metabolism and other factors

• Where chromosome damage is caused only at high levels of toxicity then predictions of potential biologically significant mutations are more complex due to factors such as apoptosis and other forms of cell death
•
However concentrations of clastogens active at high toxicity that cause chromosome aberrations in vitro are unlikely to occur in vivo



Amsacrine (M-Amsa) - Topoisomerase
II inhibitor at high toxicity
Etoposide - Topoisomerase II inhibitor at low toxicity



8-Hydroxyquinoline – causes oxidative damage at high toxicity
4NQO – Causes oxidative damage at low toxicity
Formation of highly reactive Oxygen intermediates (free radicals)
Main effect is interference with DNA replication, blocks in replication due to:
Alteration in bases (abasic sites)
Removal of bases (attacks glycosidic bond leading to depurination and strand breaks)

Examples of micronucleus studies with chemical of various toxicity

12.0
10.0
8.0
2.0
0.0
6.0
4.0
0
% bi
(cell survival)
20
Low toxicity clastogen
"profile"
30
Dose ng/ml
40
% mn/bi
(chromosome damage)
40.0
30.0
20.0
10.0
0.0
90.0
80.0
70.0
60.0
50.0
50 60

High toxicity clastogen
"profile"
2.5
% bi
(cell survival)
2.0
1.5
Significant inhibition
% mn/bi
(chromosome damage)
1.0
0.5
0.0
0 1 10 100 500
Dose ng/ml
1000 10000
40.0
35.0
30.0
25.0
20.0
15.0
10.0
5.0
0.0

Etoposide
50.0
45.0
40.0
35.0
30.0
25.0
20.0
15.0
10.0
5.0
0.0
% Bi %MN / Bi
0 0.2
0.4
0.6
dose ng/ml
Amsacrine
0.8
80.0
70.0
60.0
50.0
40.0
30.0
20.0
10.0
0.0
% Bi %MN / Bi
0 1 2 dose µg/ml
1
3
2.0
1.0
0.0
6.0
5.0
4.0
3.0
9.0
8.0
7.0
2.0
1.5
1.0
0.5
0.0
4.5
4.0
3.5
3.0
2.5

•
Using dose ranges derived from micronucleus assay data, gene expression in cultured cells can be investigated through cDNA arrays
•
Arrays can detect changes in gene expression across a large number of genes in a single experiment.
Such global expression profiling is used where specific pathways or mechanisms are unknown
• Application specific arrays are available for more focused investigations i.e. Toxicity, DNA damage signals

Principles of microarray analysis

Usually expressed due to stress from increased temperature – protect proteins from unfolding and maintain conformation
Normally expressed as result of heavy metal exposure recent evidence shows protective role during exposure to oxidative damage. Metal ion chelators

Examples of gene expression gels

6 7 8
5
1 2 3 4
1 = HSPA2
2 = HSPA1L
3 = HSPA1B
4 = HSPA1A
5 = HSPA6
Heat Shock proteins
6 = MT2A
7 = MT1H
8 = MT1A
Metallothioneins

Superarray Toxicity cDNA arrays
High toxicity chemical 8-Hydroxyquinoline (10µg/ml)
30 min 2 hours 4 hours 8 hours
MT1H HSPA6
HSPA1l

How to sum up a research career
“Life is short, science is long term, opportunity is elusive, experience is often dangerous, judgement is difficult”.
Hippocrates (460- 377 BC)
“Spotting the future is difficult and the grant giving bodies and Vice Chancellors always prefer to support the past”
Jim Parry (1940- )

What sort of activities can make it fun?

Liz and I celebrating our PhDs graduating

Liz and Jim acting as surrogate parents for the wedding of our
Indonesian students

The family team enjoying Wales beating Italy 2-1 in 2002

Acknowledgements
I would like to thank all the over 100 graduate students and post-docs who have worked with Elizabeth and myself over the past 30 years.
• It has been a great experience and considerable fun working with all my students and post-docs. It has been an enormous pleasure observing your careers develop.
•
Thanks to Sally, Margaret and Di whose loyalty, hard work and tolerance have kept the laboratory going for 35 years.
•
Thanks to Eileen for her work with Mutagenesis and her support of our bioinformatics projects.
•
Thanks to Dot, Margarets-1+2 and Liz who have understood my handwriting and typed my manuscripts over the years and even
• To all of the members of the EEMS and UKEMS for this honour and your friendships
Finally to Elizabeth (my wife and life long collaborator) without whom I would have given up long ago.