Fundamentals of Genetic Toxicology in the

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Transcript Fundamentals of Genetic Toxicology in the

Fundamentals of Genetic
Toxicology in the
Pharmaceutical Industry
Prepared by:
David Amacher, Ph.D, DABT
www.tigertox.com
What is Genetic Toxicology?
 Genetic Toxicology refers to the assessment of the
deleterious effects of chemicals or physical agents on
the hereditary material and related genetic processes of
living cells.
 By altering the integrity and function of DNA1 at the gene
or chromosomal level, the damage can lead to heritable
mutations ultimately resulting in genetic disorders,
congenital defects, or cancer.
 Targets of DNA damage include somatic cells
(detrimental to the exposed individual), germinal cells
(potentially heritable effects), and mitochondria
(detrimental to the exposed individual & progeny via
maternal inheritance).
1
DNA = deoxyribonucleic acid
2
Genotoxic Classification Scheme
Mutation
Microlesions
Base-pair substitution
mutations
[Qualitative change
in 1 or a few
nucleotide pairs]
Frameshift
mutations
[Quantitative
change in 1 or a
few nucleotide
pairs]
Macrolesions
[cytologically
visible]
Numerical
changes in
chromosomes
Stuructural changes
in chromosomes
Deletions
Rearrangements
Breaks
Diagram from Bruswick, D.J. Alterations of germ cells leading to mutagenesis and their detection. Environ.
Health Perspect. 24(1978):105-112.
3
Mechanisms for Genetic Damage
Genotoxic chemicals produce genetic damage
at subtoxic levels.
The types of DNA1 damage produced include:
 single- & double-strand breaks,
 crosslinks between DNA bases and proteins, and
 chemical additions to the DNA bases (adducts).
DNA replication itself can introduce errors via
incorrect base substitution, a process that can
be exacerbated by some genotoxic agents.
1
DNA = deoxyribonucleic acid
4
Types of Genetic Damage
 Base substitution: The replacement of the correct
nucleotide by an incorrect one.
 A transition involves a change of a purine for a purine or a
pyrimidine for a pyrimidine
 A transversion involves a change of a purine for a pyrimidine or
vice versa.
 Frame shift mutation: The addition or deletion of one
or a few base pairs (not in multiples of 3 [codon]) in
protein-coding regions.
 Structural chromosome aberrations: For nonradiomimetic chemicals, these can arise from errors of
DNA replication on a damaged template.
 Radiomimetic chemicals can directly induce strand breaks.
5
Types of Genetic Damage
 Numerical chromosome changes:
 Numerical aberrations are those involving non-diploid variations
in chromosome number in the nucleus.
 Monosomies, trisomies, & other ploidy changes arise from errors
in chromosome segregation.
 Aneuploidy (numerical deviation of the modal chromosome
number) can result from the effects of chemicals on tubulin
polymerization or spindle microtubule stability.
 Sister chromatid exchanges (SCE):
 SCE can be produced during S phase as a consequence of
errors in the replication process and are apparently reciprocal
exchanges
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DNA Repair
 DNA enzymatic repair mechanisms developed to maintain
fidelity and integrity of genetic information
 Enzymes are able to remove and replace damaged segments
of DNA
 Particularly useful during low-level exposure where excision
repair enzymes are not fully saturated by excessive DNA
damage
 Stimulation of repair activity following treatment at sublethal
concentrations can indicate presence of DNA-directed toxicity
 Cells in S phase* (DNA synthesis) are most susceptible to
genetic injury because they have less time to repair the
damage prior to mitosis.
* See supplemental slides at end of slide deck.
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DNA Repair Mechanisms
 Base excision repair: To repair DNA1 base
damages:
 A glycosylase removes the damaged base producing an apurinic or
apyrimidinic site,
 A DNA polymerase fills the gap with the appropriate base
 A ligase seals the repaired patch.
 Nucleotide excision repair: To remove bulky
lesions from DNA, a process involving as many as
30 proteins to remove damaged oligonucleotides
from DNA in steps involving damage recognition,
incision, excision, repair synthesis, and ligation.
1
DNA = deoxyribonucleic acid
8
DNA Repair Mechanisms
 Double-strand break repair: These are homologous
recombination or nonhomologous end-joining processes
which repair broken chromosomes.
 Homologous recombination steps include:
 Exonuclease or helicase activity produces a 3’-ended single-stranded tail.
 “Holliday junction DNA complex” is formed via strand invasion. This junction
is cleaved to produce two DNA molecules, neither containing a strand break.
 A second mechanism, the nonhomologous endjoining processes, involves a DNA-dependent protein
kinase.
 Unrepaired breaks result in checked cell cycle
progression or the induction of apoptosis.
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DNA Repair Mechanisms
 Mismatch repair: Mismatched bases can be formed
during DNA replication, genetic recombination, or
chemically-induced DNA damage. A specific protein
recognizes & binds to the mismatch and additional
proteins stabilize it. This is followed by excision,
resynthesis, and ligation.
 O6-methylguanine-DNA methyltransferase repair:
By transferring methyl groups from O6methylguanine in affected DNA, this repair
mechanism protects against simple aklylating
agents.
10
Testing Requirements
 The fundamental purpose of genetic toxicology testing is to safeguard the human
gene pool from chemical damage. There are two basic types of screening assays:
(1) tests for gene mutations, (2) tests for chromosomal aberrations.
 A gene mutation assay is generally considered sufficient to support all single-dose
clinical trials1. In support of multiple-dose clinical trials, 2 batteries of tests, Option 1
and Option 2, are described2. Option 2, if selected, should be completed prior to first
human use in multiple-dose studies1. The in vitro components of Option 1, if
selected, should be completed prior to first multiple-dose human studies1. The in vivo
component of Option 1 should be completed prior to Phase 21.
 If an equivocal or positive finding occurs, additional tests should be performed as
described in S2(R1)2,3 & by the FDA4.
 The standard battery of tests [S2(R1)]2 should be completed prior to the initiation of
Phase II studies. Testing must be GLP-compliant (21 CFR part 55).
1
ICH Harmonized Tripartite Guideline M3(R2) Nonclinical Safety Studies for the Conduct of Human Clinical Trials and
Marketing Authorization for Pharmaceuticals. (Final, January 2010).
(http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm073246.pdf)
2
3
ICH Harmonized Tripartite Guideline. Guideline for Industry: S2(R1) Genotoxicity Testing and Data Interpretation for
Pharmaceuticals Intended for Human Use (Step 3, March 2008). (http://www.fda.gov/cder/guidance/index.htm)
See also supplemental slides.
4 FDA
Guidance for Industry and Review Staff Recommended Approaches to Integration of Genetic Toxicology Study
Results (January, 2006). (http://www.fda.gov/cder/guidance/6848fnl.pdf).
11
Special Requirements for Testing
during Preclinical Drug Development
 If positive or equivocal findings are found in vitro, then in
vivo genetic tox tests are required prior to Phase I.
 For women of child bearing potential, pregnant women,
children, and for compounds bearing structural alerts,
both in vitro and in vivo genetic tox assays must be
completed prior to any clinical trials.
 FDA & EMA require phototoxicity testing by systemic or
cutaneous applications of drugs that absorb light &
penetrate into skin in relevant concentrations.
FDA = US Food and Drug Administration; EMA = European Medicines Agency
12
12
Examples of Genetic Toxicology
Assays
Gene Mutations
Chromosome Damage
Primary DNA Damage
Salmonella Mammalian
Microsome (Ames) Assay *‡
In vitro metaphase chromosomal
aberrations * †
Transformation Assays
(BALB/c or SHE)
Mouse Lymphoma TK Assay
(MLA) *†
In vitro micronucleus test
Comet Assay for DNA strand
breakage (in vitro & in vivo)
CHO/HGPRT Assay
In vivo metaphase chromosomal
aberrations
Alkaline Elution Assay for
DNA strand breakage
Transgenic Assays (e.g. Big
Blue® mouse & rat, Muta™
Mouse, lacZ plasmid mouse)
In vivo micronucleus test * ‡
Unscheduled DNA synthesis
(UDS) (in vitro & in vivo).
DNA covalent binding assay
CHO = Chinese Hamster Ovary, HGPRT = hypoxanthine-guanine phosphoribosyl transferase gene in CHO cells, SHE = Syrian Hamster
Embryo, BALB = BALB/3T3 cell transformation assay, Comet = also called single cell gel electrophoresis (SCGE)., DNA = deoxyribonucleic
acid.
* ICH S2B assay
‡ Good correlation with carcinogenicity (see: Regul. Toxicol. Pharmacol. 44:83-96, 2006)
† Poor correlation with carcinogenicity (see: Regul. Toxicol. Pharmacol. 44:83-96, 2006)
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The standard test battery
Assessment in a bacterial reverse mutation
assay. These assays detect the majority of
genotoxic rodent and human carcinogens.
 Bacterial mutagens are detected by selecting tester
strains that detect base substitution & frameshift point
mutations. Bacterial mutation assays for base pair
substation and frameshift point mutations include the
following base set of strains:
• TA98; TA100; TA1535; TA1537 or TA97 or TA97a; TA102 or
E. coli WP2 uvrA or E. coli WP2 uvrA (pKM101).
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The standard test battery
Evaluation in mammalian cells in vitro and/or in
vivo
 In vitro mammalian cell systems include: Mouse
lymphoma L5178Y cells, Chinese hamster cells,
primary rat hepatocytes, & human peripheral
lymphocytes.
 In vivo tests provide additional relevant ADME
factors. Commonly used in vivo systems include:
Rodent bone marrow or lymphocytes following in vivo
exposure, rat liver or other target organs following in
vivo exposure, & transgenic mice.
ADME = absorption, distribution, metabolism, and excretion
ADME = absorption, distribution, metabolism, and excretion.
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Assays for Detecting DNA Damage
& Repair
 Chromososmal aberrations are assayed in vitro by metaphase
analysis in cultured cells and in vivo by metaphase analysis of
rodent bone marrow or lymphocytes. Chromosomal structural or
numerical changes are detected in vitro via cytokinesis-blocked
micronucleus assay in human lymphocytes or mammalian cell lines
and in vivo by the micronucleus assay in rodent bone marrow or
blood.
 In mammalian cells, DNA1 repair is commonly assayed by
measuring unscheduled DNA synthesis (UDS). Also, comparisons
of a test chemical in DNA repair-deficient vs. DNA repair-proficient
bacteria strains (e.g., E. coli polA- & polA+ or Bacillis subtilis rec- &
rec+) is another indirect use of DNA repair for the detection of DNA
damage.
1
DNA = deoxyribonucleic acid.
16
Assays for Detecting DNA Damage
& Repair
 Standardized assays are used to identify germ-cell mutagens,
somatic-cell mutagens, and potential carcinogens through the
detection of gene mutations, chromosomal aberrations, and/or
aneuploidy following chemical exposure.
 Direct measures of DNA damage involve the detection of
chemical adducts or DNA strand breaks.
 Indirect assays measure DNA repair processes.
 Assays for bulky DNA adducts include:
 the 32P-postlabeling assay.
 DNA strand-breakage assays include alkaline elution assay and
electrophoretic methods.
 Single-cell gel electrophoresis (the Comet assay) is now widely
used to measure DNA damage.
17
Options for the standard battery
Option 1
A test for gene mutation in bacteria.
A cytogenetic test for chromosomal
damage (choice of three)
An in vivo test for chromosome damage
using rodent hematopoietic cells (either
micronuclei or chromosomal aberrations in
metaphase cells).
18
Options for the standard battery
Option 2
 A test for gene mutation in bacteria.
 An in vivo assessment with two tissues (e.g., micronuclei
using rodent hematopoietic cells + a second in vivo
assay (e.g., liver UDS1 assay, transgenic mouse assay,
Comet assay, etc.)
For compounds giving negative results, the
completion of either test battery (Options 1 or 2),
performed and evaluated as recommended, will
usually suffice with no further testing required.
1
UDS = unscheduled DNA synthesis.
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Outcomes & Follow-up
 If results from any of the 3 assays in the ICH1
genotoxicty standard battery are positive, complete a 4th
test from the ICH battery to confirm.
 Equivocal studies should be repeated to determine
reproducibility.
 If a positive response is seen in 1 or more assays, the
sponsor should choose from the following options: (a)
weight of evidence decision, (b) mechanism of action
decision, or (c) conduct additional supporting studies.
1
ICH = International Conference for Harmonization
20
Confounding Factors
Examples of some experimental factors that
may produce study artifacts:
 Accelerated erythropoiesis (micronucleus assay)
 Non-physiological conditions (mammalian cells)
 Positive only @ highly cytotoxic concentrations (in vitro assays)
 Presence of mutagenic impurities or precursors
 Pharmacologically related indirect threshold mechanisms
 Metabolic differences between “induced” rat S9
(overrepresentation of CYP1A & 2B enzymes) vs. human cells
 Non-DNA thresholds
S9 = metabolic activation, CYP = cytochrome P-450 enzymes, DNA = deoxyribonucleic acid.
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Rationale for Early Genetic Toxicology
Evaluation (Screening Assays)
 Frequently, presumptive mutagens are dropped from
development. If continued, potential clastogens require
disclosure & consent in clinical trials, unfavorable
labeling, & can result in diminished market potential.
 Mutagenicity pre-screening in drug discovery/lead
optimization can identify potential mutagens & remove
them from development at an early stage.
 Early in vitro clastogenicity screening can facilitate the
efficient planning of follow-up in vivo testing. The latter
can often be integrated into other toxicity studies to
expedite preclinical development and reduce cost.
22
22
Discovery/Lead Optimization
Screening Assays
Non-GLP Assays can be used at early stages in
drug discovery to select chemical candidates for
further development.
Early screening assay advantages include:
 Low cost
 Rapid turn-around time
 Require minimal amounts of test articles
 Can be highly predictive
23
23
Rapid Pre-screening Methods
 Examples of Modified or High-throughput
methods for early screening include:



Computer-assisted (in silico) SAR methods for predictive
toxicity screening1
Modified assays such as the in vitro assessment of
micronucleus induction in CHO cells2 , the Ames II assay (TA98
& TAMix), the in vitro Comet assay3, or well-based (e.g. ,96- or
384-well format) modifications of the yeast DEL assay4
Proprietary assays such as Vitotox™ (mutagenicity),
RadarScreen® (clastogenicity), & GreenScreen® GC5
1 Mohan et al. Mini Rev. Med. Chem. 7(5): 499-507, 2007.
2 Jacobson-Kram & Contrera Toxicol. Sci. 96(1): 16-20, 2007.
3 Witte et al. Toxicol. Sci. 97: 21-26, 2007
4 Schoonen et al. EXS 99: 401-452, 2009.
5 Hontzeas et al. Mutat. Res. 634(1-2): 228-234, 2007.
SAR = structure-activity relationship; CHO = Chinese Hamster Ovary.
24
Suggested sources for further
reading
 Genetic Toxicology and Cancer Risk Assessment by Wai Nang
Choy. 1st edition, 2001, Informa Healthcare.
 Genetic Toxicology by R. Julian Preston & George R. Hoffman in:
Toxicology. The Basic Science of Poisons. 7th edition, Curtis D.
Klassen editor, 2008, McGraw-Hill.
 Genetic Toxicology by Donald L. Putnam et al. in: Toxicological
Testing Handbook: Principles, Applications, and Data Interpretation.
2nd edition, David Jacobson-Kram & Kit A. Keller editors, 2006,
Informa Healthcare.
 Genetic Toxicology by David Brusick in: Principles & Methods of
Toxicology. 5th edition, Wallace A Hayes editor, 2007, Informa
Healthcare.
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Further Information
About the Author:
David Amacher is a senior investigative and biochemical toxicologist with extensive experience in
the safety evaluation of human and animal health products. Dr. Amacher is a Diplomate of the
American Board of Toxicology, a Fellow of the National Academy of Clinical Biochemistry, and
serves as an Assistant Research Professor of Toxicology and Adjunct Professor in the Graduate
School of the University of Connecticut. His professional affiliations include memberships in the
American Society for Pharmacology and Experimental Therapeutics, Society of Toxicology,
American Society of Biochemistry and Molecular Biology, International Society for the Study of
Xenobiotics, American Association of Clinical Chemistry, and the American College of Toxicology.
 An accompanying commentary on historical and current perspectives on genetic
toxicology, assay predictivity and shortcomings, regulatory guidance, and highthroughput screens to enhance preclinical drug safety can be found at ToxInsights
(www.tigertox.com).
26
Supplemental
Slides
27
Mammalian Somatic Cell Cycle
Referenced from http://bit.ly/byhKxT on
19 Sept 2010.
Meiosis, which occurs only in germ cells, is the
process of nuclear division that reduces the
number of chromosomes by half.
•
G1 (interphase) – energy stores
replenished & daughter cell
growth
•
S phase (DNA synthesis) – DNA
is duplicated in process of
replication.
•
G2 – energy reserves restored.
•
Mitosis - DNA is divided into two
identical sets before the cell
divides.
•
Cytokinesis - the division of the
cytoplasm of a parent cell. It
occurs at the end of Mitosis or the
beginning of Interphase.
28
Genotoxicity ICH Regulatory
Guidelines
ICH Harmonized Tripartite Guideline S2(R1).
Genotoxicity Testing and Data Interpretation
for Pharmaceuticals Intended for Human Use
(Step 3, March 2008).
 This guidance replaces and combines the ICH S2A &
S2B guidelines. The revised guidance describes
internationally agreed upon standards for follow-up
testing & interpretation of positive findings in vitro & in
vivo in the standard genetic toxicology battery,
including assessment of non-relevant findings.
29