Transcript Toxicogenomics

Toxicogenomics
Dr. M. Zdanowicz
WHY?
• Why didn’t everyone living the same
distance from Chernobyl develop
cancer?
• Why can some people smoke for their
entire lives and never develop lung
cancer?
• Why do certain individuals experience
toxicity from a certain level of a drug or
toxin when others don’t?
Definitions
• Toxicology:
• The study of the nature, effects and
detection of poisons and the treatment of
poisoning.
• Genomics:
• The study of genes and their function.
Genomics aims to understand the structure
of the genome, including the mapping
genes and sequencing the DNA. Genomics
examines the molecular mechanisms and
the interplay of genetic and environmental
factors in disease.
• Proteomics:
• The measurement, identification &
comparison of all proteins (and their
properties) in specific tissues and body
fluids during health, toxicity and disease
• Pharmacogenomics:
• The study of the interaction of an
individual's genetic makeup and response
to a drug.
• Branch of pharmaceutics which aims to
tailor medicines to an individual’s genetic
makeup
Toxicogenomics
• The collection, interpretation, and
storage of information about gene and
protein activity in order to identify
toxic substances in the environment,
and to help treat people at the
greatest risk of diseases caused by
environmental pollutants or toxicants.
The dose makes the
poison……
Paraclesus
∑
ENVIRONMENTAL
STRESSORS
GENETIC
INFLUENCES
X TIME =
DISEASE
Key Questions
1. How does exposure to chemicals
(toxins, drugs) effect the
expression of human genes??
2. Do we all respond the same when
exposed to chemicals, drugs and
toxins??
3. Are some of us more resistant to
their detrimental effects??
• There is a growing number of
potentially toxic compounds in the
human environment….
National Institute of
Environmental Health
• Formed the National Center for
Toxicogenomics
• http://www.niehs.nih.gov/nct/home.htm
• Charged with gathering large amounts
of gene expression data related to
toxicology.
Comparison
Classic Toxicology
• How much
exposure and for
how long a time
does the
“population” need
to be exposed to
show toxicity?
Toxicogenomics
• Identifying
agents that are
associated with
toxicity in either
the “average” or
“more sensitive”
individual.
Classic Measures of
Toxicity
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Histopathology
Clinical Chemistry
Metabolism
Physiology
Enzymology
Electron
Microscopy
New Measures
• Gene Expression:
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DNA Microarrays
RT PCR
Northern Blot
Sequencing
• Protein
Expression:
• Western Blot
• Electrophoresis
• Chips
Two Key Advances
• Human Genome Project:
• Sequences the human genome.
• Identifies genetic variations between
individuals (polymorphisms).
• DNA Microarray’s:
• Revolutionary new tool used to identify
mutations in genes.
• The “chip” consists of a small glass plate
encased in plastic.
• Each chip contains thousands of short,
synthetic, single-stranded DNA sequences
which together add up to the normal
gene(s) in question
Hand held DNA microarray
• The populations of expressed genes
(mRNA) from different cells or tissues
are labeled with fluorescent tags and
hybridized to the array of DNA
fragments.
• The array is then scanned with a laser,
at the appropriate wavelength, and
fluorescence measurements are made
at each location on the array to give a
measure of how many gene transcripts
are seen in that sample.
DNA Microarray
Each dot represents
The activity of a single gene.
The brighter the dot, the
Greater the activity!
• By comparing the levels of all these
genes between many samples, we may
understand what molecular changes are
occurring at the transcription level
during biological changes.
• The advantage over traditional methods
such as Northern blot is that you can
analyze 40,000 genes per sample
versus a dozen or so.
• 4 million soon!
Examples…
• A 24 hour exposure to ethanol in rat
liver cells induces a change in
expression of 86 hepatocyte genes
• A brief exposure to a chemical that
causes toxicity through DNA alkylation
alters the expression of 2000 genes in
a single cell!
• DNA microarrays may also be used test
a patients blood for the presence of
mutations that might be associated with
certain diseases.
• Ex. BRCA1 & BRCA2 genes and breast
cancer
Advantages of Measuring Gene
Expression in Toxicology
• Greater sensitivity:
• Can detect changes in gene expression
before gross changes such as tissue
damage, tumor formation, etc…
• Earlier Indication of a Toxic
Response:
• Before tissue is altered or tumors form.
Advantages of Measuring Gene
Expression in Toxicology
• Greater Specificity:
• Many different agents can cause a liver
tumor but they may do so through a
number of different mechanisms.
• Many toxic agents have the same endpoint
but get there by various pathways we may
now identify.
• Also may provide a way to determine the
mechanism of toxicity for a particular
agent.
Advantages of Measuring Gene
Expression in Toxicology
• Microarrays allow for study of all
toxicological endpoints in a single
assay.
• Traditionally, you need to study an agents
carcinogenicity, mutagenicity,
reproductive toxicity, immunotoxicity,
neurotoxicity, and endocrine toxicity in
separate assays.
Advantages of Measuring Gene
Expression in Toxicology
• The fact that groups of chemical with a
common toxicological mechanism produce a
characteristic pattern (“fingerprint”) of gene
expression means it may be possible to
discern the toxic potential of an agent quickly
and cheaply.
• A way to cut back on animal toxicity
testing.
Applications of Toxicogenomics
Liver Metabolizing Enzymes
• Most common and well-studied genetic
variations.
• Metabolize numerous drugs and
potential environmental toxins.
• Can persons with genetically different
enzyme activity be at increased risk for
toxicity and disease?
Nakajima, 2000
Examples
• CYP1A1:
• oxidizes polycyclic aromatic
hydrocarbons…..increased lung cancer if
reduced.
• Glutathione S-Transferases:
• Detoxify carcinogenic metabolites such as
hydrocarbon diol-epoxides and
lipoperoxidation products……increased skin
& lung cancers if reduced.
• Remember, through metabolism these
liver enzymes detoxify potentially
dangerous agents but they can also
form toxic metabolites from relatively
inert parent compounds!
• High rates of activity for certain
enzymes may likewise be a risk!!
DNA Repair Genes
• DNA Damage and DNA adducts (DNA
with covalent chemicals attached).
Large difference in DNA damage and
adduct formation have been
documented in different individuals
receiving similar exposures.
BP = Benzo[a] pyrene
A polyclycic aromatic
hydrocarbon
• DNA repair genes are polymorphic.
• They function at different levels in
different individuals.
Individuals may have significant
differences in their ability to repair DNA
damaged by exposure to toxins, uv,
radiation, chemo drugs, etc….
• What is a known outcome of DNA
damage?
• Cancer!
• How can Joe Schmoe smoke three
packs of butts a day for his entire
life and never get lung cancer???
• Cigarettes are really safe!
• High activity of genes that detoxify
carcinogens?
• Low Activity of genes that form
carcinogenic metabolites?
• High activity of DNA repair genes?
Key Point
• How might Toxicogenomics and
DNA microarrays be of value here
with respect to variability in liver
enzymes and DNA repair??
• It may be used to determine which
individuals would be at risk for longterm adverse effects following exposure
to certain toxins…..
Mechanism of Drug
Teratogenicity
• Valproic Acid:
• Avoided in pregnancy due to birth defects.
• Mechanism of teratogenicity is unknown.
• Kultima (2000) used toxicogenomics and DNA
microarrays to show that the expression of a
number of mouse fetal genes is increased
following valproic acid exposure.
• One is for metallothionein….fetal Zn+ deficiency!!
(A key cofactor)
“ToxChip”
• Chip has been developed that contains
most of the human genes known to be
involved in a toxicological response.
ToxChip Genes
Adapted from Nuwaysir, 1999
• Microarray profiling of individuals who
might be exposed to toxic agents may
act as a sensitive biomarker to define
more precisely the nature and level of
toxic exposure the individual is
experiencing…….before it is obvious
through tissue injury or disease…..
Summary
 Toxicogenomics is an emerging
area of toxicology that examines
how the genetic make-up of an
individual makes them or more less
susceptible to toxicity and
exposure related diseases.
Summary
 In contrast with classic toxicology,
toxicogenomics uses changes in
gene and protein expression to
detect subtle molecular-level
changes in organism that may
precede or predict toxic and
deleterious effects of exposure.
Summary
The development of so called “Tox-
Chips” may allow for the rapid and
sensitive detection of wide-ranging
molecular level changes in an
organism exposed to various
substances.
Summary
Toxicogenomics can provide a
powerful means of detecting
molecular level changes in an
organism before obvious
manifestations of toxicity or
disease are present.