Download The BALB/c 3T3 cell transformation assay to assess the

The BALB/c 3T3 cell transformation assay to assess the
carcinogenic activity of chemicals
Annamaria Colacci1, Maria Grazia Mascolo1, Stefania Perdichizzi1, Francesca Rotondo2, Elena Morandi2, Angela Guerrini3, Antonio Gazzilli1, Paola Silingardi1,
Sandro Grilli3 and Monica Vaccari1
1
Environmental Carcinogenesis and Risk Assessment, Environmental Protection and Health Prevention Agency Emilia-Romagna region (ER-EPA), Bologna, Italy
2 Interdepartmental Centre for Cancer Research “G. Prodi”, University of Bologna,Italy
3 Department of Experimental Pathology-Cancer Research Section, University of Bologna, Italy
The new EU regulation for chemicals, REACH, specifically requires the development of alternatives in order to reduce and eventually replace vertebrates
studies.
At the moment cell transformation assays performed on rodent cell lines (BALB/c 3T3 or C3H10T1/2) or primary cells from Syrian Hamster are regarded
as possible in vitro alternatives to animal testing for carcinogenesis studies.
Cell transformation assays (CTAs) have been proposed as screening tests for the carcinogenic potential of compounds that have no evidence of
genotoxicity and are listed among the REACH methods, as reported in EU regulation 440/2008.
THE STUDY
The BALB/c 3T3 transformation assay
For the last 20 years we have been testing many chemicals and complex mixtures by using
BALB/c 3T3 A 31 cells in different experimental protocols.
In the prevalidation study organized by ECVAM an improved protocol has been developed
that was based on BALB/c 3T3 A31-1-1 cells.
The present study was performed in the aim to compare the results obtained with the two
different clones. Cells were treated with PAHs (3-MCA, B(a)P), and aloethanes (1,2-DBE 50
µg/ml). The induction of cytotoxicity and the onset of chemically transformed foci were
evaluated by two different experimental protocols.
RESULTS
BALB/c 3T3 A31-1-1 – clonal efficiency - protocol II
mean colony number ± SE
150
The BALB/c 3T3 cells
BALB/c 3T3 A31
The original stock of BALB/c 3T3 cells, clone A31, was obtained from
the American Type Culture Collection. Cells were grown in Dulbecco's
modified Eagle's medium (D-MEM) supplemented with 10% Newborn
Calf Serum (NCS). Cells were frozen in a 10% DMSO and 90% NCS
solution.
MCA
BaP
100
**
**
**
50
**
**
**
0
0
0.5
1
mean colony number ± SE
The BALB/c 3T3 transformation assay, which is based on the
malignant transformation of immortalized embryonic mouse
fibroblasts, is one of the most commonly used CTAs.
BALB/c 3T3 cells are aneuploid, contact- inhibited cells able to grow
as a monolayer culture until confluent.
The chemical transformation of 3T3 cells results in the induction of
morphologically aberrant foci, shaped with cells that do not stop
proliferating at confluence but grow over contact-inhibited normal
cells.
Only foci that show basophilic dense multilayering of cells, random
orientation at the focus edge, invasion into the surrounding contactinhibited monolayer and domination of spindle-shaped cells are
recognized as positive transformed foci.
1,2-DBE
150
A dose-related reduction
of the clonal efficency was
observed after the
treatment with PAHs.
DBE did not exert any
toxic effect.
**
**
100
50
0
2.5
0
10
µg/ml
25
µg/ml
50
BALB/c 3T3 A31-1-1 – clonal efficiency - protocol I vs II
BALB/c 3T3 A31 1-1
protocol I
150
Day 4
Day 10
carcinogen
30
protocol I
25
1
0
10
25
DBE was toxic only when
cells were treated for 72 h.
50
1,2-DBE (µg/ml)
protocol II
**
protocol II
**
**
15
10
**
*
5
The dose-related
transforming ability of MCA
was identified, regardless of
the utilized protocol.
DBE did not induce any
significant increase in the TF
of A31-1-1 cells, so it was
not classified as a carcinogen
in this system.
protocol I
2
1
0
0
0
0.5
1
0
2.5
10
25
50
1,2-DBE (µg/ml)
3-MCA (µg/ml)
Day 1
**
0
2.5
20
TRANSFORMATION TEST
0
0.5
**
50
BALB/c 3T3 A31-1-1 – transformation frequency - protocol I vs II
fixing &
staining
seeding
(250 cells)
** **
3-MCA (µg/ml)
-4
Day 1
**
100
RCE (%)
RCE (%)
0
TF (X 10 )
CYTOTOXICITY TEST
**
0
PROTOCOL I
0
**
**
TF (X 10 -4 )
The experimental protocols
protocol II
100
50
The high susceptibility of
A31-1-1 cells to the
cytotoxicity induced by MCA
was confirmed by both
protocols.
protocol I
150
protocol II
The BALB/c 3T3 cells, clone A31 1-1, were originally selected for their
susceptibility to chemicals and ultraviolet light. The cell line was
obtained from the Health Science Research Resource Bank (Osaka,
Japan) and was grown in Minimum Essential Medium (MEM) with
10% Fetal Bovine Serum (FBS) . Cells were cryoconserved in MEM
10% FBS solution.
Day 28-35
Day 4
carcinogen
seeding
(1 x 104 cells)
medium changes
fixing &
staining
BALB/c 3T3 A31 - DBE - protocol I vs II
CLONAL EFFICIENCY
150
protocol I
TRANSFORMATION ASSAY
25
protocol II
protocol I
*
**
**
50
** **
TF (X 10-4)
100
**
**
**
15
10
**
5
0
protocol II
**
20
RCE (%)
In the originally recommended protocol, cells were seeded at 1x 104
cells/60 mm dish and exposed to chemicals in the culture medium for
72 h. At the end of the exposure, the treatment medium was
replaced with complete medium and the cultures were maintained for
a further 4–6 weeks to allow the expression of transformed foci
(Kakunaga, 1973; IARC/NCI/EPA Working Group, 1985; OECD,
2007).
**
**
0
0
24
48
1,2-DBE (µg/ml)
94
188
0
24
48
1,2-DBE (µg/ml)
94
188
The A31 cell
transformation assay
judged DBE as positive.
The classification of DBE as
a carcinogenic compound
did not depend on the
seeding density or on the
duration of the treatment
PROTOCOL II
CYTOTOXICITY TEST
0
Day 2
Day 4
Day 10
carcinogen
fixing &
staining
seeding
(250 cells)
TRANSFORMATION TEST
0
Day 2
Day 28-35
Day 4
carcinogen
seeding
(3 X1 x 104 cells)
medium changes
fixing &
staining
Aiming at reducing the toxicity of the chemical treatment, in the
modified protocol suggested by Matthews et al., the number of
seeded cells was increased from 1x104 to 3x104 per dish and the cell
treatment started two days later and lasted 48 h instead of 72 h
(Matthews et al., 1993a; Mascolo et al, 2010).
The BALB/c 3T3 A31 cells and the derived cell line A31-1-1 differed in
the response to chemicals, probably because of the different
metabolizing capacity.
The A31-1-1 cells showed a higher inherent transformation rate after
PAH treatment, but they were insensitive to 1,2-DBE.
As DBE is bioactivated to reactive forms able to bind DNA mainly
through the conjugation with intracellular glutathione (Guengerich,
2003), these results suggested a reduced activity of phase-2 enzymes
involved in gluthatione conjugation in A31-1-1 cells.
Our results seem to suggest that in vitro cell transformation protocols
performed under REACH regulation should take in account the different
sensitivity of BALB/c 3T3 clones to different classes of chemicals.