Nicnas gen tox3

Genetic Toxicology
Dr R B Cope BVSc BSc(Hon 1) PhD cGLPCP DABT ERT

Quick Review of Relevant
Basic Biology

Definitions

 Genotoxic:

 Non-genotoxic:

 Direct-acting mutagen:

 Indirect-acting mutagen:

 Pro-mutagen:

 Proximate mutagen:

Definitions

 Ultimate mutagen:

 Alkylating agent:

 Radiomimetic:

 Non-radiomimetic:

 DNA macrolesion

 DNA microlesion

Direct-acting mutagens

 By definition, these are highly DNA reactive agents that
do not require prior metabolism for direct DNA
interactions;

 Generally very reactive chemicals, often with relatively
short environmental half-lives;

 Tend to be highly reactive electrophiles;

 Typically act at the site of first contact;


 Chemical stability, transport, membrane permeability
generally determine the degree of mutagenicity
associated with these agents;

 Direct-acting mutagens are generally mutagenic at
multiple tissue sites and in essentially all species;


 Classical examples:
 Nitrogen and sulfur mustards;
 Methyl methane sulfonate;
 Propane sulfone,
 Ethyleneimine
 Beta propiolactone
 Dimethylsulfate

Classical Structural Alerts for Reactive, Electrophilic Direct-Acting
Mutagens

Cl-CH2-O-CH2-Cl

Haloethers

Epoxides Strained lactones Sulfonates

Enals

Mechanisms of Chemical Interaction
with DNA: Electrophiles

 The majority of genotoxic chemicals that
directluy interact with DNA are either
electrophiles or are metabolized to
electrophiles;
 Electrophiles are positively charged species
that are attracted to an electron rich
center i.e. a nucleophile;


 An electrophile (literally electron-lover) is a
reagent attracted to electrons that
participates in a chemical reaction by
accepting an electron pair in order to bond
to a nucleophile;
 Most electrophiles are positively charged,
have an atom that carries a partial positive
charge, or have an atom that does not
have an octet of electrons;


 Specific areas of DNA behaves like a nucleophile;
 DNA nucleophilic centers:
 Ring nitrogens of the DNA bases are generally the
most reactive centers;
 Ring oxygens of the DNA bases are also reactive
centers;
 S: groups in DNA are also targets
 Critically, the N7 of guanine (most reactive); the N3 of
adenine and the O6 of guanine are the most common
sites

Nucleophilic Centers of DNA Bases

 Electrophilic damage may also occur to the
phosphodiester backbone of DNA:

 Different electrophiles display different
preferences for the various DNA nucleophilic
sites and different spectra of damage;

 One of the intensively researched concepts is
that the DNA damage spectrum for different
electrophiles could be used as a marker of
environmental exposure: has not worked
because of the variable ability of DNA to repair
different types of DNA damage plus the often
tissue-specific nature of mutation spectra.

with DNA: Oxidization
 Oxidation of DNA bases is a normal
background biological event associated with
aerobic metabolism and other cell reactions;

 Important endogenous oxidizing agents are:
H2O2, superoxide anion, nitric oxide species, lipid
peroxides, hydroxyl radicals, Fenton reaction
products;

 Endogenous oxidative damage to DNA occurs
at a rate of about 120 occurrences per cell per
hour in NORMAL cells!
 In general, oxidative damage to DNA is
repaired with high fidelity at a maximal rate of
about 105 base pairs per hour;
 Critically, there are a number of antioxidant
pathways that limit the amount of
endogenously oxidative species that are
produced under normal circumstances;

 Oxidative damage to DNA occurs most readily
at guanine residues due to the high oxidation
potential of this base relative to cytosine,
thymine, and adenine;

 Most common oxidized DNA adduct is 8-
hydroxyguanine followed by thymine glycol.

with DNA: Hydrolysis - deamination,
depurination, and depyrimidination
 Deamination of cytosine:
 Spontaneous deamination is the hydrolysis
reaction of cytosine into uracil,
 Corrected for by the removal of by uracil-DNA
glycosylase, generating an abasic (AP) site;
 The AP site is then corrected by base excision
repair;

with DNA: Hydrolysis -
deamination, depurination, and
depyrimidination
 5-methylcytosine: Spontaneous deamination of
5-methylcytosine results in thymine;
 This is the most common single nucleotide
mutation. In DNA, this reaction can be corrected
by the enzyme thymine-DNA glycosylase;

 Guanine: Deamination of guanine results in the
formation of xanthine.
 Xanthine, in a manner analogous to the enol
tautomer of guanine, selectively base pairs with
thymine instead of cytosine. This results in a post-
replicative transition mutation, where the original
G-C base pair transforms into an A-T base pair.
Correction of this mutation involves the use of
alkyladenine glycosylase during base excision
repair;

 Adenine: Deamination of adenine results in the
formation of hypoxanthine.
 Hypoxanthine, in a manner analogous to the
imine tautomer of adenine, selectively base pairs
with cytosine instead of thymine. This results in a
post-replicative transition mutation, where the
original A-T base pair transforms into a G-C base
pair;

with DNA: Hydrolysis -
deamination, depurination, and
depyrimidination
 Depurination is a chemical reaction of purine
deoxyribonucleosides, deoxyadenosine and
deoxyguanosine, in which the β-N-glycosidic bond is
hydrolytically cleaved releasing a nucleic base,
adenine or guanine, respectively.
 Deoxyribonucleosides and their derivatives are
substantially more prone to depurination than their
corresponding ribonucleoside counterparts.
 Loss of pyrimidine bases (Cytosine and Thymine)
occurs by a similar mechanism, but at a
substantially lower rate.

 When depurination occurs with DNA, it leads to the
formation of apurinic site and results in an alteration
of the structure;

 As many as 5,000 purines are lost this way each day
in a typical human cell;

 [In cells, one of the main causes of depurination is
the presence of endogenous metabolites
undergoing chemical reactions;

 Apurinic sites in double-stranded DNA are efficiently
repaired by portions of the base excision repair
(BER) pathway;
 Depurinated bases in single-stranded DNA
undergoing replication can lead to mutations,
because in the absence of information from the
complementary strand, BER can add an incorrect
base at the apurinic site, resulting in either a
transition or transversion mutation;
 Depurination is known to play a major role in cancer
initiation.

with DNA: Intercalation
 Intercalation occurs when ligands of an
appropriate size and chemical nature fit
themselves in between base pairs of DNA;

 Intercalating agents are generally
polycyclic, aromatic, and planar, and good
DNA stains;

 Important examples:
 Ethidium bromide (DNA stain);
 Anticancer agents:
proflavine, daunorubicin, doxorubicin, dactinomy
cin, thalidomide.

Molecular structure of ethidium intercalated between two pairs of adenine-
uracil base pairs.

with DNA: Intercalation
 In order for an intercalator to fit between base pairs,
the DNA must dynamically open a space between
its base pairs by unwinding;
 The amount of unwinding depends on the specific
agent;
 This unwinding induces local structural changes to
the DNA strand resulting in functional changes:
inhibition of transcription and replication and DNA
repair processes,
 Intercalating agents are commonly potent
mutagen.

with DNA: Intercalating agents
 Important examples:
 8,9 epoxide of aflatoxin B1;
 Acridine dyes.

with DNA: DNA cross linking
 Crosslinks occur when exogenous or endogenous
agents react with two different positions in the DNA;

 Crosslinks can occur in the same DNA strand
(intrastrand crosslink) or between opposite strands
(interstrand crosslink);

 Crosslinks between DNA and protein can occur;

 Crosslinks impair DNA replication if the crosslink is not
repaired;

 Mechanisms:
 Bifunctional alkylating agents (e.g. methylene
dimethanesulphonate, sulphur mustard, methyl
methanesulphonate): mostly act on adjacent N7-
guanine bases;
 Cisplatin: 1,2-intrastrand d(GpG) adducts (via N7-
guanine bases);

 Mechanisms:
 Nitrous acid is formed in the stomach from
dietary nitrites (meat prreservatives): forms
interstrand DNA crosslinks at the aminogroup
of N2 of guanine at CG sequences;

 Malondialdehyde from lipid peroxidation:
forms etheno adduct-derived interstrand
crosslinks;

 Mechanisms:
 Psoralens: photoactivated by UVA  form
covalent adducts with thymine, one type of
which is an intrastrand crosslinking reaction
targets TA sequences intercalating in DNA
and linking one base of the DNA with the one
below it. Psoralen adducts cause replication
arrest and is used in the treatment of psoriasis
and vitiligo;

 Mechanisms:
 Aldehydes such as acrolein and
crotonaldehyde found in tobacco smoke or
automotive exhaust can form DNA
interstrand crosslinks in DNA.

 Formaldehyde (HCHO) induces protein-DNA
and protein-protein crosslinks;

with DNA: DNA single strand breaks
 Single strand breaks are an extremely common
phenomenon – thousands of incidents per cell
per day;

 Mechanisms of production:
 Free radical attack (notably with radiation-
induced DNA damage);
 DNA alykylation (i.e. electrophylic attack);
 Many of the mechanisms are poorly
understood;

with DNA: DNA double strand breaks
 Particularly hazardous to the cell because they can
lead to genome rearrangements: regarded as the
most dangerous of DNA lesions;
 Mechanisms are poorly understood, however
oxidization and alkylating agents are able to
produce these DNA lesions;
 DSBs are induced by a number of different
mechanisms, including exposure to ionizing
radiation, radiomimetic drugs, collapse of
replication forks when the replication machinery
encounters single-stranded breaks (SSBs) in the
template DNA,

Micro-DNA Lesions:
Small damage with BIG outcomes.

The Ability to Chemically Interact
With DNA is Not Enough
 For a mutation to occur:
 Exposure of DNA generally must occur at the right
time in the cell cycle;

 A DNA lesion must be chemically produced;

 The DNA lesion must not be so gross as to prevent
DNA replication and/or produce cell death;

The Ability to Chemically Interact
With DNA is Not Enough
 For a mutation to occur:
 The lesion must persist in the DNA long enough for at
least 1 cell division (i.e. the “fixing” of the mutation
within the cell genome; often referred to as the
“expression time” in genetox assays);

 The DNA lesion must not trigger the G1/S checkpoint
(i.e. apoptosis or senescence; more on this in the
carcinogenesis sildes);

 The DNA lesion must not trigger the intra-S phase
checkpoint (if it does, repair is the likely outcome);

The Cell Cycle and Mutation:
The vulnerability of the S phase.
 Small mutations are most likely to occur when
the DNA is being copied i.e. S phase;

 Reason for this is that the DNA double helix is
unwound and the 2 strands are separated 
single DNA strands are particularly vulnerable to
chemical attack;

(A) Nucleoside triphosphates serve as a substrate for DNA polymerase, according to the
mechanism shown on the top strand. Each nucleoside triphosphate is made up of three
phosphates (represented here by yellow spheres), a deoxyribose sugar (beige rectangle)
and one of four bases (differently colored cylinders). The three phosphates are joined to
each other by high-energy bonds, and the cleavage of these bonds during the
polymerization reaction releases the free energy needed to drive the incorporation of
each nucleotide into the growing DNA chain. The reaction shown on the bottom strand,
which would cause DNA chain growth in the 3' to 5' chemical direction, does not occur in
nature. (B) DNA polymerases catalyse chain growth only in the 5' to 3' chemical direction,
but both new daughter strands grow at the fork. The leading strand grows continuously,
whereas the lagging strand is synthesized by a DNA polymerase through the backstitching
mechanism illustrated. Thus, both strands are produced by DNA synthesis in the 5' to 3'
direction.

Proteins at the Y-shaped DNA replication fork: These proteins are illustrated schematically in panel a of the figure below, but in reality, the
fork is folded in three dimensions, producing a structure resembling that of the diagram in the inset b. Focusing on the schematic illustration
in a, two DNA polymerase molecules are active at the fork at any one time. One moves continuously to produce the new daughter DNA
molecule on the leading strand, whereas the other produces a long series of short Okazaki DNA fragments on the lagging strand. Both
polymerases are anchored to their template by polymerase accessory proteins, in the form of a sliding clamp and a clamp loader. A DNA
helicase, powered by ATP hydrolysis, propels itself rapidly along one of the template DNA strands (here the lagging strand), forcing open
the DNA helix ahead of the replication fork. The helicase exposes the bases of the DNA helix for the leading-strand polymerase to copy.
DNA topoisomerase enzymes facilitate DNA helix unwinding. In addition to the template, DNA polymerases need a pre-existing DNA or
RNA chain end (a primer) onto which to add each nucleotide. For this reason, the lagging strand polymerase requires the action of a DNA
primase enzyme before it can start each Okazaki fragment. The primase produces a very short RNA molecule (an RNA primer) at the 58
end of each Okazaki fragment onto which the DNA polymerase adds nucleotides. Finally, the single-stranded regions of DNA at the fork
are covered by multiple copies of a single-strand DNA-binding protein, which hold the DNA template strands open with their bases
exposed. In the folded fork structure shown in the inset, the lagging-strand DNA polymerase remains tied to the leading-strand DNA
polymerase. This allows the lagging-strand polymerase to remain at the fork after it finishes the synthesis of each Okazaki fragment. As a
result, this polymerase can be used over and over again to synthesize the large number of Okazaki fragments that are needed to produce
a new DNA chain on the lagging strand. In addition to the above group of core proteins, other proteins (not shown) are needed for DNA
replication. These include a set of initiator proteins to begin each new replication fork at a replication origin, an RNAseH enzyme to remove
the RNA primers from the Okazaki fragments, and a DNA ligase to seal the adjacent Okazaki fragments together to form a continuous DNA
strand.

The S phase checkpoint.
 The S-phase checkpoint is a surveillance
mechanism, mediated by the protein kinases ATR
and Chk2 in human cells;
 Responds to to DNA damage by co-ordinating a
global cellular response necessary to maintain
genome integrity;
 A key aspect of this response is the stabilization of
DNA replication forks, which is critical for cell
survival;
 A defective checkpoint causes irreversible
replication-fork collapse and leads to genomic
instability, a hallmark of cancer cells.

If DNA replication is blocked (e.g. by a DNA adduct) ssDNA regions at stalled
forks continue to grow because MCM (minichromosome maintenance complex)
helicase continues DNA unwinding, although uncoupled from DNA synthesis.

The ssDNA binds RPA (replication protein A), which triggers the activation of the
checkpoint response. This process is initiated by the recruitment of the Mec1/ATR
sensor to RPA-coated ssDNA at stalled forks by its regulatory subunit, Ddc2 (ATRIP
in human cells). Mec1 then phosphorylates Mrc1 (the homologue of human
Claspin), a mediator that transduces the signal from Mec1 to the effector kinase
Rad53, which becomes phosphorylated and activated.

The S phase checkpoints.
 The S-phase checkpoint response co-ordinates DNA
replication, DNA repair and cell-cycle progression and
regulates processes such as firing of replication origins,
stabilization of DNA replication forks in response to DNA
damage or replicative stress, resumption of stalled DNA
replication forks, transcriptional induction of DNA
damage response genes, choice of the repair pathway
and inhibition of mitosis until replication is completed;

 The S-phase checkpoint is required for cellular viability in
DNA damage or replicative-stress conditions

Types of Mutations: Point mutations.

 Point mutation = single base substitution = the
replacement of a single base nucleotide with another
nucleotide of the genetic material (either DNA or RNA);

 Point mutations most commonly occur during S phase
(i.e. DNA replication);

 The term also includes insertions or deletions of a single
base pair which will result in a frame shift mutation;

 Classifications:
 By type: deletion, transition, insertion, or transversion;
 By the effect on function: nonsense, missense and silent;


 Transversions (beta mutations):
 The substitution of a purine for a pyrimidine or vice versa;
 Can only be repaired by a spontaneous reversion;
 Transitions produce large chemical changes to DNA
structure; thus the consequences of this change tend to be
more drastic than those of transitions.
 Transversions are classically caused by ionizing radiation and
alkylating agents.

 Transitions (alpha mutations):
 A point mutation that changes a purine nucleotide to another
purine (A ↔ G) or a pyrimidine nucleotide to another pyrimidine (C
↔ T);
 Approximately two out of three single nucleotide polymorphisms
(SNPs) are transitions;
 Transitions can be caused by oxidative deamination and
tautomerization;
 Although there are twice as many possible transversions, transitions
appear more often in genomes, possibly due to the molecular
mechanisms that generate them;
 5-Methylcytosine is more prone to transition than unmethylated
cytosine, due to spontaneous deamination.


 Nonsense mutations are point mutations in a sequence of DNA
that results in a premature stop codon, or a nonsense codon in
the transcribed mRNA, and in a truncated, incomplete, and
usually nonfunctional protein product;

 Missense mutations are point mutations in which a single
nucleotide is changed, resulting in a codon that codes for a
different amino acid i.e. a non-synonymous change (mutations
that change an amino acid to a stop codon are considered
nonsense mutations, rather than missense mutations). There are
2 possible outcomes:
 Conservative mutations: Result in an amino acid change.
However, the properties of the amino acid remain the same
(e.g., hydrophobic, hydrophilic, etc). At times, a change to one
amino acid in the protein is not detrimental to the organism as a
whole. Most proteins can withstand one or two point mutations
before their functioning changes;
 Non-conservative mutations: Result in an amino acid change that
has different properties than the wild type. The protein may lose its
function, which can result in a disease in the organism.

Types of Mutations:
Frame shift mutations.
 Silent mutations: Code for the same amino acid.
 A silent mutation has no effect on the functioning of the protein;
 A single nucleotide can change, but the new codon specifies
the same amino acid, resulting in an non-mutated protein;
 This type of change is also called synonymous change, since the
old and new codon code for the same amino acid;
 This is possible because 64 codons specify only 20 amino acids;
 Different codons can lead to differential protein expression
levels.

 A frame shift mutation (also called a framing error or a reading
frame shift) is a genetic mutation caused by indels (insertions or
deletions) of a number of nucleotides that is not evenly divisible by
three from a DNA sequence;

 Remember that it takes 3 DNA and RNA nucleotides to code for a
specific amino acid in a protein i.e. the “reading frames” consist of
3 nucleic acid residues;

 Thus insertion or deletion of a DNA nucleotide can change the
reading frame (i.e. the grouping “3s” of the codons), resulting in a
completely different translation from the original;The earlier in the
sequence the deletion or insertion occurs, the more altered the
protein produced is.

 Frame shift mutations;

 The earlier in the sequence the deletion or insertion
occurs, the greater the effect on protein structure. This is
because all of the reading frames after the insertion or
deletion will be altered i.e. earlier in the relevant DNA coding
sequence the error is, the bigger the overall change to the
downstream amino acid composition of the protein;

 Frame shift mutations;
 Frameshift mutations will also alter the first stop codon ("UAA",
"UGA" or "UAG") encountered in the sequence. The
polypeptide being created could be abnormally short or
abnormally long, and will most likely not be functional;

 Frameshift mutations frequently result in severe genetic
diseases

DNA Repair Mechanisms.
 High level overview only;

 The self-repair of DNA is unique amongst biological molecules;

 Major types of mammalian DNA repair:
 Base excision repair;
 Nucleotide excision repair;
 Mismatch repair;
 Recombinational repair.

 DNA repair capacity varies by mechanism, tissue, organ,
individual and species;

 Not all DNA adduct types are equal: some are more easily
repaired than others and some types cannot be repaired;

 Because of the above 2 points, the use of the number of DNA
adducts per cell is NOT a reliable predictor of genetic hazard
UNLESS there is very detailed information regarding the kinetics
of DNA adduct removal in the specific target tissue and target
species!


 In general terms, DNA repair are saturatable mechanisms i.e.
there is maximal threshold for the amount of DNA that can be
repaired within a given set of parameters;

 All DNA repair mechanisms have the ability to detect and repair
DNA, and if the DNA repair is successful, the impact of the
original DNA damage on the animal is reduced or eliminated;

DNA Repair Mechanisms:
 In general terms, when low levels of DNA damage occur (i.e.
below saturation for repair), error-free (high fidelity) repair
occurs. When there are large amounts of DNA damage (i.e.
above the saturation for repair), error prone DNA repair
predominates;

 However: there are big species, sex, age, tissue, and organ
differences;
 The type of DNA adduct has a significant influence over
which type of repair predominates;

 Intrinsic variability within human populations
 There is a very large variability to repair DNA
damage between individuals: up to 65% of
average rate in populations without inherited DNA
repair defects (e.g. XP);
 People with XP have DNA repair capacity of ~1-
2% of normal;
 There are differences in DNA repair capacity
between rodents and humans: you cannot directly
extrapolate results unless you accurate kinetics
across the relevant species;

Mechanisms where only one strand is
damaged
 When only one of the two strands of a double helix
has a defect (i.e. only one of the 2 strands has a
damaged/missing nucleotide), the other strand can
be used as a template to guide the correction of the
damaged strand;

 These types of DNA repair are called excision repair:
remove the damaged nucleotide and replace it with
an undamaged nucleotide complementary to that
found in the undamaged DNA strand

Base excision repair
 Base excision repair:
 Repairs damage to a single base caused by oxidation,
alkylation, hydrolysis, or deamination;
 The damaged base is removed by a DNA glycosylase;
 The DNA base is then recognized by an enzyme called
AP endonuclease, which cuts the phosphodiester
bond;
 The missing part is then resynthesized by a DNA
polymerase, and a DNA ligase performs the final nick-
sealing step;

Base excision repair
 Base excision repair:
 BER is the major repair pathway involved in the removal
of non-bulky damaged nucleotides;
 In general BER is a high fidelity process i.e. not error
prone;
 BER protects both nuclear and mitochondrial DNA;
 Heritable defects in BER (particularly DNA polymerase)
are associated with cancer.

Nucleotide excision repair
 Nucleotide excision repair (NER), which recognizes
bulky, helix-distorting lesions;

 A specialized form of NER known as transcription-
coupled repair deploys NER enzymes to genes that
are being actively transcribed;

1) 3 protein complexes
are involved in DNA-
damage recognition:
XPA, XPC-HR23 and RPA.
2) These proteins recruits
Transcription factor II
(TFIIH) that incorporate
two helicases: XPB and
XPD that unwinds a 30 bp
DNA fragment around
the DNA damage.
3) After DNA unwinding,
damaged-DNA strand is
excised by XPG and the
XPF-ERCC1 complex at 3'
and 5' sites respectively.

4) After excision,
damaged-DNA strand is
removed and replaced
by re-synthesizing the
template complementary
DNA strand by
polymerase complex (Pol.
E/D, replication protein A
(RPA) and replication
factor C).

Nucleotide excision repair
 Mismatch repair (MMR), which corrects errors of DNA
replication and recombination that result in
mispaired (but undamaged) nucleotides;

 MMR functions primarily as a “proof reader” following
DNA replication

Macro DNA Damage:
 Macro DNA damage = damage to chromosomes =
clastenogenesis e.g. single strand breaks, double
strand breaks, sister chromatid exchange, non-
homologous end joining, changes in ploidy;
 Abnormal chromosome number = aneuploidy;
 Increased chromosome number = polyploidy;

Macro DNA Damage:
Sister chromatid exchange
 Exchange of genetic material between two identical
sister chromatids or between chromosomes with identical
mutations;

 Primarily occurs during S phase;

 Four to five sister chromatid exchange/chromosome
pair/mitosis is within the normal range, 14-100 exchanges
is not normal and presents a danger to the organis;

 Mediated by the homologous end-joining mechanism fo
DNA;

Macro DNA Damage:
Sister chromatid exchange
(equal cross over)
 SCEs can be induced by various genotoxic treatments
that result in double DNA strand breaks, suggesting that
SCEs reflect a DNA repair process i.e. they are measure of
DNA damage;

 This process is considered to be conservative and error-
free, since no information is generally altered during
reciprocal interchange by homologous recombination.

 Most forms of DNA damage induce chromatid exchange
upon replication fork collapse: Holliday model;

 Occurs during prophase I of meiosis (pachytene) in a
process called synapsis.

Macro DNA Damage:
Other potential outcomes of
DNA double strand breaks
 SCE (equal cross over) is the least harmful outcome
of a DSB;

 Other potentially catestrophic outcomes include due
to misrepair of DSBs include:
 Inversions;
 Interstitial deletions;
 Terminal deletions;
 Translocations;
 Unequal crossovers.

Macro DNA Damage:
 Inversions:
 Inversions that
involve the
centromere are
called pericentric
inversions;
 Those that do not
involve the
centromere are
called paracentric
inversions;
 Inversions potentially
have massive effects
on gene function.

Macro DNA Damage:
 Interstitial deletions:
 Causes include the
following: Losses
from translocation;
chromosomal
crossovers within a
chromosomal
inversion; unequal
crossing over;
breaking without
rejoining

Macro DNA Damage:

Macro DNA Damage:
 Translocation
 = a chromosome abnormality caused by rearrangement of
parts between nonhomologous chromosomes;

 Gene fusion may be created when the translocation joins
two otherwise separated genes: very important in
carcinogenesis as it may place the coding region of a
relatively inactive gene with normally very active gene
promoter region  inappropriate upregulation of a gene
(alternatively gene silencing may occur);

Macro DNA Damage:
 Translocation:
 There are two main types, reciprocal (also known as non-
Robertsonian) and non-reciprocal (Robertsonian);

 Also, translocations can be balanced (in an even exchange
of material with no genetic information extra or missing, and
ideally full functionality) or unbalanced (where the exchange
of chromosome material is unequal resulting in extra or
missing genes).

Macro DNA Damage: Other potential outcomes of

 Reciprocal translocations:
 Reciprocal translocations are usually an exchange of
material between nonhomologous chromosomes;
 Estimates of incidence range from about 1 in 500 human
newborns;
 Such translocations are usually harmless and may be found
through prenatal diagnosis;
 However, carriers of balanced reciprocal translocations have
increased risks of creating gametes with unbalanced
chromosome translocations leading to miscarriages or
children with abnormalities.
 Most balanced translocation carriers are healthy and do not
have any symptoms.

Macro DNA Damage: Other potential
outcomes of DNA double strand breaks

 Nonreciprocal (Robertsonian) translocations:
 This type of rearrangement involves two acrocentric
chromosomes that fuse near the centromere region with loss
of the short arms
 The resulting karyotype in humans leaves only 45
chromosomes since two chromosomes have fused together


 Nonreciprocal (Robertsonian) translocations:
 This has no direct effect on the phenotype since the only
genes on the short arms of acrocentrics are common to all of
them and are present in variable copy number (nucleolar
organiser genes). Robertsonian translocations have been
seen involving all combinations of acrocentric chromosomes.
The most common translocation in humans involves
chromosomes 13 and 14 and is seen in about 0.97 / 1000
newborns.
 Carriers of Robertsonian translocations are not associated
with any phenotypic abnormalities, but there is a risk of
unbalanced gametes which lead to miscarriages or
abnormal offspring.


 Balanced translocations: no genetic material is lost


 Unbalanced translocation: genetic material is lost from one
chromosome but gained by another. This means that the
progeny will either have missing genetic material or extra
genetic material

Macro DNA Damage:

 Acentric fragments: a segment of a chromosome
that lacks a centromere;

Macro DNA Damage:

 Acentric fragments:
 Because acentric fragments lack a centromere, the cannot attach
to the mitotic spindle i.e. acentric fragments are not evenly
distributed to the daughter cells in cell division (mitosis and meiosis).
As a result one of the daughters will lack the acentric fragment;
 Lack of the acentric fragment in one of the daughter cells
may have deleterious consequences, depending on the
function of the DNA in this region of the chromosome;
 In the case of a gamete, it will be fatal if essential DNA is
contained in that DNA segment;
 In the case of a diploid cell, the daughter cell lacking the
acetric fragment will show expression of any recessive genes
found in the homologous chromosome.

Acentric fragments are lost from the nuclei of cells following
mitosis or meiosis. They form a micronucleus.

Macro DNA Damage: Changes in
chromosome number

 Changes to chromosome number can result from:
 Nonreciprocal (Robertsonian) translocations;
 Errors in chromosomal segregation i.e. a whole
chromosome is left behind during anaphase of
mitosis or meiosis;
 Damage to the mitotic spindle: e.g. griseofulvin,
pcalitaxel, colecemid, vinblastine;

chromosome number

 Kinetocore:
 The protein structure on chromatids where the
spindle fibers attach during cell division to pull sister
chromatids apart;
 The kinetochore forms in eukaryotes, assembles on
the centromere and links the chromosome to
microtubule polymers from the mitotic spindle
during mitosis and meiosis;
 Kinetochores start, control and supervise the
striking movements of chromosomes during cell
division;

chromosome number

 Kinetocore:

 Kinetochores are critical in initiating/avoiding the
spindle checkpoint

chromosome number

 The spindle checkpoint:
 The spindle checkpoint (= spindle assembly checkpoint = mitotic
checkpoint), is a cellular mechanism responsible for detection of:
 Correct assembly of the mitotic spindle
 Attachment of all chromosomes to the mitotic spindle in a
bipolar manner
 Congression of all chromosomes at the metaphase plate.
 When just one chromosome (for any reason) remains lagging during
congression, the spindle checkpoint machinery generates a delay
in cell cycle progression: the cell is arrested, allowing time for repair
mechanisms to solve the detected problem. After some time, if the
problem has not been solved, the cell will be targeted for apoptosis
(programmed cell death), a safety mechanism to avoid the
generation of aneuploidy, a situation which generally has dramatic
consequences for the organism.

chromosome number

 Micronuclei can also contain whole chromosomes that
were improperly attached to the mitotic spindle during
anaphase: these are detectable by staining the
micronuclei for kinetocores;

 There are at least major causes of micronuclei:
 The formation of chromosome fragments that lack a
centromere (and thus a kinetocore  cannot attach o the
mitotic spindle);
 The loss of a whole chromosome which has failed to attach
to the mitotic spindle or has broken off the mitotic spindle.
This effect is produced by spindle agents;
 Extrachromosomal double minutes.

chromosome number

 So what the heck is a double minute you ask (no it is not 120
seconds):
 Double minutes are small fragments of extrachromosomal DNA,
which have been observed in a large number of human tumors;
 They are a manifestation of gene amplification during the
development of tumors, which give the cells selective advantages
for growth and survival;
 They frequently harbor amplified oncogenes and genes involved in
drug resistance;
 Double minutes, like actual chromosomes, are composed of
chromatin and replicate in the nucleus of the cell during cell
division;
 Unlike typical chromosomes, they are composed of circular
fragments of DNA, up to only a few million base pairs in size and
contain no centromere or telomere.

Micronucleus
fluorescently labeled
for kinetocores

Mechanisms of micronuclei formation. (A)
Aneugenic agents prevent the formation of
the spindle apparatus during mitosis. The use of
these agents generates micronuclei as a
consequence of whole chromosomes lagging
behind at anaphase. These chromosomes are
left out of the cell nucleus at the end of mitosis;
The DNA in the micronuclei could balance the
nuclear DNA and result in a complete
genome, or be additional to the cell's
genome.
(B) Clastogenic agents induce micronuclei by
breaking the double helix of DNA, thereby
forming acentric fragments. These fragments
are incapable of adhering to the spindle fibres
and integrating in the daughter nuclei, and
are thus left behind during mitosis.
(C) Micronuclei can also contain highly
amplified gene sequences, derived from
extrachromosomal double minutes (DM)
(yellow dots indicate the presence of a DM).
(D(I)) Torsion between the two centromeres of
a dicentric chromosome would give rise to the
formation of an anaphase bridge that is
frequently resolved by breakage. The bridge
breakage often results in the formation of
acentric fragments that are not included in
any of the daughter cell nuclei and form one
or more micronuclei at the end of mitosis. (D
(II)) It has also been described that, instead of
breaking, dicentric chromosomes involved in
anaphase bridges are sometimes detached
from the two centrosomes, left behind at
anaphase and sequestered into micronuclei.

US EPA Testing Requirements & Tiers
Tier Test Types Assessment Function

Rapid, low cost screening,
Ames (bacterial reverse mutation)
typically for agents where
In vitro mammalian cell mutation (e.g.
human exposure is low.

Hazard Identification
1 mouse lymphoma TK)
If all results are negative,
In vitro chromosome aberration or
generally no further
micronucleus assay
testing is required

In vivo testing: at least one or more of: in Required if there is
vivo micronucleus, comet assay, in vivo significant human
2 DNA binding, in vivo unscheduled DNA exposure or +ve results in
synthesis, transgenic mouse models Tier 1
Required if +ve results in
In vivo tests in germ cells (i.e. dominant
Tier 2. Provides a basis for
3 lethal, germ cell micronucleus, germ cell
hazard assessment of
DNA binding, germ cell USD,
germ line effects
Provides a quantitative
Quantitative in vivo tests for germ cell
assessment of germ cell
mutation (specific locus test, visible or
4 biochemical markers [mouse spot],
mutations for use in
quantitative risk
heritable translocation test in mice)
assessments

Germ Cell Versus Somatic Mutation:
Female germ cell
 The timing of exposure is critically important:

 Mutation is most likely to occur during the S phase
(during DNA replication) i.e. mitosis and meiosis;

Female germ cell
 The timing of exposure is critically important:
 In humans (and most mammals), this means:
 During mitosis of oogonium during fetal
development (between weeks 4 and 30 in
humans; between days 14.5 and 18.5 in the rat
and between days 10.5-12.5 in mice);
 There are no further S phases until after
fertilization and the formation of a zygote;

 Remember: DNA repair occurs in oocytes!

Female germ cell

Female germ cell
 Consequences in terms of germ cell mutation:
 The critical timing for female germ cell mutation in
mammals occurs at the prenatal stage of
development!

 Ooctyes are RESISTANT to mutation by non-
radiomimetic chemicals (i.e. chemicals that do not
produce chromosome or chromatid breaks);

 Oocytes are susceptible to radiation and
radiomimetic chemicals;

Male germ cells
 Same basic principle holds true: S phase of cell
division processes is most susceptible to
mutagenesis;
 S phase in spermatogenesis occurs during
spermatogonial stem cell stage of development;
 Unlike in females, spermatogonial stem cell
mitosis occurs throughout the life time of males!
 Late spermatids and spermatozoa lack DNA
repair – also susceptible to unrepaired DNA
damage!

Germ Cell Versus Somatic Mutation
 Overall, males are generally regarded as being
at greater risk of generating germ cell mutations
than females because of the continuous, life-
time replication of spermatogonial stem cells
(where as oogonium are only generated in large
numbers during prenatal development in
females).

Classical Assays for Genotoxicity:
Bacterial reverse mutation assays
 Fundamental principles:
 These assays are for micro-DNA damage;

 Mutant bacterial test strains are created/selected (typically
loose the capacity for synthesis of an amino acid) so they
require some form of additional supplementation (usually an
amino acid) in order to grow – these are called auxotrophs
(auxotrophy is the inability of an organism to synthesize a
particular organic compound required for its growth);


 The mutation required to produce the auxotrophic strain is
generally either a point mutation or a frame shift mutation i.e.
a small DNA sequence change.

 In order for an auxotroph to grow in medium where the
supplement is NOT present, they must undergo a reverse
mutation in order to regain the capacity to synthesize the
essential substance for growth;

 Once the bacteria have undergone a reverse mutaiton, they
are able to grow in media that DO NOT contain the
supplement (typically an amino acid;

 These reverse mutations are typically point mutations or
frame shifts.

 Many test strains have features that make them more
sensitive for the detection of mutations:
 Specific responsive DNA sequences at the reversion sites;
 Increased cell permeability to large molecules
 Lack of DNA repair or enhancement of error-prone DNA
repair;

 Advantages:
 Fast;
 Cheap;
 Quick screening for micro-DNA damage;
 Extensive database/library of the effects of a very diverse
array of chemicals;
 Generally has acceptable false positive/false negative levels
(i.e. good sensitivity, specificity, precision and predictive
value)
 Although many compounds that are positive in this test are
mammalian carcinogens, the correlation is not absolute. It is
dependent on chemical class and there are carcinogens
that are notdetected by this test because they act through
other, non-genotoxic mechanisms or mechanismsabsent in
bacterial cells.


 Although many compounds that are positive in this test are
mammalian carcinogens, the correlation is not absolute. It is
dependent on chemical class and there are carcinogens that
are not detected by this test because they act through other,
non-genotoxic mechanisms or mechanismsabsent in bacterial
cells.

 Disadvantages:
 Utilizes prokaryotic cells, which differ from mammalian cells in
such factors as uptake, metabolism, chromosome structure
and DNA repair processes;
 Tests conducted in vitro generally require the use of an
exogenous source of metabolic activation. In vitro metabolic
activation systems cannot mimic entirely the mammalian in
vivo conditions;
 The test does not provide direct information on the
mutagenic and carcinogenic potency of a substance in
mammals.

 Limitations:

 Difficult to use with substances that are potent bacteriocides
or bacteriostats (i.e. substances that block mitosis);

 Culture media are hydrophilic i.e. requires specialized
techniques for highly lipophilic substances (e.g. petroleum
distillates);

 Special techniques are required for gases, vapors or
substances that evaporate at 37OC;

 Metabolic activation systems:
 The objective is to replicate at least some of the major
biotransformation pathways in vitro;
 Classical system is liver S9 fraction:
 Rats are dosed with Arochlor 1254 (a PCB mixture that acts as a
potent inducer of liver CYP and UGT enzymes via the AhR
pathway)  livers are homogenized  centrifuged at 9000 g for
20 minutes  supernatant is collected;
 S9 contains cytosol and microsomes (= smooth endoplasmic
reticulum): microsomes component contains cytochrome P450
isoforms (phase I metabolism);cytosolic portion contains the
major part of the activities of transferases (phase II metabolism)

 Metabolic activation systems:
 Classical system is liver S9 fraction:

 A NADPH-regenerating system or NADPH solution is required to
supply the energy demand of the CYP enzymes (powers the CYP
cycle);

 For the catalytic activity of phase II enzymes, addition of
exogenous cofactors is necessary: UDPGA and alamethicin for
UGT; acetyl CoA, DTT, and acetyl CoA regenerating g system for
NAT; PAPS for ST; and GT for GST;

 Bacteria and strains required for OECD 471:
 Salmonella typhimurium TA1535 rfa+ uvrB+ hisG46:
 S. typhimurium TA1537 rfa+ uvrB+ hisC3076;
 S. typhimurium TA98 rfa+ uvrB+ hisD3052 pKM101;
 S. typhimurium TA100 rfa+ uvrB+ hisG46 pKM101;
 Escherichia coli WP2 trp+ uvrA;
 In order to detect cross-linking mutagens it may be preferable to
include TA102 or to add a DNA repair-proficient strain of E.coli [e.g.
E.coli WP2 or E.coli WP2 (pKM101)]

 Bacteria and strains required for OECD 471:So what does this all
mean?
 His genotype: location of the deletion mutation in the histidine
operon (operon is a functioning unit of genomic DNA containing a
cluster of genes under the control of a single regulatory signal or
promoter. Net result is that the bacterial carrying this mutation
cannot synthesize histidine and are auxotrophs);

 rfa mutation: A mutation (rfa) in all strains that leads to a defective
lipopolysaccharide (LPS) layer that coats the bacterial surface,
making the bacteria more permeable to bulky chemicals;

 uvrB and uvrA mutations: The uvrB deletion mutation eliminates the
accurate excision repair mechanism, thereby allowing more DNA
lesions to be repaired by the error-prone DNA repairmechanism.
The deletion through the biotin gene makes the bacteria biotin
dependent.

 Bacteria and strains required for OECD 471:So what does this all
mean?
 Plasmid pKM101: present in strains TA1535 and TA1538 resulting in
the corresponding isogenic strains TA100, TA98, TA97, TA102 and
TA104. Plasmid pKM101 enhances chemical and UV-induced
mutagenesis via an increase in the recombination DNA repair
pathway. The plasmid confers ampicillin resistance, which is a
convenient marker to detect the presence of the plasmid;

 Insertion of the mutation hisG428 on the multi-copy plasmid pAQl
which was introduced in strain TA102 with the aim of amplifying the
number of target sites. To enhance the ability of this strain to detect
DNA crosslinking agents, the uvrB gene was retained making the
bacterium DNA repair proficient

 Bacteria and strains required for OECD 471: So what does this all
mean?
 trp+: Trp operon is that codes for the production of tryptophan. trp+
E. coli are auxotrophs for tryptophan;

 Note: all strains except S. typhimurium TA102 are also biotin
dependent (i.e. are both histidine and biotin auxotrophs).

Notes:
• A trace of histidine (or
tryptophan
depending on the
auxotroph) + biotin
are incorporated to
allow for 1 or 2 rounds
of cell replication in
order to fix any
mutations present;

• The plate
incorporation method
is reputed to increase
the assay sensitivity
and to allow the
testing of suspensions
as well as solutions of
test article

 Modifications to the standard plate incorporation assay:

 The preincubation assay: the tester strains are exposed to the
chemical for a short time (20 to 30min) in a small volume (0.5ml) of
either buffer or S-9 mix, prior to plating on glucose agar minimal
medium (GM agar) supplemented with a trace amount of histidine.
With few exceptions it is believed that this assay is more sensitive
than the plate incorporation assay, because short-lived mutagenic
metabolites may have a better chance reacting with the tester
strains in the small volume of preincubation mixture, and the
effective concentration of S-9 mix in the preincubation volume is
higher than that on the plate.


 The desiccator assay for liquids and gases: the use of a closed
chamber is recommended for testing highly volatile chemicals and
gases;
 The Kado Salmonella microsuspension assay for testing samples of
small volumes;

 Testing chemicals in a reduced oxygen atmosphere: anaerobic
environments, such as anaerobic chambers, have been used to
study mutagenicity of chemicals and fecal samples under reduced
oxygen levels.


 Fluctuation method: The fluctuation method is performed entirely in liquid
culture and is scored by counting the number of wells that turn yellow from
purple in a 96-well microplate. If bacteria are able to revert back to
metabolic competence they will continue to replicate and turn the liquid
media acid. By including a pH indicator in the media, the frequency of
mutation is counted as the number of wells out of 96 which have changed
color.

 The fluctuation method is comparable to the traditional pour plate method
in terms of sensitivity and accuracy, however, it does have a number of
advantages, namely, allowing for the testing of higher concentrations of
sample (up to 75% v/v), increasing the sensitivity and extending its
application to low-level environmental mutagens.[20]

 The fluctuation method also has a simple colorimetric endpoint; counting
the number of positive wells out of a possible 96 wells is much less time
consuming than counting individual colonies on an agar plate.

 Strain checks:
 Histidine dependence (his): streak a loopful of the culture across a
GM agar plate supplemented with an excess of biotin. Because all
the Salmonella strains are histidine dependent, there should be no
growth on the plates.

 Biotin dependence (bio): streak a loopful of the culture across a
GM agar plate supplemented with an excess of histidine. There
should be no growth on the plate except for strain TA102 which is
biotin independent.
 Biotin and histidine dependence (bio, his): streak a loopful of the
culture across a GM agar plate supplemented with an excess of
biotin and histidine. Growth should be observed with all strains.

 Strain checks:
 rfa marker: streak a loopful of the culture across a GM agar plate
supplemented with an excess of biotin and histidine. Apply 10 µl of
a sterile 0.1% crystal violet solution. All Salmonella strains should
show a zone of growth inhibition (crystal violet is a relatively large
bacteriocidal molecule [mw = 407.979] which cannot penetrate
the bacteria if a normal cell wall is present);

 Presence of plasmid pKM101 (ampicilline resistance): apply in the
center of a plate 10 µl of ampicilline solution. Streak a loopful of the
pKM101-carrying Salmonella culture across an agar plate
supplemented with an excess of histidine and biotin. Growth should
be observed.

 Strain checks:
 Spontaneous mutant frequency: use the standard plate
incorporation assay procedure without the inclusion of a solvent for
determining the spontaneous mutant frequency (negative control)
of each of the tester strains. When the spontaneous control values
fall outside an acceptable range the genetic integrity of the strain is
considered compromised, and a new culture should be isolated.

 Controls:
 Must have: suitable positive control for non-metabolic activation
and metabolic activation + solvent/vehicle negative control;

For tests with metabolic activation:

 Evaluation of results:
 Surviving populations: usually a 2 – 3-log range of doses are used
and the highest of these doses is selected to show some degree of
bacterial toxicity (background clearing or reduction in the number
of spontaneous mutants);
 Dose-response phenomena: a mutagen should display a clear
dose-related increase in revertant colonies (can be influenced by
poor dose range selection);

 Evaluation of results:

 Generally speaking a mutagen will produce a positive dose
response over at least 3 different concentrations with the hiighest
increase in revertants being 2 – 3 times that of the level of
spontaneous revertants in the negative control plates;

 Pattern: TA-1535 and TA-100 are derived from the same parental
strain, thus commonly the responses of these two strains should be
nearly identical;

 The results (particularly the pattern of reversion) should be
repeatable and consistent;

 Specific gene sequencing may be of use in some cases.

Mouse lymphoma L5178Y tk+/- forward
mutation assay (OECD 476)
 Basic principle of the assay:

 Cells deficient in thymidine kinase (TK) due to the mutation
TK+/- → TK-/- are resistant to the cytotoxic effects of the
pyrimidine analogue trifluorothymidine (TFT);

 TFT is converted to TFT-monophosphate by thymidine kinase
 TFT-triphosphate (TFT-TP) is incorporated into DNA, resulting
in cytocidal effects.


 Thymidine kinase proficient cells are sensitive to TFT, which
causes the inhibition of cellular metabolism and halts further
cell division;

 Thus mutant cells are able to proliferate in the presence of
TFT, whereas normal cells, which contain thymidine kinase,
are not.


 The TK mutations have no effect on the growth of cells in
normal media because normal DNA synthesis does not
involve the TK pathway;

 The L5178Y cells also have point mutations in both p53
alleles,. Because p53 is an important protein in the DNA
damage response in the cell, the L5178Y cell line is
inadequate in its response to DNA damage, which is
arguably important for enhanced assay sensitivity for the
detection of genotoxic compounds.

 Assay is usually conducted with and without S9 metabolic
activation;

 Controls:
 Vehicle/solvent negative control;
 Positive controls: methylmethanesulfonate for studies without
metabolic activation; cyclophosphamide, benzo(a)pyrene
and 3-methylcholanthrene for tests with metabolic
activation;

 Dose range:
 Selected so that the test doses span the range from 0% to
80% reduction in cell growth with or without S9 activation.

 Assay acceptance criteria:
 The average absolute cloning efficiency must be in the
range of 65 – 120% (ability of single cells to form a new
colony);

 The average increase in the vehicle control cell population
should be 8 -32 fold over 2 days;

 Background forward mutation frequency should be
acceptable;

 The frequency of forward mutation in the positive controls
must be consistent with historical data for the lab;

 The assay must include applied concentrations that reach 5
mg/mL or 10 mM (which ever is the lower) for test articles that
cause little or no cytotoxicity;

 The assay must include applied concentrations that reduce
the relative cell growth by approximately 20%; OR

 Reach a concentration that exceeds the solubility limit.

 Interpretation:
 Cell colony size on agar: large mutant colonies have few
genetic changes other than the tk+/-  tk-/- forward mutation;
small mutant colonies generally represent more extensive
genetic modification of the cells;

 A mutant frequency of at least 2 times that in the negative
control is suggestive of a positive control (induction of
mutation is an additive process and not a multiple over
background – the global evaluation factor should be used);

 Interpretation:
 Global evaluation factor = the mean of each vehicle control
mutant frequency distribution + 1 SD across multiple test labs;
 The GEF for the agar assay method = 90;
 The GEF for 96 well plate assays = 126;

 A positive assay is one where the mutant frequency for the
test article equals or exceeds the GEF PLUS there is a
statistically demonstrable dose trend present;

 A negative assay is one where the mutant frequency for the
test article is below the GEF PLUS there is no statistically
demonstrable dose trend present

 Interpretation:
 If only one criterion is met, additional studies are needed to
clarify the test outcome

Other mammalian cell forward
mutation tests(OECD 476)

 The HRPT assay:
 Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is
a transferase that catalyzes conversion of hypoxanthine to
inosine monophosphate and guanine to guanosine
monophosphate;

 Test cell lines are HRPT+/- i.e. have a single functional HRPT
allele that is lost with mutation;

 Multiple different mammalian cell lines with this genotype
are available: V79 Chinese hamster cells, AS52 Chinese
hamster cells, Chinese hamster ovary cells (CHO) and human
TK6 human lymphoblastoid cells.


 The HRPT assay:
 Basic principles: HRPT converts 6-thioguanine (TG) or 8-
azaguanine (AG) to non-toxic metabolites;

 Cells that have HRPT function are able to survive and
replicate in the presence of TG or AG;

 Those cells that lack HRPT function due to a forward mutation
die in the presence of TG or AG;

 The assay is conducted and assessed in a manner that is
similar to the TK assay.


 The XHRPT assay:

 Transgene of xanthineguanine phosphoribosyl transferase
(XHRPT);
 Works on the same principle as the HRPT assay;
 XHRPT is located on autosomal chromosomes where as HRPT
is located on the X chromosome.


 The TK, HPRT and XPRT mutation tests detect different spectra of
genetic events. The autosomal location of TK and XPRT may
allow the detection of genetic events (e.g. large deletions) not
detected at the HPRT locus on X-chromosomes.

In vivo mammalian forward mutation
assays in transgenic animals.

 The Big Blue mouse assay:
 The genome of these mice has been manipulated such
that every cell contains, stably integrated into the
DNA, multiple tandem copies of a bacterial lac I repressor
gene;

 lf the mice are exposed to mutagens, there is a small
probability that a mutation will occur somewhere along
the inserted sequence;

 Any mutation will lead to an inactive lac I gene and lac
repressor protein, meaning the gene (lacZ) for beta-
galactosidase will no longer be repressed;


 The DNA is extracted from the tissues of the treated mouse 
the vector is isolated and used to make functional
bacteriophages  E. coli cells are mixed with the
bacteriophage and spread on a solid culture medium  the
bacteriophages infect and destroy ("lyze") the E. coli cells --?
this causes clear circular zones, called plaques, to appear in
a "lawn" of bacteria  Before they die, cells that have been
infected by bacteriophages carrying a mutated lac I will
produce beta-galactosidase  This reacts with a substrate in
the culture medium turning it blue Count both colorless
and blue plaques  The number of blue plaques divided by
the total number of plaques gives the mutation frequency.


 Bacteriophages with non-mutated genes produce colorless
plaques because no beta-galactosidase is synthesized;


 The muta-mouse assay operates in a manner similar to the
Big Blue except it uses a LacZ gene


 In vivo forward mutation assays offer very substantial
advantages:
 The complete array of metabolic processes are present;
 Tissue specific metabolism is taken into account rather than
just the over simplified metabolism that occurs with S9;
 Tissue and even cell-type specific mutation rates can be
measured. This includes germ cell (spermatogonial)
mutations in males!;
 Takes into account toxicokinetic and toxicodynamic
differences between different organs, tissues and cell types;
 In general, tissue specific mutation frequencies generally
match the distribution of mutation in live animals!

In vitro chromosomal aberration
assay
(OECD 473)
 Typically Chinese hamster ovary cells strain CHO-WBL ATCC
CCL61 are used (fibroblastic cell line);

 Chromosomal number of 21 with a low frequency of
spontaneous mutations;

 Alternatively human peripheral blood lymphocytes that have
been stimulated to divide using a mitogen (PHA) are used;

assay
(OECD 473)
 Cell cultures are exposed to the test substance both with
and without metabolic activation

 At predetermined intervals after exposure of cell cultures to
the test substance, they are treated with a metaphase-
arresting substance (e.g. Colcemid® or colchicine),
harvested, stained and metaphase

 Cells are analysed microscopically for the presence of
chromosome aberrations.

assay
(OECD 473)
 Cell cultures are exposed to the test substance both with
and without metabolic activation

 At predetermined intervals after exposure of cell cultures to
the test substance, they are treated with a metaphase-
arresting substance (e.g. Colcemid® or
colchicine), harvested, stained and metaphase

 Cells are analysed microscopically for the presence of
chromosome aberrations.

assay
(OECD 473)
 Measurement of results:
 This assay does NOT determine aneuploidy (i.e. increased or
decreased number of chromosomes; this is because of
artifacts associated with the cell analysis preparation): only
cells with a normal chromosome number are analysed;
 The types of chromosomal aberrations that are detected
are:
 Simple breaks;
 Complex exchanges;
 Gaps;
 Dicentric chromosomes;
 Ring chromosomes.

assay
(OECD 473)
 Advantages:
 Accurate identification of all the different chromosome
mutation types;
 Possible co-detection of mitotic indices;
 No full automatic but interactive scoring possible;

 Disadvantages:
 High false positive rate;
 Labor intensive and time consuming;
 Heavily dependent on operator/reader skill.

In vivo bone marrow chromosomal
aberration assay (OECD 475)

 Advantages over the in vitro method:
 Takes into account in vivo TK and TD to a certain extent;

 Can test different routes of exposure;

 Full in vivo metabolism (but with some limitations, particularly
given that the tissue analyzed is bone marrow);


 Disadvantages over the in vitro method:
 Exposure of the bone marrow must occur in order for the test
to be valid – this may need to be demonstrated by a TK
study;

 If there is evidence that the test substance, or a reactive
metabolite, will not reach the targettissue, it is not
appropriate to use this test;


 Disadvantages over the in vitro method:

 If metabolism to an ultimate mutagen is required, then the
blood and tissue T½ must be long enough for bone marrow
exposure to occur unless local metabolism in the bone
marrow occurs;

 Risk of excessive toxicity producing distorted results: dose
ranging studies are often needed.


 Principle of the assay:
 Animals (typically rats or Chinese hamsters) are exposed to
the test substance by an appropriate route of exposure and
are sacrificed at appropriate times after treatment;

 Prior to sacrifice, animals are treated with a metaphase-
arresting agent (e.g., colchicine or Colcemid®);

 Chromosome preparations are then made from the bone
marrow cells and stained, and metaphase cells are analysed
for chromosome aberrations.

In vivo spermatogonial chromosomal

 Assay is similar in principle to OECD 475;

 Important difference is that it tests for GERM CELL
chromosomal damage;

 If there is evidence that the test substance, or a reactive
metabolite, will not reach the target tissue, it is not
appropriate to use this assay.

Unscheduled DNA Synthesis OECD428

 This is essentially a test of DNA repair that occurs
outside of the normal period of DNA synthesis in cells;

 Measures DNA synthesis in cells which are not in the S
phase of the cell cycle;

 The assay measures global genomic nucleotide
excision repair (NER);


 The classical OECD assay measures DNA synthesis by
measuring the incorporation of 3H-thymidine or BrDU
into DNA;

 More modern techniques use specific DNA dyes and
flow cytometry: faster, more accurate, provides
more information;


 The test requirements are very broad:
 Can be done in vitro or in vivo;
 Primary cell cultures or established cell lines can be used;
 Test is performed with or without metabolic activation;
 In order to discriminate between UDS and normal semi-
conservative DNA replication, cell replication is inhibited or
minimized using an arginine-deficient medium, low serum
content, or by hydroxyurea in the culture medium;
 For flow cytometric methods, inhibition of the cell cycle is not
required.


 Given that the assay measures nucleotide excision repair, it is
critical that:
 At least some of the DNA lesions produced by the test article
are repairable by nucleotide excision repair;
 The test cell line/animal line is capable of relatively normal
nucleotide excision repair

Mammalian Erythrocyte Micronucleus
Test OECD 474

 It is an assay of macro-chromosomal damage (chromosome
fragments lacking a kinetocore/centromere) OR damage to
the mitotic spindle (whole chromosomes chromosome
fragments containing a kinetocore);
 The assay detects lagging chromosome fragments or
lagging chromosomes;

Test OECD 474

 Takes advantage of the fact that when bone marrow
erythroblast develops into a polychromatic erythrocyte the
cell nucleus is extruded from the cell however DNA
containing micronuclei are not extruded from the cell;

 Micronuclei can be detected by cell staining and flow
cytometry. The lack of a normal cell nucleus makes the
detection of micronuclei much easier.

Test OECD 474

 This assay is regarded as the highest tier assay of the commonly
conducted genetic toxicology assays for chemicals and other
agents;

Test OECD 474

 Important features:
 Classically outbred rats and mice are used in preference to
inbred strains in order to reduce the likelihood of strain
specific responses (classically CD-1 [ICR] BR mice and/or
Sprague-Dawley CD [SD] IGS BR rats);
 Animals are usually 8 -10 weeks old at the start of the study;
 Various routes of administration, including parenteral routes,
are available. Unless there are specific reasons not to, the
study route of exposure should match those of the likely
human routes of exposure;
 Parenteral routes (IV, IP) are used if absorption is poor or if
there are other technical constraints regarding dosing.

Test OECD 474

 The IV route of exposure has a particular advantage in that it
guarantees exposure of the bone marrow;

 If non-IV routes of exposure are used, there must be sufficient
TK data that demonstrates that bone marrow exposure
occurs or is likely to occur;

 If the test article is a pro-mutagen (i.e. requires metabolic
activation), then there must be reasonable certainty that
appropriate metabolic activation can occur in the bone
marrow OR the active metabolites have a chemical half-life
that is long enough for them to reach the bone marrow!

Test OECD 474

 Ideally, 2 positive controls should be used:
 Cyclophosphamide: requires metabolic activation;
 A substance that does not require metabolic activation
(e.g. ethyl methanesulfonate)

Test OECD 474

 Positive controls:
 Can be administered by a different route than the test
article;
 Can be administered as a single dose;

 Negative controls: typically a solvent or vehicle control

Test OECD 474

 “No standard treatment schedule (i.e. 1, 2, or more treatments
at 24 h intervals) can be recommended. The samples from
extended dose regimens are acceptable as long as a positive
effect has been demonstrated for this study or, for a negative
study, as long as toxicity has been demonstrated or the limit
dose has been used, and dosing continued until the time of
sampling. Test substances may also be administered as a split
dose, i.e., two treatments on the same day separated by no
more than a few hours, to facilitate administering a large
volume of material.”

Test OECD 474

 The test can be performed in 2 ways:

 “Animals are treated with the test substance once. Samples of
bone marrow are taken at least twice, starting not earlier than
24 hours after treatment, but not extending beyond 48 hours
after treatment with appropriate interval(s) between samples.
The use of sampling times earlier than 24 hours after treatment
should be justified. Samples of peripheral blood are taken at
least twice, starting not earlier than 36 hours after treatment,
with appropriate intervals following the first sample, but not
extending beyond 72 hours. When a positive response is
recognized at one sampling time, additional sampling is not
required.”

Test OECD 474

 The test can be performed in 2 ways:

 “If 2 or more daily treatments are used (e.g. two or more
treatments at 24 hour intervals), samples should be collected
once between 18 and 24 hours following the final treatment for
the bone marrow and once between 36 and 48 hours following
the final treatment for the peripheral blood (12).”

Test OECD 474

 Dose rates:
 “If a range finding study is performed because there are no suitable
data available, it should be performed in the same laboratory,
using the same species, strain, sex, and treatment regimen to be
used in the main study (13). If there is toxicity, three dose levels are
used for the first sampling time. These dose levels should cover a
range from the maximum to little or no toxicity. At the later
sampling time only the highest dose needs to be used. The highest
dose is defined as the dose producing signs of toxicity such that
higher dose levels, based on the same dosing regimen, would be
expected to produce lethality. “

 Toxicity can take any form. A commonly used measure is a
reduction in the ratio of polychromatic to normochromatic
erythrocytes (what does this say about the bone marrow?);

Test OECD 474

 Dose rates:
 “Substances with specific biological activities at low non-toxic doses
(such as hormones and mitogens) may be exceptions to the dose-
setting criteria and should be evaluated on a case-by-case basis.
The highest dose may also be defined as a dose that produces
some indication of toxicity of the bone marrow (e.g. a reduction in
the proportion of immature erythrocytes among total erythrocytes
in the bone marrow or peripheral blood).”

 In general, a maximum limit dose of 2 g/kg is used for substances
with low toxicity

Test OECD 474

 Sexes:
 In general, if there is data that indicates that there are no sex
differences in toxicity, only males are used;

 If there is data indicating sex differences, BOTH sexes MUST
be used.

Test OECD 474

 % micronucleated PCE in the negative control group must lie
within the historical normal range for the lab and must be
below 0.4%;

 Must be a statistically significant increase in the %
micronucleated PCE relative to the negative controls in the
positive control group and this increase should be consisted
with historical data for the lab;

 Either the limit dose is reached or at least some non-leathal
toxicity must be present in the highest test article dose

Test OECD 474

 What constitutes a positive response?
 A statistically significant increase in micronucleated
PCE in at least one of the test article doses relative to
the negative controls; OR

 A statistically significant dose response relationship is
present;

 The results make biological sense.

Rare Assays

 Mouse Spot Test (OECD 484)
 Now extremely difficult to perform because the appropriate
mouse strains are no longer available (lost);
 It is a high tier assay in terms of risk assessment/hazard
classification;
 Often now replaced by Big Blue or Mutamouse
 An in vivo test in mice in which developing embryos are
exposed to a test agent.
 The target cells are melanoblasts;
 The target genes are those which control the pigmentation
of the coat hairs;

Rare Assays

 Mouse Spot Test (OECD 484)
 The developing embryos are heterozygous for a number of these
coat colour genes;

 A mutation in, or loss of (by a variety of genetic events), the
dominant allele of such a gene in a melanoblast results in the
expression of the recessive phenotype in its descendant
cells, constituting a spot of changed colour in the coat of the
resulting mouse.

 The number of offspring with these spots, mutations, are scored and
their frequency is compared with that among offspring resulting
from embryos treated with the solvent only. The mouse spot test
detects presumed somatic mutations in foetal cells.

Figure 2Defects in melanocyte development cause white spotting, while the
stem cell defect results hair graying.

(A) A Ednrbs-l/Ednrbs-l mouse demonstrating extensive piebald spotting. (B) A
KitW-2J/+ mouse demonstrating a white head blaze, and small dorsal spot on the
back. (C) A Sox10Lacz/+ mouse exhibiting the characteristic white belly spot. (D)
A Mitfvit/vit mouse (upper) exhibits gradual hair graying. A lower mouse is age-
matched control. Adapted from the WEB site of the European Society for
Pigment Cell Research (ESPCR), “Color genes”: http://www.espcr.org/micemut/.

Rare Assays

 Mouse Heritable Translocation Assay (OECD 485)
 This is a particularly useful assay because it is an in vivo
measure of germ cell macro-chromosomal damage;
 Currently there is no commonly used in vivo equivalent to this
assay;
 There are 2 forms of the assay:
 Detection of changes in fertility of the F1 progeny of
exposed animals;
 Cytogenetic examination of the F1 progeny of exposed
animals;

Rare Assays

 The assay primarily measures the frequency of structural
chromosome assays that do not result in a loss of
genetic material i.e. reciprocal translocations;

 Reciprocal translocations = fetal survival + no
malformations;

 Non-reciprocal translocations usually mean fetal death;

Rare Assays

 Reciprocal translocation, however, result in a loss of fertility in
the F1 generation due to the production of unbalanced
chromosomes in the gametes (i.e. unbalanced
translocations);

Rare Assays

 Insect and yeast OECD assays: rarely if ever used. You will
occasionally come across them in historical data sets.

Treat them with caution!

Other Common Genotoxicity Assays:
The comet assay

 = Single Cell Electrophoresis Assay;

 Simple, reliable and particularly useful!;

 Well accepted assay;

 Can also be used as a very simple, but effective and
accurate assay of DNA repair;

Other Common Genotoxicity Assays:
The comet assay

 Principle:
 Measures DNA strand breaks (i.e. clastenogenesis);
 Can be done in vitro or in vivo;
 Can be used for specific tissues or even specific cells;
 Cells embedded in agarose on a microscope slide are lysed with
detergent and high salt to form nucleoids containing supercoiled
loops of DNA linked to the nuclear matrix;
 Electrophoresis at high pH results in structures resembling comets,
observed by fluorescence microscopy;
 The intensity of the comet tail relative to the head reflects the
number of DNA breaks

A Few Slides of Gratuitous Silyness

After 228 power point slides on genetic toxicology, I figured you
were entitled…..

Before Genetic Tox After Genetic Tox

Nicnas gen tox3

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