1. Role of Genetic Polymorphisms in Responses to Toxic Agents
Definitions
“Forward genetics” and toxicology
“Reverse genetics” and toxicology
Genetic markers
SNPs and their use in toxicology
Ethical, Legal and Social Issues (ELSI)

2. “Toxicology is concerned with the interaction between xenobiotics and biological molecules directly or indirectly coded in the DNA, and can be regarded as a branch of GENETICS.”
Michael F.W. Festing (2001)
Gregor Mendel (1822 – 1884)

3. TERMINOLOGY Gene: A sequence of DNA bases that encodes a protein
Allele: A sequence of DNA bases
Locus: Physical location of an allele on a chromosome
Linkage: Proximity of two alleles on a chromosome
Marker: An allele of known position on a chromosome
Distance: Number of base-pairs between two alleles
centiMorgan: Probabilistic distance of two alleles
Phenotype: An outward, observable character (trait)
Genotype: The internally coded, inheritable information
Penetrance: No. with phenotype / No. with allele
Modified from M.F. Ramoni, Harvard Medical School

4. the Human Genome Project
The 80s Revolution and
the Human Genome Project
Genetic Polymorphisms: naturally occurring DNA markers that identify regions of the genome and vary among individuals
The intuition that polymorphisms could be used as markers sparkled the revolution
On February 12, 2001 the Human Genome
Project announced the completion of a first
draft of the human genome and declared:
“A SNP map promises to revolutionize both
mapping diseases and tracing human history”
SNP are Single Nucleotide Polymorphisms – subtle
variations of the human genome across individuals
Modified from M.F. Ramoni, Harvard Medical School

5. DISTANCES ON A GENETIC MAP
Physical distances between alleles are base-pairs
But the recombination frequency is not constant
A useful measure of distance is based on the probability of recombination: the Morgan
A distance of 1 centiMorgan (cM) between two alleles means that they have 1% chance of being separated by recombination
A genetic distance of 1 cM is roughly equal to a physical distance of 1 million base pairs (1Mb)
Modified from M.F. Ramoni, Harvard Medical School

6. MORE TERMINOLOGY Physical Maps: maps in base-pairs
Human physical map: 3000Mb (Mega-bases)
Genetic Maps: maps in centiMorgan
Human Male Map Length: 2851cM
Human Female Map Length: 4296cM
Correspondence between maps:
Male cM ~ 1.05 Mb; Female cM ~ 0.88Mb
Modified from M.F. Ramoni, Harvard Medical School

7. Simple and Complex Traits
Single Gene (Mendelian) diseases:
Autosomal dominant (Huntington)
Autosomal recessive (Cystic Fibrosis)
X-linked dominant (Rett)
X-linked recessive (Lesch-Nyhan)
Today, over 400 single-gene diseases have been identified
Problem: traits don’t always follow single-gene models
Complex Trait: phenotype/genotype interaction
Multiple cause: multiple genes in several loci determine a phenotype in conjunction with non-genetic factors (accidents of development, social factors, environment, infections, other factors)
Multiple effect: gene causes more than one phenotype
Modified from M.F. Ramoni, Harvard Medical School

8. Classes of polymorphic genetic markers:
Even though we share most DNA, there are variations (polymorphisms)
Polymorphic: two or more forms of the same gene, or genetic marker exist with each form being too common in a population to be merely attributable to a new mutation
Classes of polymorphic genetic markers:
Single Nucleotide Polymorphisms (SNP): single base differences in population
Microsatellites: short tandem repeat (e.g. GATA, 2 – 6 bp long)
Minisatellites: simple sequence repeats (10 – 40 bp long)
Variable Number of Tandem Repeats: the number of repeats may vary
Restriction Fragment Length Polymorphisms: presence/absence of a site
Deletions, Duplications, Insertions: alterations on a chromosome level
Complex haplotypes: combinations of the above

9. Genetic Markers Coding: Non-coding: Single Nucleotide Polymorphisms
Restriction Fragment Length Polymorphisms
Deletions, Duplications, Insertions
Non-coding:
Microsatellites
Minisatellites
Variable Number of Tandem Repeats

10. Genetic Markers Polymorphisms (allelic variations) are essential to:
Study inheritance patterns
Map phenotypes and anchor genes to the genetic map by co-segregation analysis
Determine change in function: resistant/sensitive populations
Genetically determined variability among humans is due to a difference in 0.1% of the genomic sequence!
Polymorphisms can be silent, or be exhibited at levels of:
Morphology
Protein
DNA

11. Chromosomal rearrangements: Deletions, Duplications, Insertions
Deletions: a certain part is lost, for example abc  ac
Insertions: a part is added, for example ac  abc
Duplications: can be tandem, for example abc  abbc, or not, for example
abc  abcabc
Reversals: a part is turned around, head to tail abc  cba
Transpositions: two parts change places, for example abcd  acbd
Insertion Deletion

12. Copy Number Variability (CNVs)
CNV are DNA segments at 1 kb or larger with a variable number of copies in comparison with a reference genome. CNV can have dramatic phenotypic consequences as a result of altering gene dosage, disrupting coding sequences, or perturbing long-range gene regulation.
There are several well-known examples of CNV, including CYP2A6, CYP2D6, GSTM1, GSTT1, SULT1A1, SULT1A3, UGT2B17, and also the nearby UGT2B7, UGT2B10 and UGT2B11 genes. All these genes are deleted at a relatively high frequency in at least one ethnic group. In addition, CYP2A6, CYP2D6, SULT1A1and SULT1A3 can also present duplications and even multiduplications.
Pharmacology & Therapeutics 116, Issue 3, 2007, Pages 496–526

13. Minisatellites Original DNA fingerprinting technique
Relies on stretches of tandemly repeated sequences (usually bp)
Alleles show high variability in numbers of repeats
Genotyping using minisatellites:
Digest genomic DNA
Run out on gel
Southern blot and probe with radiolabelled repeat DNA
Individuals appear with a set of bands unique to them, although each band is shared with one of their parents

14. Microsatellites Number of repeats varies greatly between individuals
Make up to 10-15% of the mammalian genome
Believed to have no function
Have high mutation rates
Used in forensic analysis
Can be amplified by PCR – fragments that are generated have different length due to different number of repeats

15. Microsatellites are highly polymorphic due to potential for
“skipping” during DNA replication

16. Restriction Fragment Length Polymorphisms (RFLPs)
Consider two alleles having slightly different sequences
GAATTC GCATTC
CTTAAG CGTAAG EcoRI will cut the first but not the second

17. Single nucleotide polymorphisms (SNPs)
Variations of a single base between individuals:
A most common form of genetic variation in humans
Thought to be a major cause of genetic diversities among different individuals in drug response, disease susceptibility...
A SNP must occur in at least 1% of the population
Occur every bp
About 50,000 – 100,000 SNPs in coding sequences
SNPs may occur in coding regions:
cSNP: SNP occurring in a coding region
rSNP: SNP occurring in a regulatory region
sSNP: Coding SNP with no change on amino acid
Modified from M.F. Ramoni, Harvard Medical School

18. Single nucleotide polymorphisms (SNPs)
Two bases (one for chromosome) for each locus
Because of the A-T C-G complement, a SNP can have only two variants: (AT) or (CG)
A SNP is a variable with two states:
Major allele: Allele (AT) or (CG) more frequent
Minor allele: Allele (AT) or (CG) less frequent
An individual can be, for each polymorphic locus:
Homozygous on major allele
Heterozygous on major/minor allele
Homozygous on minor allele

19. The role of SNP analysis through all stages of drug development
Target Identification: disease association studies identify SNPs in candidate genes. The proteins encoded by such genes may represent novel drug targets
Target Validation: population analysis determines the level of variation within a candidate gene. The presence of several SNPs will generate a large number of potential variants and such candidates can be eliminated
Lead Identification: screens can be developed to identify lead compounds that interact with each variant of the drug target
Lead Validation: biological assays can be performed that incorporate different lead compounds and all variants of the target protein
Lead Optimization: knowledge of polymorphisms affecting the target can be used to develop drugs that work more efficiently over a broader group of patients or to identify drugs that work more efficiently in specific genotypes
Preclinical Testing: animal models can be developed incorporating all known variants of the target to provide more accurate predictions of drug efficacy in humans
Clinical Trials: trials can be carried out with groups of patients selected on the basis of genotype, to specifically test for adverse drug reactions at particular doses

20. SNP discovery and SNP genotyping
SNP discovery: detection of novel polymorphisms
DNA sequencing
In silico: comparing the sequences of genomic clones or ESTs deposited in public and proprietary databases
Single strand conformational polymorphisms
SNP genotyping: identification of specific alleles in a known polymorphism
Allele discrimination:
allele-specific PCR, allele-specific single-base primer extension (mini-sequencing), allele-specific ligation, allele-specific enzymatic cleavage, etc.
2. Presence of allele(s) of interest in a given DNA sample:
Fluorescence detection, fluorescence resonance energy transfer, fluorescence polarization, mass spectrometry, etc.
See details in: Twyman RM & Primrose SB, Techniques patents for SNP genotyping. Pharmacogenomics 4:67-79 (2003)

21. Toxicology ≈ Genetics
There is substantial polymorphism in genes that determine the response to xenobiotics both in humans and animals
This has important implications for toxicology and pharmacology:
adverse reactions to drugs cause thousands of deaths each year and many of those are associated with susceptible phenotypes
are we protecting the most sensitive in human population when occupational/environmental limits of exposure are established?
how to account for strain differences in susceptibility in animal studies (1000-fold differences have been reported for TCDD LD50 in rats)?
genotyping of individuals from a sample of blood DNA is becoming increasingly easy so it is possible to genotype people for loci that are thought to control susceptibility to certain drugs/xenobiotics
Adapted, in part, from M.F.W. Festing, Tox. Lett. 120: (2001)

22. …loci that are thought to control susceptibility to certain drugs/xenobiotics:
Before we can correctly interpret genotyping results we need to:
gain a much better understanding of the genetics of susceptibility
know the mode of action of xenobiotics
Problem: relatively little research is done on the genetics of susceptibility and toxicologists in general seem to be unaware of the extent of genetic variation in response among the experimental animals that are being used
Problem: modes of action of an overwhelming majority of established toxic substances are still largely unknown (not even worth mentioning scores of compounds that are being newly developed)
Adapted, in part, from M.F.W. Festing, Tox. Lett. 120: (2001)

23. Genotype-Phenotype Interactions in Complex Biological Systems
Age
Environment
Adapted from: Huang, 2002

24. “The classical interaction of exposure with phase I and phase II XME metabolism, and risk of developing cancer. High exposure to a foreign chemical, combined with rapid metabolic activation and slow conjugation, should put an individual at a high risk of developing cancer. Low or negligible exposure, in combination with slow rates of activation and rapid rates of conjugation, should lead to a low risk of developing environmentally caused cancer.”

26. Reactive metabolites (acetoxy-derivatives)
Hetero-cyclic amines
Aromatic amines
N-oxidation
O-acetylation:
NAT1 and NAT2
Reactive metabolites (acetoxy-derivatives)
cancer
From: Hulla et al. Toxc. Sci. (1999)
Rapid acetylator
Intermediate acetylator
Slow acetylator

27. J Cancer Res Clin Oncol. 2011 Nov;137(11):1661-7
Lung Cancer. 2011 Aug;73(2):153-7. 

28. Several GST gene families have been identified
Survival in women with epithelial ovarian cancer
From: Introduction to Biochemical Toxicology 3rd Edition (2001) p. 128
From: Strange et al. Toxc. Lett. (2000)
Several GST gene families have been identified
Null-phenotypes are detoxification-deficient and more likely to suffer formation of carcinogen-DNA adducts and/or mutations
In general, GSTM1- and GSTT1-null are considered high-risk

29. Genetics in Toxicology
Phenotype (e.g., toxic symptoms, cancer)
Genes that control susceptibility/resistance
Studying mechanisms
of action
“Forward Genetics”
Genotype (gene knockout, polymorphism, etc.)
Phenotype
Studying mechanisms
of action
“Reverse Genetics”
Adapted, in part, from M.F.W. Festing, Tox. Lett. 120: (2001)

30. “Forward Genetics” and Toxicology
Different animal strains nearly always respond differently to the same agent/dose unless the toxic insult is so dramatic that all the animals die very quickly
Examples of strain differences (rats) in response to xenobiotics:
3,2’-dimethyl-4-aminobiphenyl  prostate tumors
48% F344, 41% ACI, 13% LEW, 7% CD, 0% Wistar
N-methyl-N-nitro-N-nitrosoguanidine(MNNG)  stomach adenocarcinomas
67% WKY, 60% S-D, 53% LEW, 23% Wistar, 6% F344
There is no such thing as an “animal strain that is particularly susceptible/resistant to carcinogenesis” !
Adapted, in part, from M.F.W. Festing, Tox. Lett. 120: (2001)

31. Current Approach: Animal studies  Human population
CD-1
Pharmaceutical Industry
Single genome-based
risk prediction
B6C3F1
National Toxicology Program
Genetically Diverse Human Population

32. “Forward Genetics” and Toxicology
Designing an IDEAL “forward genetics” animal study for investigating genetic variability in response to a toxic agent:
Survey the known facts about susceptibility in different strains of rodents
Small numbers of animals (4-6 per strain) of several strains should be used to characterize the response to the toxic agent “X”
At least 5 strains should be studied
Dose levels should be selected to elicit a suitable response
Endpoints should be quantitative (e.g., number of tumors)
Adapted, in part, from M.F.W. Festing, Tox. Lett. 120: (2001)

33. Parental strains and derivation of five major types of mouse genetic resources
Each of the sequenced strains is shown in a different color depending on the origin. The four wild-derived strains, denoted by asterisks, are CAST/EiJ (M. m. cataneus) in red, PWD/PhJ (M. m. muculus) in blue, MOLF/EiJ (M. m. molossinus) in purple, and WSB/EiJ (M. m. domesticus) in green. The remaining 12 classical laboratory strains are shown in green reflecting the predominant contribution of the M. m. domesticus subspecies to these strains. The shade of green denotes the different origin of the classical strains, with the darker shades denoting strains of Swiss origin (FVB/NJ and NOD/LtJ), the yellow-green denoting a strain of Asian origin (KK/HlJ), and intermediate shade denoting Castle or C57-related strains (129S1/SvImJ, A/J, AKR/J, BALB/cBy, C3H/HeJ, DBA/2J, BTBR T+tf/J, and NZW/LacJ).
The figure also shows schematically the derivation process for five types of resources, recombinant inbred lines (BXD); chromosome substitution strains (B.P), Collaborative Cross (CC), heterogeneous stocks (Northport HS), and laboratory strain diversity panel (LSDP)
Mamm Genome July; 18(6): 473–481

34. Recombinant inbred strains (RIs)
+ … +
BXD1
BXD2
C57BL/6J (B)
DBA/2J (D) F1
20 generations brother-sister matings
BXD80 F2
BXD RI
Strain set
fully
inbred
isogenic
hetero-
geneous
Recombined chromosomes are needed for mapping
female
male
chromosome pair
Inbred
Isogenic
siblings
BXD
The next few slides provide a short introduction to mapping the loci that are responsible for variation in a trait such as App expression level. These modulatory regions of the genome are sometimes called quantitative trait loci or QTLs. You may want to do some independent reading on this topic if this is your first exposure to QTL analysis.
The genetic reference population (GRP) of BXD recombinant inbred strains were originally generated about 25 years ago by Benjamin Taylor at The Jackson Laboratory. He crossed female C57BL/6J mice with male DBA/2J mice to generate the F1 and F2 progeny. At the bottom of this slide we have schematized one chromosome pair from three of the BXD RI strains. The dashed vertical lines that lead to the final BXD RI lines involve 21 full sib matings (about 7 years of breeding). Some lines die out during inbreeding. For example, there is no longer any BXD3 strain.
Notes:
1. Over the last decade, our group (Lu Lu and Rob Williams) and Jeremy Peirce and Lee Silver at Princeton have enlarged Ben Taylor’s set. There are now just over 80 BXD strains. They have all been genotyped using about 13,700 markers (SNPs and microsatellites). These markers are used to define the “blue” and “red” regions of the chromosomes as shown in the figure above.
2. Chromosomes of RI GRPs usually have about 4 times as many recombinations as those of F2 animals. However, unlike an F2, both chromosomes of an RI are identical. Therefore, 50 RI strains contain as many recombinations as 100 F2 animals.
3. BXD43 through BXD100 were generated using a special method that resulted in a further doubling of the average recombination density per chromosome. The entire set of 80 BXDs therefore contains as many recombinations as about 260 F2 animals.
Image Credit: genenetwork.org

35. A set of RI strains can be tested for the susceptibility to agent “X”
Once a susceptible/resistant strains have been identified, loci can be mapped
In mice, Recombinant Inbread strains (susceptible x resistant) can be generated
A set of RI strains can be tested for the susceptibility to agent “X”
Once the phenotype have been established, mice can be genotyped to determine which loci segregated with susceptibility/resistance
From Zhou et al. (2005)
Problems: large number of animals ( , or more)
resolution of the genetic mapping is only about ± 20 cM (mouse genome is ~50K genes and 1900 cM  1cM ≈ 0.5 Mb) so the identified locus can contain ~500 genes
Adapted, in part, from M.F.W. Festing, Tox. Lett. 120: (2001)

36. “Collaborative Cross” The Resource for Forward Genetics Research
Images from Threadgill DW

37. Single Strain: Constant Genotype Many Strains: Varied Genotype
Population-based studies: a mouse model fills critical gaps
Single Strain: Constant Genotype
Control
10 mg
25 mg
50 mg
100 mg
Vary the environment (e.g., treatment)
Many Strains: Varied Genotype
Strain 1
Strain 2
Strain 3
Strain 4
Strain 5
Strain 6
Strain 7
Fix the environment (same treatment), vary the genotype
Total mouse SNPs = ~40M
(M.m.musculus, M.m.domesticus, M.m.castaneous)
Total human SNPs = ~20M

38. Profiling Liver Toxicity to APAP in a Genetically Diverse Population
Dose response to liver injury: ALT (24 h)
Multi-strain profiling of APAP-induced liver injury:
% liver necrosis (24h), reduced GSH (4h), ALT (24h), ALT (4h)
Dose response to liver injury (4 h) vs survival (24 h)

39. “Reverse Genetics” and Toxicology
A knockout or over-expressor animal strain, or animals with a known polymorphism(s) in important genetic regions
Dose with a chemical(s)
Evaluate the phenotype
Looks MUCH easier than “Forward Genetics” experiment! Let’s do it!
Problems: if mutant to non-mutant comparison is being made, the genetic backgrounds MUST be identical !
if the strains have been crossed, care is needed to ensure that the observed differences are not due to a gene closely linked to the gene of interest
genes do not act alone! Several alleles may be important, their effects can be additive or epistatic
Adapted, in part, from M.F.W. Festing, Tox. Lett. 120: (2001)

40. PPARa (+/+) + WY-14,643 (11 months) PPARa (-/-)
Peters et al., Carcinogenesis, 1997

41. Peroxisome Proliferators: Species Differences
Mouse and rat: highly responsive
Marmoset: does not respond
Guinea Pig: no peroxisome proliferation, but have hypolipidaemia
Humans: believed to be unresponsive, but have hypolipidaemia
PPARa exists in mouse, rat, guinea pig and human
In humans: Lower hepatic levels of PPARa
Lower ligand binding activity
Different structure (polymorphisms)
Different PP Response Elements in DNA
Presence of competing proteins for PPRE
Expression of dominant-negative form of PPARa
across mouse inbred strains
Palmer et al., Molecular Pharmacology, 1998

42. Activation of PPARα in mouse and human hepatocytes
Untreated (6, 24 hr in culture)
Limited overlap in response to Wy-14,643 at individual gene level but major overlap at pathway level
Treated (6, 24 hr in culture)
Downregulated genes
Upregulated genes
Wy-14,643 treatment causes major changes in gene expression in human and mouse hepatocytes
Gene Ontology
Gene Set Analysis

44. Well-studied genetic variants in human disease
From Taylor et al. Trends Mol Med 7: (2001)

45. Most drug-metabolizing enzymes exhibit clinically relevant genetic polymorphisms. Essentially all of the major human enzymes responsible for modification of functional groups [phase I reactions (left)] or conjugation with endogenous substituents [phase II reactions (right)] exhibit common polymorphisms at the genomic level; those enzyme polymorphisms that have already been associated with changes in drug effects are separated from the corresponding pie charts. The percentage of phase I and phase II metabolism of drugs that each enzyme contributes is estimated by the relative size of each section of the corresponding chart. ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; CYP, cytochrome P450; DPD, dihydropyrimidine dehydrogenase; NQO1, NADPH:quinone oxidoreductase or DT diaphorase; COMT, catechol O-methyltransferase; GST, glutathione S-transferase; HMT, histamine methyltransferase; NAT, N-acetyltransferase; STs, sulfotransferases; TPMT, thiopurine methyltransferase; UGTs, uridine 5'-triphosphate glucuronosyltransferases. From Evans WE and Relling MV Science 286:487 (1999).

46. Cytochrome P450 genotyping
From: Flockhart DA and Webb DJ. Lancet (1998)

48. Human Cytochrome P450 2C9 with bound Warfarin
FDA OKs Genetic Test Linked to Warfarin
Sep WASHINGTON (AP) - A genetic test that can reveal what patients are especially sensitive to the blood-thinner warfarin won federal approval Monday. Such screenings could prevent thousands of complications each year, health officials estimate.
The approval of the test comes a month after warfarin, sold under the brand name Coumadin and in generic forms, became the first widely used drug to include genetic testing information on its label. The information can help doctors determine how best to prescribe the drug.
An estimated one-third of patients process the drug differently than do most others, exposing them to a higher risk of bleeding. Research suggests that most of that sensitivity is due to variations in two genes. The new test, made by Nanosphere Inc. of Northbrook, Ill., can detect some of those variants.
One of the genes produces an enzyme that helps the body metabolize warfarin and other medicines; the second produces the blood-clotting protein that warfarin blocks.
Human Cytochrome P450 2C9 with bound Warfarin
Nature 424, (2003)
Image Source:

49. Population-based GWAS and Toxicology:
Drug-induced Adverse Effect studies
Daly et al. HLA-B*5701 genotype is a major determinant of drug-induced liver injury due to flucloxacillin. Nat Genet Jul;41(7):816-9.
866,399 markers
51 cases of flucloxacillin DILI
282 matched controls

50. The FDA Abacavir Warning (July 24, 2008) 
Abacavir (marketed as Ziagen) and Abacavir-containing Medications
FDA reviewed data from two studies that support a recommendation for pre-therapy screening for the presence of the HLA-B*5701 allele and the selection of alternative therapy in positive subjects. Genetic tests for HLA-B*5701 are available and all patients should be screened for the HLA-B*5701 allele before starting or restarting treatment with abacavir or abacavir-containing medications. Development of clinically suspected abacavir HSR requires immediate and permanent discontinuation of abacavir therapy in all patients, including patients negative for HLA-B*5701.

51. Genomenewsnetworks.org
The genomes of more than 180 organisms have been sequenced since The Quick Guide includes descriptions of these organisms and has links to sequencing centers and scientific abstracts.

52. Ultra High Throughput Sequencing – Towards the “$1,000 Genome”
Illumina® “SOLEXA” Genome Analyzer
Roche® 454 Genome Sequencer
DNA Sequencing
Transcriptome analysis
Gene regulation and control
Roche.com & Nature Biotechnology
Seqanswer.com
Illimina.com
DNA Sequencing
Transcriptome analysis
Gene regulation and control

53. Ultra High Throughput Sequencing – Enabling GWAS Studies
amateurbrainsurgery.com
Genome-wide plots of available GWAS results for all associations P = (BMC Medical Genetics 2009)
Epidemiological studies: hundreds of individuals could now be genotyped for ~106 SNPs
ornl.gov
In vivo animal studies: dozens of inbred mouse strains have been genotyped for ~107 SNPs
compgen.unc.edu
In vitro studies:
tens to hundreds of cell lines have been genotyped for ~107 SNPs

54. From: Strange et al. Toxc. Lett. (2000)

55. Toxicogenetics: what’s next?
Goal: When we find all polymorphisms in genes important for metabolism/detoxification of xenobiotics, we can link them to particular drug or chemical toxicity and identify susceptible populations
Problem: Simple research questions generate erroneous results (e.g. CYP2D6 polymorphisms and lung cancer, CYP2E1 polymorphisms and alcoholic liver disease)
Problem: Biological complexity of mechanisms, ethnic variation, clinical heterogeneity, etc…  both positive and negative results are true?
Linking complex trait diseases to genetic polymorphisms requires (Todd, 1999):
large sample sizes and small p-values
Initial study + several replications
Genetic associations should make biological sense
Physiologically meaningful data should support a functional role of the polymorphism in question

56. Ethical, Legal and Social Issues in toxicogenetics are as complex as the studies of polymorphisms themselves