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: Genomics: 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 ∑ DISEASE X TIME = GENETIC INFLUENCES

Key Questions How does exposure to chemicals (toxins, drugs) effect the expression of human genes?? Do we all respond the same when exposed to chemicals, drugs and toxins?? 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 Histopathology Clinical Chemistry Metabolism Physiology Enzymology Electron Microscopy New Measures Gene Expression: 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: Glutathione S-Transferases: 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 long-term 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.