Title: Genomic and Post-genomic Analysis as Tools in Risk Assessment: Toxicogenomic analysis of human umbilical cords to detect potential high risk group from the views of exposure level and susceptibility
1 Genomic and Post-genomic Analysis as Tools in
Risk Assessment Toxicogenomic analysis of human
umbilical cords to detect potential high risk
group from the views of exposure level and
susceptibility Chisato Mori, M.D.,
D.Med.Sc Department of Bioenvironmental
Medicine, Graduate School of Medicine, Chiba
University, Japan,
ASPH Environmental Health Conference at
University of Minnesota July 11-13, 2004,
Minneapolis, MN, USA
2- Key issues in multiple chemical exposure to human
- High-risk group and High-risk life stage
- High-risk group depends on the following two
factors - Exposure level
- Susceptibility
- High-risk life stage is fetal and/or neonatal
periods -
-
-
Mori, Reprod Med Biol 351-58, 2004
3The main goal of our projects is to establish a
new method for risk assessment of fetal exposure
to multiple chemicals to reduce human health
risk for future generation
4Outline of Todays Talk
1, Summary of exposure assessment of
multiple chemicals in human fetus
(High-risk life stage) 2, Application of
toxicogenomic analysis of human umbilical cords
for development of a new risk assessment based on
human fetus 3, Our attempts of risk reduction
5Endocrine Disruptors (EDs)
An exogenous agent that interferes with the
synthesis, secretion, transport, binding, action,
or elimination of natural hormones in the body
which are responsible for maintenance or
homeostasis, reproduction, development and/or
behavior.
EDs are thought to have - a variety of effects
on the reproductive system, immune system,
and nervous system, etc. - a possibility to
affect at low dose - sensitive window(fetus,
neonate, infant) - delayed long-term effcts on
the next generation
6Concentration of chemicals including man-made and
natural in umbilical cords/cord blood from
newborns in Japan
Dioxins
Mori C., Acta Anat. Nippon. 76 361-368 (2001)
PCBs
DDT
DDE
HCH
Chlordane
HCB
Endosulfan
Bisphenol A
Phytoestrogens
(g/g-wet tissue or g/ml)
10-12
10-10
10-14
10-8
7Detection ratio of each chemical in human
umbilical cord
Dioxin 100 PCBs 100 DDE
100 HCH 100 Chlorden
80 Endosulfan 90
Bisphenol A 55 TBT 100 Pb
100 Phytoestrogens 100
Concentration 1st baby gtgt 2nd or 3rd baby
Todaka Mori. Congenit Anom Kyoto 4287-93
(2002)
8Exposure Assessment Sum of the concentration of
persistent chemicals detected in human umbilical
cords
(ng/g-fat)
Concentration
Umbilical cords
9Correlations between PCB conc. and other chemical
conc.
Modified from Mori et al., EHP 2003, 111, 803-809.
10High Risk Group Fetuses highly exposed to
chemicals
highly susceptible
- Our exposure assessment suggests that
there are fetuses highly exposed to multiple
chemicals including man-made and natural in
Japan. - This means that there is the presence
of high risk group exposed to multiple
chemicals, when we carry out a risk
assessment of human. - Exposure assessment find
some babies highly exposed to chemicals, but
in this method we cant find babies who are
highly susceptible. Mori High-risk group and
high-risk life stage Key issues in adverse
effects of environmental agents on human health.
Reprod Med Biol 351-58, 2004
11Outline of Todays Talk
2, Application of toxicogenomic and epigenetic
analysis to a new risk assessment 1)
Animal data 2) Our toxicogenomic analysis
of human umbilical
cords
12Necessity of a new evaluation method of health
risk
Current risk assessment target adult
method assess to risk of each single
chemical Lack of Consideration Susceptibility,
Multiple chemical exposure Sensitive window
Todaka Mori, Congenit. Anom. Kyoto 42 87-93
(2002)
13New Methodsfor the Toxicological Evaluation
DNA
Disorder of spermatogenesis and reproduction
genome
methylome
Post Genome Project
Gene expression (DNA microarray)
Protein expression (Protein chip)
RLGS
Solution of problems - susceptibility - time
- multiple exposure
Biomarkers, Toxicogenomic analysis
14Our Challenge Projects
Animal Studies Effects of neonatal exposure of
DES, Genistein or bisphenol A on gene
expression in mouse testis
The final goal is to apply the animal
experimental data on human study
(From bench-side to bed-side)
15Endocrine Disruptors
DES Bisphenol A Genistein
Diethylstilbestrol a synthetic estrogen A
material of resin-based and plastic products One
of the phytoestrogens
1
10-3
10-6
(Nishihara 1999)
16Experimental Protocol
12 weeks
DES 0.5-50 µg/day Genistein 10-1000
µg/day Bisphenol A 20-200 µg/day
Conventional Toxicological Parameters for the
Male Reproduction
Testis weightSperm countsSperm motility
17Effects of DES
Adachi et al., Chiba Med. J. 77 151-158, 2001
18Effects of Genistein
G10 Genistein 10 µg/day G100 Genistein
100 µg/day G1000 Genistein 1000 µg/day
Adachi et al., Chiba Med. J. 77 151-158, 2001
19Effects of Bisphenol A
Adachi et al., Chiba Med. J. 77 151-158, 2001
20Effects of Neonatal Exposure to Genistein
B
ERa
AR
1
1
0.8
0.8
0.6
0.6
ERa mRNA
AR mRNA
0.4
0.4
0.2
0.2
0
0
4 w
8 w
12 w
4 w
8 w
12 w
Time course
Time course
Shibayama et al., Endocrine J. 48655-663, 2001
21Effects of Neonatal Exposure to Genistein
Western blot analysis
Shibayama et al., Endocrine J. 48655-663, 2001
22Evalutation of effects by neonatal exposure to
DES or Genisten at three different
endpoints in mice Endpoints Gene
expression Reproductive disorder Cancer
level level level
4-12 weeks 12 weeks 18
monthsEvaluation Toxicogenomic Conventional
Toxicological Delayed long-term
methods Analysis Assessment
Effect
DES Alteration
of Adverse effects Adenocarcinoma
gene expression Genistein Alteration
of No adverse effects Adenocarcinoma
gene expression Term after neonatal
exposure to chemicals Newbold et al.
Mori et al., EHP 111, 803-809, 2003
23Toxicogenomics
An emerging scientific field combines studies of
genetics, genome-wide mRNA expression, cell and
tissue-wide protein expression, bioinformatics
and toxicology to understand the roles of
genes-environment interactions in disease.
24Animal Studies to develop a new health risk
assessment derived from fetal exposure to
multiple chemicals
1, Gene expression analysis
using cDNA microarray 2, Methylation analysis
(Epigenetic toxicology) using
RLGS
25Experimental Protocol
Neonatal 5 days
12 weeks
RNA from testes
DES (50 mg/day) Genistein (1 mg/day) Bisphenol A
(0.2 mg/day)
In conventional toxicological test, adverse
effects were detected at 50 mg/day DES and 0.2
mg/day BPA.
cDNA microarray(Mouse GEM I, Incyte) 8800 genes
26Scatter Plots of cDNA Microarray Analysis
Adachi et al., Chiba Med. J. 77 151-158, 2001
27Clustering Analysis of Gene Expression Profiles
Gene expression profiles might be helpful for the
grouping of endocrine disruptors from a view
point of their effects on organs.
Komiyama et al., Toxicogenomics pp156-162, 2003
28Transition of Gene ExpressionsAffected by DES
Neonatal 5 days
4 w
8 w
12 w
DES (50 mg/day)
RNA from testes
In house cDNA microarray (Mouse 14.5 day Fetus,
Helix) 2304 genes
29Transition of Gene Expressions
If the genes up/down-regulated continuously in
long term, they might be the candidates of the
biomarker to indicate the exposure of individuals
to Eds.
Komiyama et al., Toxicogenomics pp156-162, 2003
30Long-Term Effects of Chemicals
- DNA sequence changes are one of the most
important mechanism to explain long-term effects
of chemicals. - Many of endocrine disruptors (EDs) have
- no or weak mutagenic activity.
- ? do not change DNA sequence
- Some of EDs show long-term effects and/or late
effects. - Therefore, some of EDs may have epigenetic
mutagenicity. - ? Necessity to understand the mechanism of
long-term - effects of EDs from a new angle such as
epigenetic - toxicology
31 Trials of Epigenetic Toxicology
Restriction landmark genomic scanning (RLGS) is a
genome scanning method based on two-dimensional
gel electrophoresis using restriction enzyme
sites as landmarks.
32Experimental Design for Screening of Methylome
using RLGS
C57BL/6
30-day-old
DES 0 (mg/mouse/day)0.003 0.3
3 Vehicle Sesame Oil
Epididymis, Uterus
Genomic DNA
RLGS
33Number of Spots Changed by DES (Uterus)
2.5
Total Number of Spots 914
2.0
20
1.5
1.0
0.5
6
4
0
0 ? 0.003
0.003 ? 0.3
0.3 ? 3
DES (mg/mouse/day)
Epididymis vs. Uterus 0.4 , Placenta vs.
Kidney 2.3
Fukata and Mori, Reprod Med Biol 3, 2004 (in
press)
34Application of toxicogenomic analysis
to a new risk assessment
From Animal studies ( From bench-side ) -
Gene expression profiles might be helpful for the
grouping of endocrine disruptors from a view
point of their effects on organs. - If the
genes up/down-regulated continuously in long
term, they can be the candidates of the
biomarker to indicate the exposure of individuals
to endocrine disruptors. - DNA methylation was
changed depending on the dose of DES. Long-term
effects and/or late effects of DES might be due
to the epigenetic modification. To
Toxicogenomic analysis of human umbilical cords
( To bed-side )
35Our challenging framework to establish a new risk
assessment of human fetal exposure to multiple
chamicals
Toxicogenomic Analysis
Step 2
Combined Analysis
Step 1
Step 4
Umbilical Cord
Gene Expression Analysis of Umbilical Cord
Integrated Analysis
Step 3
Gene Expression Analysis of HUCCs
Human Umbilical Cord- derived Cells (HUCCs)
Modified from Mori et al., EHP 2003, 111, 803-809.
Comprehensive Studies - animal experiments
- human prospective studies - etc.
New Risk Assessment
36Concentrations of chemicals in each umbilical cord
Umbilical cord
Compound Total PCBs HCB HCH cis-Chlordane trans-Ch
lordane Oxychlordane trans-Nonachlor p,p-DDT o,p
-DDT p,p-DDE p,p-DDD Dieldrin Endosulfan Heptach
lor Heptachlor epoxide Total
A 73.0 25.0 16.0 0.32 0.59 3.1 8.0 3.1 nd
28.0 nd 7.0 2.8 0.56 2.4 169.9
B 28.0 17.0 18.0 0.48 1.3 1.6 3.4 1.6 nd
28.0 0.53 2.8 3.1 0.82 1.2 107.8
C 33.0 15.0 17.0 0.80 1.3 nd 5.9 nd nd 6
7.0 nd 4.1 nd 1.3 1.3 146.7
D 19.0 9.0 15.0 nd nd nd nd 2.0 nd 16.0
nd 2.6 3.0 nd nd 66.6
E 160.0 42.0 78.0 0.43 1.0 3.6 11.0 11.0
0.78 180.0 1.1 12.0 2.8 0.74 3.3 507.8
F 43.0 23.0 26.0 0.73 1.0 nd 4.2 3.4 nd
47.0 3.6 3.1 4.2 1.3 1.1 161.6
G 53.0 24.0 32.0 0.75 1.2 3.2 7.4 2.3 0.3
6 59.0 0.48 4.4 nd 1.1 2.6 191.8
H 130.0 27.0 21.0 0.54 0.67 2.4 7.6 11.0
0.77 140.0 0.62 3.9 2.0 0.28 1.3 349.1
I 78.0 20.0 10.0 0.59 0.95 3.1 7.7 3.7 nd
56.0 nd 4.3 1.8 0.73 1.3 188.2
nd, not detectable.
(ng/g-fat weight)
37The sum of concentrations of chemicals
38Step 1 Gene expression analysis of umbilical
cord Hieralchal cluster analysis (cut-off at
2-fold 1,976 genes)
E
D
H
G
I
F
A
C
B
39Step 2 Combined Analysis Relationship between
chemical concentrations and gene expression
Hierarchal cluster analysis (cut-off at 2-fold
1,976 genes)
E
D
H
G
I
A
F
C
B
Concentration
Umbilical cords
Gene expression profiles were different between
high exposure gr low exposure gr. Pay
attention to an exceptional case like the
umbilical cord D,
40In the risk assessment, we need to consider the
susceptibility of the people, in addition to the
exposure level.
Umbilical cord sample D
Exposure level was the lowest, but its gene
expression profile was quite similar to that of
high exposure group.
Possible causes
- The sample D was highly exposed to other
chemicals which were not measured in the present
study. - The sample D was very susceptible to
chemicals even at low doses. - Etc.
Sample D is potential high risk group?
Both of them might be regarded as higher health
risk to the individual.
Using gene expression profiles, we can see some
different parameters for the evaluation of
health risk which are undetectable in exp.
assessment
41Conclusions (Toxicogenomics)
- Gene expression profiles of highly exposed
umbilical cords were different from those of
umbilical cords with lower exposure levels. -
There was an exceptional case in which total
concentration of the chemicals was lowest, but
its gene expression profile was quite similar to
those of the umbilical cords with higher total
chemical concentration levels. - These results
suggest that the gene expression profile of
umbilical cord can be used for evaluation of
exposure levels during fetal period. - Moreover,
it might be useful to detect potential high risk
group, because both of the actually higher
exposure and genetic susceptibility of an
individual might be regarded as health risk to
the individual. Mori, Reprod
Med Biol 351-58, 2004
42Outline of Todays Talk
3, Our attempts of risk reduction for
future generation 1) Using risk communication
to reduce exposure level 2) A clinical
strategy to reduce accumulation level of
persistent chemicals in human body
43Strategy of risk reduction
Exposure from environment
Avoid multiple chemical exposure
Intake
Risk communication Environmental education
Human Body
Reduce the concentration level of persistent
chemicals in human body
Clinical treatment
Elimination of accumulated chemicals
44 Framework of risk avoidance of chemicals (based
on fetus)
Exposure Assessment
Fetal Exposure to multiple chemicals
Convenience Comfortableness, etc.
Risk Assessment of Chemicals
benefit
Risk Assessment
Risk
High or Clear
Low or Unclear
Dioxin
Mixture of chemicals Endocrine Disruptors
Administration ? Industry ?
Strategy for Risk Avoidance
Administration Industry
Preventive Medicine
Primary prevention Secondary prevention Tertiar
y prevention
Preventive Environmental Medicine
Pre-Primary Prevention
Risk Management
Risk Communication
Todaka Mori (2002)
45Necessity to establish a new risk communication
method
Risk communication can be the most effective
strategy to reduce the risk for human fetus (even
if multiple chemical exposure risk is not clear)
Information disclosure and discussion Environmenta
l education Follow-up counseling system
46Reduction of the concentration level of dioxin
in human body
Sakurai et al., Internal Medicine 2004 (in press)
47Conclusion
- Human fetuses are exposed to numerous chemicals
including man-made and natural. - Toxicogenomic analysis could be used as a
powerful and effective tool for developing a new
risk assessment to understand and hopefully
prevent long-term effects caused by fetal
exposure to multiple chemicals in high risk
group. - It is necessary to develop a risk reduction
method to avoid multiple chemical exposure and to
reduce the concentration of persistent chemicals
in human body - Worldwide cooperation is necessary to reduce the
risk for the future generations.