David Kirkland

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In the early stages of drug development it is necessary to obtain sufficient safety data to be able to dose healthy volunteers and patients, but not spend large amounts of time and money when it is not known if the drug (a) is bioavailable, (b) is effective.

Carcinogenicity studies take 3 years to complete and cost up to $2,000,000 so surrogate information from genotoxicity studies is obtained in early development

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Many industrial chemicals, food additives, household products, cosmetics etc. are not manufactured in sufficient quantity to require carcinogenicity testing◦ Genotoxicity testing provides valuable information on likely

cancer risk for these substances◦ Cosmetic ingredients cannot now be tested in animals in the

EU Agrochemicals usually are tested for carcinogenicity,

but screening for genotoxicity provides information at an early stage on whether the substance should be developed

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Initiation event for many tumours is a mutation in one or a few DNA bases

There are 4 main types of gene involved in cell division. Most tumours have faulty copies of >1 of these:◦ oncogenes (e.g. Harvey-ras, c-MYC, c-ABL)◦ tumour suppressor genes (e.g. p53, retinoblastoma gene, Wilm’s

tumour gene)◦ suicide genes◦ DNA-repair genes

Other conditions believed to originate from mutation (atherosclerosis, inborn errors of metabolism)

Tumour progression involves loss/gain of chromosomal material

Many spontaneous abortions and birth defects result from chromosome loss/gain

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Mutations: What Do They Do?

Human Impact

CV diseases e.g. atherosclerosis

Aging

Teratogenesis - disrupted development

Decreased fertility foetal wastage

CancerHeritable defects

inborn errors

genetic susceptibility to disease

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No single test can measure all types of genotoxic damage

Battery of in vitro tests for hazard identification◦ mutation in bacteria (Ames test)◦ mutation in mammalian cells (mouse lymphoma assay)◦ chromosomal aberrations (or micronuclei) in cultured human or

Chinese hamster cells Test to high concentrations/extreme conditions

◦ can lead to artefacts Include metabolising mixture (rat liver S9) to mimic

mammalian liver metabolism

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Bacteria and established cell lines do not contain the enzyme systems which, in mammals, transform many mutagens and carcinogens to DNA-reactive electrophiles

A post-mitochondrial 9000xg supernatant (S9) from the livers of rats induced with Aroclor 1254 or phenobarbitone/ß-naphthoflavone is used◦ supplemented with co-factors and an energy source

S9 contains monooxygenases, oxidases, amidases, esterases, acyl and methyl transferases, dehydrogenases, peroxidases

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Battery of in vivo tests available for hazard characterisation and to check there are no unique in vivo effects (usually 1 or 2 from the following)◦ chromosomal aberrations (usually as micronuclei) in rodent

bone marrow, blood or liver◦ induction of DNA repair (UDS) in hepatocytes of treated rats◦ DNA damage (e.g. Comet assay) in appropriate tissues◦ DNA adducts in appropriate tissues◦ mutation in target genes of transgenic animals (e.g.

MutaMouse)

Although the genotoxicity tests we use are sensitive, they can give “false negative” results if robust protocols are not followed

They can also give “misleading positive” results (discussed later)

Decision making therefore revolves around several key questions

Are negative results a reliable indicator of lack of hazard?◦ Limitations of test system or protocol

Are positive results a true indicator of hazard, or are they misleading?◦ Artefacts◦ Thresholds

With mixed positive and negative results, which is indicative of true properties of chemical?

Has appropriate follow-up testing been done to resolve questionable data?

If a “true” positive, do exposures fall below a threshold of toxicological concern (TTC)?

If a threshold mechanism, is there an acceptable safety margin?

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Reversion assay – bacteria already mutant at a locus whose phenotypic effects are easily detected◦ Detect reversion from growth-dependence on a particular amino

acid to growth in its absence (auxotrophy to prototrophy) Genetic target is small, specific and selective Several bacterial strains with different markers are required

to accommodate mutagen specificity Sensitivity increased by addition of several other traits◦ DNA repair deficiencies◦ Increased permeability of the cell wall to bulky hydrophobic

chemicals◦ Introduction of plasmids that confer increased susceptibility to

mutation without concomitant increase in sensitivity to lethal effects of chemical

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Need basic 4 strains of Salmonella typhimurium (G-C sites) plus 1 or more strains to detect mutagens acting at A-T rich sites:◦ TA1535, TA1537 (or 97 or 97a), TA98, TA100 ◦ plus either TA102 or E. coli WP2 uvrA or E. coli WP2 uvrA

pKM101 Each strain detects different effect (see next slide)◦ +ve only in 1 strain indicates hazard

Plate incorporation and pre-incubation methods available(some unique mutagens with pre-inc.)

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Typically test up to 5000 μg/plate◦ Can test insoluble concentrations as long as ppt does not interfere with

scoring - useful for detecting mutagenic impurities 3 replicate plates per concentration At least 5 concentrations with colonies to score With and without S9 mix In most cases results need to be confirmed:◦ not identical repeat◦ can change conditions (e.g. from plate incorporation to pre-incubation)

or concentrations (of chemical and/or S9)◦ if full range-finder is done (with full revertant counts and positive

control) plus main experiment, this is acceptable◦ for pharmaceuticals now proposed single expt. only

For pharmaceuticals, Ames test required even for anti-microbial substances (mutagenic nitrofurans)

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Overlay Onto Minimal Agar

Bacterial Culture

Test Article Solution

S9 Mix or Buffer

Molten Soft Agar(+ his or tryp)

Mix

Incubate for 2-3 Days

Score Colonies Using Automated Counter

37oC

Number of colonies = 7

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Bacteria need a trace of histidine (or tryptophan) to undergo a few divisions after treatment in order to “fix” the mutations◦When amino acid used up only mutants continue to grow

If test substance contains/releases his or tryp, treated bacteria undergo more divisions than controls before amino acid supply exhausted◦ Each division has a defined chance of a spontaneous mutation,

therefore more mutants on treated than control plates, but these result from “feeding” and not from interaction with DNA

If “feeding effect” is suspected, can do “treat and plate” test where chemical is washed out after (say) 1 hour treatment

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Weak responses by plate incorporation◦ revertants <3-fold (often used for TA1537 positives)◦ statistical significance at 1 conc. in Expt. 1, & 3 concs. in Expt

2, but no dose response in Expt. 2◦ probably not considered biologically relevant

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Weak responses by plate incorporation◦ revertants <3-fold (often used for TA1537 positives)◦ statistical significance at 1 conc. in Expt. 1, & 3 concs. in Expt 2,

but no dose response in Expt. 2◦ probably not considered biologically relevant

Massive +ve response by pre-incubation◦ anthraquinone class, so might expect TA1537 response, but why

not with plate-incorporation?◦ inhibited by agar or short half-life metabolite?

Single expt. by plate incorporation would have “missed” problem

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L5178Y mouse lymphoma cells, heterozygous for the thymidine kinase (tk) gene i.e. tk+/- ◦ Inactive gene on chromosome 11a, active gene on 11b

Cells with active tk gene express the enzyme which can convert trifluorothymidine (TFT) to a lethal form

If the active tk+ gene is mutated to tk- (i.e. the cells become tk-/-) the cells are not killed by TFT◦ Simple selective system

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Point mutations Intragenic deletions Deletion of the tk+ allele Deletion of the tk+ allele and mitotic nondisjunction of

11a Translocations involving 11b Mitotic recombination and gene conversion also

postulated

This is the range of genetic alterations found in tumour cells

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Test up to 10 mM or 5000 µg/ml◦Maximum of 1 insoluble concentration because cells grow in

suspension and ppt cannot be removed Usually 4 concentrations with duplicate treatments

per concentration◦Need to test more concentrations if single replicates

With and without S9 (3-6-hour treatments) plus a 24-hour treatment without S9◦ Latter needed to detect nucleoside analogues and aneugens

where exposure for full cell cycle required

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Large colonymutant

Smallcolonymutant

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CA can be measured in cultured cells (established cell lines or primary cells) in vitro or in certain cells/tissues in vivo

Damage only visualised when chromosomes are visible (during metaphase stage of mitosis or meiosis), therefore cells need to be dividing, or be made to divide after treatment

Metaphase chromosomes can appear in many different ways, therefore extensive training to distinguish abnormal chromosomes from all the different ways normal chromosomes can appear

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Treat exponentially growing cells for short (3-6 hr) and long (e.g. 20-24 hr) periods◦ Short treatments - and + S9 but longer treatments only -S9 (prolonged

exposure to S9 is toxic) Sample 1.5 cell cycles after start of treatment (plus delayed

sample in certain circumstances) Usually 3 concentrations (up to 10 mM or 5000 µg/ml) with

scorable cells Score visible damage in metaphase chromosomes◦ 100 cells/replicate, 2 replicates per concentration

Need to achieve at least 50% toxicity (currently)

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MN are fragments or whole chromosomes not incorporated into daughter nuclei ◦ Enveloped in nuclear membrane, they look like a “micro”

“nucleus” Can be determined in vitro or in vivo Cells need to have divided either during or after

treatment MN are quicker and easier to score than CA◦ Less training, easier to automate, more cells per sample can

be scored

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Important to know cells have divided (negative result otherwise questionable)

Common to use cytochalasin B (essential for blood cultures) which blocks cytoplasmic division◦ Cells that have divided therefore have 2 nuclei

All the cell types used for CA can be used for MN Treatment period same as for CA, but sample slightly

later ◦ Cells need to progress to next interphase i.e. beyond the

metaphase that would have been scored in CA

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In 2005 we published analysis of correlations between in vitro genotoxicity and rodent carcinogenicity results for >900 chemicals [Mutation Research 584 (2005) 1-256]◦ 553 rodent carcinogens had genotoxicity results◦ 177 chemicals that were -ve for tumours in male and female,

rats & mice had genotoxicity results◦Ames + MLA + in vitro chrom abs (CA) or MN was the

genotoxicity battery we studied◦ Sensitivity was high, particularly when tests were combined

(>90%)

Sensitivity of individual tests ranged from 60-80% This improved to 80 to >90% if tests combined in

pairs and if either test was positive There was a marginal increase if 3 tests were

combined in a battery High sensitivity for the in vitro tests in terms of

detecting in vivo genotoxins (not in the carcinogens database) has also recently been shown

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Ames MLA MN CA

% sensitivity (+ve with carcinogen)

No. of chemicals 542 246 89 353

Specificity in the Ames test was reasonable (>70%) but in the mammalian cell tests was poor (only 35-45%)

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Ames MLA MN CA

Clear -ve

% specificity (-ve results with non-carcinogens)

No of chemicals 176 105 26 136

Specificity in the Ames test was reasonable (>70%) but in the mammalian cell tests was poor (only 35-45%)

Specificity deteriorated (10-35%) when tests combined in pairs, because both tests needed to be negative ◦ i.e. >2 in 3 chance of wrong prediction

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Ames +MLA

Ames +MN

Ames +CA

MLA +MN

MLA +CA

Clear -ve

% specificity (-ve in both tests)

No. of chemicals 105 25 136 20 96

Specificity in the Ames test was reasonable (>70%) but in the mammalian cell tests was poor (only 35-45%)

Specificity deteriorated (10-35%) when tests combined in pairs, because both tests needed to be negative ◦ i.e. >2 in 3 chance of wrong prediction

Specificity was very poor when 3 tests battery was used (<25%)◦ i.e. >3 in 4 chance of wrong prediction

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Ames + MLA + MN Ames + MLA + CA

Clear -ve

No. of chemicals 20 96

% specificity (-ve in all 3 tests)

Sensitivity/specificity trends with in vitro assays

Black = actual; red = desirable

Comments that the chemicals in the Gold database are not representative of e.g. new pharmaceuticals◦Matthews et al (2006) confirmed findings with a wider

database and using conservative weight of evidence criteria to classify chemical carcinogens

15% of non-carcinogenic pharmaceuticals in PDR are +ve in CA (Snyder & Green, 2001)◦How many compounds were dropped from development

because of +ve CA or MLA results that would also have been non-carcinogenic?

804 mammalian cell studies submitted between 1995 and 2005

(testing of 596 compounds)

242 MLA(30%)

161 CHO(20%)

71 V79(9%)

50 CHL (6%)

280 huly(35%)

219 of 804 studies positive = 27%181 of 596 compounds positive in

at least 1 in vitro clastogenicity test = 30%

Comparison of rate of positives among the cell systems currently in use

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huly MLA CHO V79 CHL

n =71

n =70

n =42

n =18

n =18

Data kindly provided by Peter Kasper, BfArM

The high false +ve rate, particularly with the rodent cells, is unacceptable◦ Lack of functional p53, impaired DNA repair and karyotypic

instability are probably key factors There is some evidence that human lymphocytes may be

less prone to false +ves◦ p53 expression increases dramatically when blood cultures

stimulated with PHA◦ Currently limited to measuring toxicity by reduction in mitotic

index (affected by cell cycle disruption as well as lethal events) Some early data from BfArM and Galloway’s group

suggested primary human lymphocytes less susceptible to misleading positives than hamster cell lines

Comparison of V79, CHO, CHL, human lymphocytes, TK6 and HepG2 cells with 19 “false positive” non-carcinogens

8 were negative (i.e. published positive results not confirmed)

11 were positive in 1 or more of the p53-deficient hamster cells, but negative (or very weak) in the p53-competent human cells

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V79 3+21 hours -S-9CHL 3+21 hours -S-9CHO 3+21 hours -S-9HuLy 3+21 hours -S-9HepG2 3+45 hours +S-9TK6 3+21 hours +S-9

** *** p≥0.001

******

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p≥0.01

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V79 24+0 hours -S-9CHL 24+0 hours -S-9CHO 24+0 hours -S-9HuLy 24+0 hours -S-9TK6 24+0 hours -S-9

** *** p≥0.001

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low cell numbers

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CHL 4-nitrophenol

CHL Eugenol

CHL Propyl gallate

TK6 4-nitrophenol

TK6 Eugenol

TK6 Propyl gallate

HLM 4-nitrophenol

HLM Eugenol

HLM Propyl gallate

p≥0.001

**p≥0.05p≥0.01

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TK6 4-nitrophenol

TK6 Eugenol

TK6 Propyl gallate

HLM 4-nitrophenol

HLM Eugenol

HLM Propyl gallate

****

p≥0.001

**p≥0.05p≥0.01

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CHL 4-nitrophenol

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TK6 4-nitrophenol

TK6 Eugenol

TK6 Propyl gallate

HLM 4-nitrophenol

HLM Eugenol

HLM Propyl gallate

***

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** ****

p≥0.001

**p≥0.05p≥0.01

***

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Does it make a difference where cells originate? Are there differences between batches of the same

cells? How stable are cell stocks over time in culture? Do we need to make sure we are all using the same

cells?

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One of the 19 “false +ves” not confirmed in CHO, CHL, V79 etc. However, published +ve was in MLA.

Confirmed +ve in L5178Y cells (MN induction) with cells that are commercially available from ATCC.

Repeated with “Don Clive” cells (sourced from AZ) used for routine MLA assays.

Very different results seen ATCC cells showed toxicity and MN increases Don Clive cells non toxic and negative for MN Anthranilic acid is cell line- and source-specific +ve

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ATCC L5178Y

Don Clive L5178Y

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p≥0.001

**** p≥0.05

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Typically test to 1400 µg/mL (10mM), top dose in this case (55% toxicity) around 350 µg/mL (2.5 mM)

Very different toxicity and MN responses depending on source of cells.

Both same clone TK+/- 3.7.2c Differences in karyotype also seen, ATCC cell line had

very high frequency of translocations making identification of chromosomes almost impossible.

FISH painting showed ATCC derived stocks had ~30% cells with 3 copies of Chromosome 11

In this lecture I have attempted to highlight some problems with some of the in vitro tests

In the next talk I will present further examples of problems with in vitro tests, but also discuss in vivo tests and some issues to be considered there.