Using Mammalian cells to

Screen for Hazard

Cell killing & growth inhibition
Flasks, microplates, microscopy, flow cytometry

Genotoxicity
Carcinogenesis, mutation induction, chromosome
damage, DNA damage

Lots of different assays for

different cellular endpoints
Contact Barbara.sanderson@flinders.edu.au 8204 5788

What kinds of changes

might be detected
at the cellular level?
Interested in effect on us
therefore want as close to testing
people as possible without using
humans or other animals

Changes that can occur to/in cells

Programmed cell death (Apoptosis)
Necrosis
Disrupted metabolism
Disrupted mitosis
Loss of whole chromosomes
Chromosome breakage crossing over
DNA deletion
DNA base-pair changes

Screening for Chemical or

Environmental Hazard
Complex mixtures = mixtures of chemicals

Multiple damage mechanisms

Multiple damage endpoints

Suite of bioassays

Range of changes = Human cell

phenotype & genotype changes
Phenotype
Severe and Moderate: Some examples?

Genotype
Chromosome level and DNA level:
Some examples?

Detect if the hazard

= Toxic to human cells
Flasks (death and chronic growth inhibition)
Microplates (acute cell death)
Microscopy & flow cytometry assays (apoptosis)
Commercial kits

Cytotoxicity
Induce cell death
metabolism stops

Inhibit population growth

cell division stops

Induce apoptosis

Carrying out the assays

Use aseptic technique
UV light sterilisation
70% MeOH washing
not talking
using laminar flows correctly

cell self-destructs

Haemocytometer

Survival in flasks
Use aseptic technique
Supply nutrient media
Some need growth factors
particularly normal cells eg. IL2 for
t-lymphocytes

Use a measure of viability

eg. Active dye exclusion - Trypan blue - dead
cells become leaky due to increase in plasma
membrane permeability and hence stain blue

http://homepages.gac.edu/~cellab/chpts/chpt1/Images/fig1_8.gif

Assaying toxicity in microplates

Survival of cells
Ability to proliferate and form clones
One 96-well microplate per treatment
condition
Plate 1-10 cells/well
Use inverted microscope to score wells for
growth
Positive well

Negative Well

What type of cell killing?

Example of an Apoptosis Assay

(Prepare slides, stain and score)
Other Apoptosis assays
are for DNA
fragmentation
(laddering on
electrophoresis) or of
specific enzymes
involved in apoptosis
or of cell markers
using flow cytometry

120
6 hr
24 hr

100

48 hr

Detects presence of metabolic activity of

mitochondria
Treat cells and incubate at 37oC
Plate out cells in 96-well microwell plates
Add MTT dye, incubate then add SDS
Read in an ELISA plate reader
Convert OD readings to viable cells/well (read off
std curve with known numbers of cell/well)
Convert to % survival relative to untreated control

MTT assay of Titanium Dioxide <100 nm

80
Cell Viability (%)

Assaying for acute toxicity using

the MTT Assay

**

60
40
20

*
**

0
0

26

65

130

Dose of particles (g/ml)

Used in the production of paints, paper, plastics

and welding-rod-coating material or in cosmetics

Apoptosis

Apoptosis
Some of the kits available from OncogeneTM:
Suicide Track DNA Ladder isolation
Annexin flow cytometry for externalized
phosphatidylserine
Nucleosome ELISA
Mitocapture (mitochondrial membrane potential)
Caspase-3 Assays flourometric/colourimetric
(Also see the Roche site on the Web)

Detection of hazards that =

Genotoxic to mammalian cells

Unscheduled DNA Synthesis

In vitro assays
Increased DNA repair (Unscheduled DNA
synthesis)
Cytogenetic (SCEs, Micronucleus &
chromosome aberrations)
DNA breakage (Comet assay)
Mutation assays (HPRT & HLA)

DNA synthesis occurs in S-phase in cells in

culture
Screen for increased DNA repair not in Sphase as indirect indicator of DNA damage
Uses 3H-thymidine incorporation

Sister Chromatid Exchanges

SCEs

(Evidence of cross-over events - BrdU

incorporated into DNA then counterstain)

http://www2.mrc-lmb.cam.ac.uk/PNAC/Sale_J/images/js2.jpg

Induction of SCEs by arsenite

NaAsO2

SCEs (+SE)

0
M

100

3.24 (0.14)

2.5
M

92

5.23 (0.18)*

5
M

35

6.20 (0.41)*

N = number of metaphases analysed

* Significantly different from control group p<0.001
Arsenic is a metalloid found in water, soil and air.
Inorganic arsenic can be ingested as arsenite.

http://www.genetichealthindia.com/images/genotoxicity.jpg

Micronucleus assay
CBMN provides a readily measurable
index of chromosome breakage and loss
Micronuclei originate from either lagging
chromosomes or chromosome fragments
not included in main nucleus at anaphase
On same slides as score for micronuclei
can score for nucleoplasmic bridges as
evidence of formation of a dicentric
chromosome

Mourn et al., Toxicology in Vitro, 20: 279-285, 2006

Cytokinesis Block Micronucleus Assay

Stained cells from a Micronucleus assay

Micronucleus

By adding Cytochalasin-B, a cytokinesis blocking

agent, cells that have completed one nuclear division
accumulate and can be identified as binucleate cells
Chromosome Fragment
at anaphase

Micronucleus

Cytokinesis block

Bridge

a. Nucleoplasmic bridge arising from a damaged chromosome

having 2 centromeres, resulting in joining daughter nuclei
b. Binucleate cell with one micronucleus

Micronuclei induction by nanoparticles

(<100nM) from Iron Ore mines
16
14

0 g/ml
75g/ml

150g/ml

Frequency of MNBNCs

**

12

Chromosome Aberrations
Checking for abnormal karyotype
ie. altered chromosome number or shape

**

10
8
6
4
2
0
1002

HG1

HG2

HG4

Dust

Exposed cells to 0 control + two doses of dust from 4 sites

(10 hr exposure, 3 replicates Mean/1000 BN cells + SE)

Comet Assay Detects s- or d-strand

DNA breaks or alkali labile lesions

Mutation Assays
HPRT- Select for loss of purine salvage
enzyme using a purine analogue
straight forward to set up and score

HLA - Select for loss of cell surface

markers via antibody selection

Expose cells to different doses of damaging agent

(increasing from A-D), put in agarose on a microscope slide,
run electrophoresis, stain, visualise, computer analyse tail area

technically complex and time consuming to

carry out
was used for research not commercial screening

Basis of HPRT Assay is

DNA Synthesis Pathways
De novo
Synthesis

Wild type cell

De novo
Synthesis
DNA

DNA
Salvage
Pathway

Salvage
Pathway

WIL2NS
FH25
FH6
FH9
FH10

10

-5

Induced Mutant Frequency (x10 )

De novo

Cell division

GSTM1 deficient

GSTM1 positive

0
0.0

Salvage

6-Thioguanine

Mutation induction by Styrene Oxide

HPRT deficient mutant cell

DNA

Cell Death

Cell growth

0.5

1.0

1.5

2.0

2.5

SO Concentration (mM)

SO is used in the manufacture of epoxy resins, an intermediate in

the preparation of various agricultural chemicals, cosmetics, and
plastics and in the processing of textiles and fibers