Health Canada Genetic Tox Lecture Part 2

David Kirkland

Could a +ve in vitro result be explained by:◦ Production of reactive oxygen species by reaction between test

substance and culture medium?* [*next slide]

◦ Degradation of test chemical in medium?◦ Confounding effects of apoptosis or necrosis?◦ Possible effects on kinases?◦ Topoisomerase II or DNA synthesis inhibition?◦ Interference by stray UV light?◦ Nucleotide pool imbalance?◦ Metabolic overload?

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DMEM F-10 F-12 RPMI RPMI McCoys Williams Hepes 5A E

M of H2O2 produced

- + - + - + - + - + - + - + EGCG

Sig. clastogenicdose of H2O2

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McCoy's %CAF-10 %CA McCoy's % survivalF-10 % survival

% cells with CA (solid line) Relative cell count (%)(dashed line)

Incubation time (hr) of EGCG in medium before stopping reaction

McCoy’s 5A

F-10

Km for most biochemical reactions is <100 μM yet top concentration for testing is 10 mM◦ ICH will probably agree 1 mM acceptable for pharms but will

this be accepted for other chemicals? Recent review (Parry et al) identified 24 rodent carcs –ve

in Ames, only +ve in mammalian cells at >1 mM◦ Some are non-genotoxic carcs so not expected to be detected◦ 9 other chemicals re-tested, and either negative up to cytotoxic

concs or 10 mM, or positive at 2 mM (200 μg/ml) and below Proposal for new upper limit of 1 mM or 500 μg/ml,

whichever is the higher

Chemical Previous LEC New LEC

Allyl isovalerate 2.81 mM in MLA +ve at 0.55 mM in CA (3 hr +S9)

Benzofuran 1.27 mM in MLA -ve at in MLA up to 1 mM (64-99% toxicity ). Increased MF at 2 mM but almost 100% toxicity

Caffeic acid 1.11 mM in MLA +ve 0.4 mM in MLA (24 hr –S9) and 1 mM in CA (20 hr –S9)

Chlorobenzene 1.11 mM in MLA +ve at 0.6 mM in MLA (3 hr +S9)

Daminozide 13.75 mM in CA; 11.25 mM in MLA

-ve up to 10 mM (CA & MLA)

Furan 1.47 mM in CA +ve at 0.8 mM in MLA (3 hr +S9) and at 4 mM in CA (3 hr +S9)

Methylolacrylamide 2.94 mM in CA +ve at 2 mM in CA (20 hr –S9) and MLA (24 hr –S9)

Toluene 2.44 mM in MLA -ve up to toxic doses (10% RTG) in MLA (not tested in CA)

Monuron 6.54 mM in CA -ve in CA up to 4.25 mM which induced >50% toxicity (- and + S9)

Many different measures of cytotoxicity can be used and are acceptable under OECD◦ They may not all give the same conc for 50% toxicity◦ Greenwood et al (2004) showed several non-DNA reactive

clastogens were -ve if 50% toxic conc chosen by population doubling instead of cell counts

◦ Further evidence of this from COLIPA trial at Covance using in vitro MN test on 19 “false positive” chemicals

A rigorous comparison (collaborative trial) of different measures of cytotoxicity is needed◦ Cell counts, population doubling, replication index, RTG, ATP

levels, LDH leakage, Alamar Blue etc.◦ Rodent cells, human lymphocytes, TK6 etc.◦ Need measures other than mitotic index for lymphocytes

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0 59.469.982.296.7 114 134 158 185 218 249 257 302 355 389 486

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120CARel cell countRel PD

% cells with CASurvival measurerelative to control

Conc. of drug(g/ml)

50% toxicity

COLIPA trial has looked at relative increase in cell count (RICC) or relative population doubling (RPD) with 6 confirmed false +ves◦ Relative cell count (RCC) picked higher concs for 50%

toxicity than RICC or RPD and therefore more prone to give false positive results with non-carcinogens

Recent collaborative trial on in vitro MN test showed that range of 14 “real +ves” all detected as +ve if RICC or RPD used◦ No false -ves

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Concentration (µg/mL)

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Caspase 3/7

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*** p≥0.001

Negative

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*** p≥0.001

Bacterial-specific metabolites Nitroreduction Rat liver S9 In vitro specific effects

In addition compounds may disrupt non-DNA targets (e.g. spindle) and exhibit a threshold

Sodium azide is converted to mutagenic form (azidoalanine) by O-acetylserine(thio)lyase in bacteria

Mammalian cells express enzyme but azidoalanine is not formed◦ different sub-type of enzyme?◦ is azidoalanine rapidly converted to non-mutagenic

intermediate? Sodium azide is not mutagenic for mammalian cells

Bacteria are very efficient at nitroreduction Mammalian nitroreductase is oxygen-labile

◦ where intestinal bacteria may not be exposed, there may be no risk to humans

Metronidazole is bacterial mutagen (via nitroreduction) but -ve in mammalian cells◦ mammalian metabolism is via side chain (NO2 intact)◦ lung tumours not consistent with bacterial nitroreduction◦ is reduced metronidazole unable to pass from the bacteria

into surrounding mammalian tissue?

Rat may produce metabolites not produced by humans “Induction” of rat livers with Aroclor or phenobarbital/ ß-

naphthoflavone results mainly in elevation of Phase I (activation) enzymes rather then Phase II (detoxification) enzymes◦ CYP1A and 2B enzymes at 40x levels found in normal liver◦ In normal testing we do not add cofactors such as glutathione,

acetyl CoA (for acetylation), PAPS (for sulphation), or UDPGA (for glucuronidation) for detoxification pathways

Understanding relative importance of Phase I & II metabolism can determine whether +ve results may have threshold of detoxification which may not be exceeded under normal human exposure

Quercetin is clearly genotoxic in vitro◦ formation of active oxygen by redox cycling

All in vivo genotoxicity studies are -ve 10 carcinogenicity studies are -ve Humans take 50 mg/day in food In vivo quercetin is rapidly methylated via catechol-

O-methyltransferase, and redox cycling is negligible

Interaction with non-DNA targets DNA-reactive chemicals/metabolites at high

concentrations, but which, at low concs, are effectively conjugated and unable to form adducts

Repair mechanisms that enable cells to tolerate low levels of potentially mutagenic DNA adducts without biological consequences◦ Difficult to demonstrate and quantify, but some DNA-

reactive carcinogens have been shown to exhibit thresholds

Non-relevant mechanisms for +ves in vitro would not be expected to occur in animals in vivo, or only under certain conditions

Chemicals inducing DNA damage via a threshold mechanism can produce +ve results in vivo if a high enough exposure can be achieved

Safety evaluation of a threshold genotoxin is therefore quantitative rather than qualitative, and more difficult to manage without animal data

Enzyme induction Imbalance of DNA

precursors Energy depletion Production of active

oxygen species Lipid peroxidation Sulphydryl depletion

Nuclease release from lysosomes

Inhibition of protein synthesis

Protein denaturation Ionic imbalance High osmolality Low pH

* High cytotoxicity was not identified at this time

Inhibitors of DNA gyrase can also cause other changes both in bacteria and mammalian cells◦ Inhibitors of topo I or II do not bind to DNA but seem to

increase no. of strand breaks or prevent their resealing ◦ Activity can produce point mutations, but predominantly

chrom abs, recombination and aneuploidy Chemicals primarily inhibiting mammalian topo

enzymes (m-AMSA, etoposide) will be genotoxic at human exposures◦ Critical to determine whether primary or secondary effect &

concs at which topo inhibition occurs

Deoxyribonucleotide pool imbalances aberrant DNA replication/repair multitude of genetic effects - mutation, DNA breaks, chrom abs and MN

Should lead to genotoxicity without DNA interaction - should be a threshold

Methotrexate, other antifolate drugs, hydroxyurea and FUdR disrupt nucleotide triphosphate levels◦ Can effects be reversed by restoring nucleotide pools

balance? Chrom abs induced by excessive thymidine are reduced by

simultaneous addition of deoxycytidine ◦ If genotoxic response only partially reversed, 2nd (non-

threshold) mechanism may be involved?

Paracetamol (acetaminophen) inhibits ribonucleotide reductase, but genotoxicity probably due to NAPQI metabolite◦ NAPQI conjugates to glutathione◦ If conjugation saturated, chom abs & DNA damage induced

threshold of conjugation Other situations may be similar

◦ Determine conjugation threshold and absence of DNA adducts and genotoxicity below this level

◦ Compare threshold with human exposures and employ conservative safety factors

DNA-damage by various chemicals, or enhancement of others, accompanied by glutathione depletion ◦ Acrolein, chloropropanones, some acrylate esters, 1,3-butadiene,

isobutene, various metals, dichlorvos, diethylmaleate Obtain supporting evidence of threshold

◦ Look for different responses in different cell types with different constitutive levels of glutathione

◦ Add glutathione to treated cultures to see how the genotoxic response is modified or eliminated

Multi-fibre nature of mitotic spindle means it can function effectively even when experiencing some damage

Widely accepted that spindle inhibitors exhibit threshold (non-DNA target)◦ Identify induction of polyploidy or centromere +ve micronuclei◦ Non-disjunction between daughter nuclei is most sensitive

measure of threshold (Elhajouji, Marshall, Zijno)

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MN that contain whole chromosomes (that failed to attach to spindle) will contain centromeres

Can look for these using fluorescently-tagged DNA probe against 1 or all (pan) centromeres

Induction of predominantly centromere +ve MN indicates aneuploidy mode of action◦ Will have a threshold because results from damage to

spindle and not to DNA◦ Important in risk assessment and establishing safe levels of

exposure

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If no convincing experimental evidence of threshold or non-relevant mechanism, evidence of absence of direct DNA-mediated effect can help◦ Should be no DNA adducts if chemical interacts with non-DNA

target◦ Can 14C-labelled chemical bind to DNA of cells/bacteria at

concs. that are genotoxic? 24 hr treatments with “hot” chemical may not be possible

◦ 32P-postlabelling is more sensitive, but selection of solvents/conditions affected by type of adduct (but if no adduct expected????) How convincing is absence of spots?

Concept acknowledges human exposure threshold (including those of unknown toxicity) below which no significant human health risk exists

Based on analysis of carcinogenic potencies initially of 343 (and subsequently >700) chemicals from Gold et al database (CPDB)

FDA threshold of regulation for food contact materials determined that, for most carcinogens, lifetime exposure to 1.5 μg/day would increase cancer risk by 1 in 106

Gold database contained genotoxic and non-genotoxic carcinogens

ILSI Europe Task Group (Kroes et al, 2004) determined that for genotoxic chemicals (high potency carcinogens, structural alerts), TTC would be 0.15 μg/day for lifetime risk of 1 in 106

For drug substances, providing health benefits, argued that lifetime cancer risk of 1 in 105 would be acceptable, therefore TTC of 1.5 μg/day proposed for genotoxic impurities

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MN are usually measured in bone marrow cells Erythrocyte precursor cells at the time of their last

division are the target When the nucleus is expelled (to form an erythrocyte)

any chromosome fragments or non-incorporated whole chromosomes are left behind and appear as micronuclei

MN can also be measured in reticulocytes in peripheral blood

Centromeric probes can be used (aneuploidy)

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In vivo micronucleus assayIn vivo micronucleus assay

6-8 h6-8 h 18-22 h18-22 h

ErythroblastsErythroblastsErythroblastsErythroblasts

ImmatureerythrocytesImmatureerythrocytes

MatureerythrocytesMatureerythrocytes

Bone marrowBone marrowBone marrowBone marrow

Peripheral bloodPeripheral bloodClastogenClastogen

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Acute study most common for bone marrow◦ 1 administration with 2 sampling times (24 & 48 hr) or 2

administrations with 1 sampling time (24 hr) 5 animals/sex per dose group 3 dose levels if toxic (or 1 if non-toxic) 2000 polychromatic erythrocytes (PCE) per animal

scored for MN Ratio of PCE to normochromatic erythrocytes gives

measure of bone marrow toxicity Measure concentration of chemical in plasma

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Micronuclei can also be measured in young reticulocytes (CD71 expressing) in peripheral blood

Because spleen of many species removes micronucleated erythrocytes, need to score larger numbers of cells to detect a +ve effect◦ Successfully done for mice, rats, dogs & humans

This can be done by flow cytometry where 20,000 to 2 million cells per sample can be scored

Animals can be bled at different intervals (don’t have to sacrifice as for bone marrow – non-invasive) and pre-dosing bleeds provide additional control data

FITC

PI

488nm laser

NCE (No Fluorescence)

Reticulocyte (Green)

MN-Reticulocyte (Green + Red)

Platelet (Yellow)

Key

PE

Peripheral Blood MN MethodologyInside the Cytometer

EMS - 3 hr

Peripheral Blood MN Methodology

Data Output

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MN can also be measured in liver Surgery (partial hepatectomy) is needed to make

cells divide Alternative recommendation is to test in young rats

(before liver is fully grown) but metabolism is also not mature◦ Results not relevant for adult rats may be obtained

Techniques also being developed to measure MN in skin, colon, testes etc.

Howell-Jolly bodies are small nuclear remnants that have the colour of a pyknotic nucleus & contain DNA◦ Spherical in shape; usually only 1/cell but may be numerous

Usually removed by the spleen (present in blood after splenectomy & in hyposplenic state, in haemolytic anaemia, megaloblastic anaemia)

During normal erythrocyte maturation they arise from nuclear fragmentation (karyorrhexis) or incomplete expulsion of the nucleus

Stimulate erythropoiesis and increased karyorrhexis will lead to a transient increase in MN◦ erythropoiesis is a response to secretion of erythropoietin (EPO)

by the kidney

Administration of EPO Hypoxia leads to secretion of EPO

◦ iron deficiency◦ thyroid dysfunction◦ thyroid, adrenocortical and human growth hormones◦ hypothermia? - reduced O2 uptake by Hb?◦ hyperthermia? - cancer patients on hyperthermic therapy

show increased EPO

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MN %

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In MN test at 200 mg/kg, body temperature dropped from 37oC to 24oC within 6 hr

Temperature was still as low as 29oC at 48 hr 10oC decreases in temperature for several hours,

induced by reserpine & chlorpromazine, have resulted in 2-3-fold increases in MN at late sampling times

Hypothermia rescue study therefore performed with CNS Drug X

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Most widely used is the single cell gel electrophoresis (Comet) assay◦ Measures DNA damage as it migrates from lysed cells in an

electrical field (electrophoresis) - the more damaged the DNA, the more of it and the further it migrates

◦ Any cell culture, or any tissue from which single cell suspensions can be made can be used

Cells do not have to be dividing By using alkaline conditions any lesions in DNA are

converted to strand breaks

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• Cells mixed with agarose

• Spread on microscope slides

• Lysis

• Unwinding (pH): expresses different types of DNA damage as strand breaks – next slides

• Electrophoresis

• Neutralization

• Image analysis: Automated, Manual

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More SBs Smaller DNA fragments

Increased DNA migration

Unwinding DNA at different pH expresses different types of DNA damage as strand breaks (SBs), resulting in fragments of DNA. ◦ 7-8 - Double Strand Breaks, Crosslinks◦ 12.1 - Strand Breaks, Excision-repair Sites, Crosslinks ◦ >13 - Strand Breaks, Alkali-labile Sites, Excision-repair Sites,

Crosslinks These fragments of DNA are then subjected to

electrophoresis and the DNA migration pattern analysed.

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Data Collected by Image Analysis

• Length of DNA migration (smallest detectable DNA)

• % Migrated DNA (amount of migrating DNA)

• Olive Tail Moment(migrated DNA x tail length)

Head Tail

Length of DNAmigration

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Cells with DNA migration Cells without DNA migration

Correlation of in vivo Comet assay results with rodent carcinogenicity very high◦ Particularly useful with compounds negative in bone marrow

MN study (Kirkland & Speit, 2008) Unclear whether DNA strand breaks can result from

cytotoxic effects Most published results with in vivo Comet assay from 1

lab, therefore consistency and ability to “travel” not well established

Collaborative trials on-going to try to establish OECD guideline

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Mice have been engineered to carry a bacterial lacI or lacZ transgene on a lambda bacteriophage vector

Multiple copies of the transgene exist in each cell The transgene is neutral and confers no advantage or

disadvantage to the mouse After treatment of the mice, the DNA is extracted,

“packaged” into viable lambda bacteriophage, which are assayed on sensitive bacteria where mutations in the transgene can be detected

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MUTATION IN TRANSGENES

Treat mice

take tissues

isolate DNA

packagecolour selection (LacI)

positive selection (LacZ)

(cut at cos sites:1 lambda vector per phage)

1 lacZ or lacI gene

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Cohesive

end

Bam

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amH

1Bam

H1 B

amH

1XH

ol

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coR1

EcoR

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end

LacZ

0 50 kb

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The lambda phage will lyse the sensitive bacteria and form plaques

By growing the bacteria on agar containing a particular substrate for the ß-galactosidase enzyme, mutant plaques can form a colour whereas wild-type plaques are colourless

For the lacZ system a positive selection system has been devised which means wild-type phage do not form plaques (reduces size of assay)

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MutaTMMouse (lacZ) and Big Blue (lacI) most widely used Treatment period is usually 28 days (followed by further

3-day expression period) to accommodate slow-acting mutagens◦ Mutations from fast-acting mutagens plateau

At least 3 dose levels and 5 animals/sex/group Mutations can be detected in any tissue, therefore useful

for site-of-contact mutagens (skin, GI tract, lungs) Limitation - not sensitive to chemicals inducing large DNA

deletions (delete through cos sites; will not package)◦ Gpt-delta or spi mutation models (see papers by Nohmi et al) are

able to detect deletion mutations

Is there a credible mechanistic/metabolic reason why +ve results in vitro are expected to have threshold, or only occur in conditions to be irrelevant for human exposure?

Are there -ve results from 2 appropriate in vivo tests?◦ need toxicokinetic data to relate exposure to the genotoxic

concentrations in vitro and to human exposure Are exposure (or safety) margins acceptable?

◦ human plasma Cmax compared with highest no-effect concentration (NOEC) in +ve in vitro test(s)

◦ human exposure (area under conc. curve, AUC) compared with AUC in -ve in vivo tests

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CRITERIA FOR POSITIVES

•Statistical significance at 1 or more dose pointssensitive e.g. p<0.01 for 1.4-fold increase in Ames mutants

(Dunnett's test) •Fold increase over control

conservative e.g. get significant dose responses without breaching pre-set level

•Frequencies exceed historical control rangegood for low frequency events (chromosomal aberrations,

micronuclei), not so for Ames or tk mutations•Frequencies exceed pre-set level

Global evaluation factor used for mouse lymphoma assay•Dose response

probably most relevant in terms of biological effect•Reproducibility

would be required for publication in peer-reviewed journal

I hope I have been able to show that decision making with genotoxicity data is not easy

There are many factors involving study design and potential for artefacts that need to be considered in order to understand whether a test result truly indicates lack of, or potential for, human hazard

Thank you for your attention