Mutagenesis vol. 27 no. 3 pp. 257–266, 2012
Advance Access Publication 6 December 2011
doi:10.1093/mutage/ger086
REVIEW
The Syrian hamster embryo (SHE) assay (pH 6.7): mechanisms of cell transformation
and application of vibrational spectroscopy to objectively score endpoint alterations
Abdullah A. Ahmadzai, Júlio Trevisan, Nigel J. Fullwood1,
Paul L. Carmichael2, Andrew D. Scott2 and
Francis L. Martin*
Centre for Biophotonics, Lancaster Environment Centre and 1Division of
Biomedical and Life Sciences, School of Health and Medicine, Lancaster
University, Lancaster LA1 4YQ, UK, 2Safety and Environmental Assurance
Centre, Unilever Colworth Science Park, Bedfordshire MK44 1LQ, UK.
*
To whom correspondence should be addressed. Centre for Biophotonics,
Lancaster Environment Centre, Lancaster University, Lancaster LA1 4YQ,
UK. Tel: þ44 (0) 1524 510206; Fax: þ44 (0) 1524 510217; Email: f.martin@
lancaster.ac.uk
Received on August 18, 2011; revised on October 6, 2011;
accepted on October 19, 2011
Using morphological transformation as an endpoint, the
Syrian hamster embryo (SHE) cell transformation assay
(pH 6.7) is an in vitro system with a high sensitivity and
specificity for testing the carcinogenic potential of test
agents. Advantages of the assay are that SHE cells are
metabolically competent, genetically stable and acquire
spontaneous transformation with a low frequency; additionally, it detects both genotoxic and non-genotoxic
carcinogens. However, in comparison with other shortterm mammalian cell assays, it is time consuming,
laborious and, most importantly, the visual scoring of
morphological transformation might be subjective. In this
review, we examine the background to the test and why it
has the potential for use in safety risk assessment.
Additionally, we propose a novel approach to objectively
interrogate and classify SHE colonies using vibrational
spectroscopy coupled to a mathematical framework for
high-throughput screening. It is our view that this
alternative approach has the potential to improve the
sensitivity and specificity of the in vitro SHE assay.
Introduction
Initially described by Berwald and Sachs in 1963, the Syrian
hamster embryo (SHE) cell transformation assay is an in vitro
cell culture system that has been used to test the carcinogenic
potential of biological, physical and chemical agents (1–3). To
date, SHE cell neoplastic transformation is the most relevant
and truest in vitro simulation of the in vivo neoplastic process
(3,4). SHE cells are primary diploid cells, isolated from 13day-old embryos, with a finite lifespan (4). Following test agent
exposure, SHE cells may undergo morphological transformation as a result of a block in cellular differentiation (Figure 1)
(3,5). Additional mutations in morphologically transformed
SHE cells lead to immortal, tumorigenic and then malignant
cells (3,6,7).
The absolute goal of short-term genotoxicity testing is the
risk detection of human carcinogens rather than identifying
positives in rodents (8). Such chemicals cannot be tested in
humans, so the rodent bioassay is the current standard by
which the potential human carcinogenicity of different test
agents is established (8). The SHE cell transformation assay is
a unique in vitro assay that detects both genotoxic and nongenotoxic rodent carcinogens (2,9); as suggested later, this
uniqueness critically underpins the rationale for harnessing this
assay to spectroscopy interrogation with computational analysis to allow one to discriminate test agent effects based on
mechanisms of action. SHE cells, under standard culture
conditions, like human cells and unlike mouse cells, are
genetically very stable and acquire spontaneous transformation
with a low frequency (4,10,11). However, when treated with
carcinogenic agents, the immortality rate increases within SHE
cell populations with a frequency dependant on the particular
test agent being investigated (4,12,13), thus making this assay
a useful tool to study the multistage carcinogenic process (4).
Since its first introduction in 1963, many modifications
have been made to the SHE assay in order to improve its
performance. The reduction of SHE cell culture media from
pH 7.3 to pH 6.7 by LeBoeuf and Kerckaert (13) improved the
assay sensitivity (ability to detect true positives) to 87% from
82% and specificity (ability to classify true negatives) to 83%
from 69% (2,8). Thus far, in the pH 6.7 version of the SHE cell
transformation assay, .200 physical and chemical agents have
been tested (5,8). With an overall concordance of 85% with
rodent bioassays, the SHE assay has been reported to
outperform the standard battery of in vitro genotoxicity tests
(Salmonella typhimurium bacterial mutagenicity assay, mouse
lymphoma assay and in vitro chromosome aberrations test) in
terms of their ability to predict carcinogenic potential (8).
However, the SHE assay has limitations including the
subjectivity of scoring morphologically transformed colonies
and its low rate of increased transformation in response to
carcinogenic agents (2,14). In this review, we examine the
background to the assay and why it still has potential as an
important test for safety risk assessment. Additionally, we
propose a novel approach to objectively interrogate and
classify SHE colonies based on infrared (IR) spectroscopy
coupled to a mathematical framework for high-throughput
screening. Such an approach offers the possibility to objectively discriminate agents based on mechanism of action
according to the post-spectroscopy classification of altered IR
spectra (14). With IR spectroscopy, staining is not necessary
and results are produced computationally, based on a previously
built knowledge base. This knowledge base consists basically
of data and classification models trained on these data. Because
of the non-destructive nature of this approach, SHE colonies
can be subsequently stained, and conventionally visually scored
for comparative purposes and towards the long-term validation
of this novel approach.
Ó The Author 2011. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.
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Fig. 1. Schematic overview of the SHE cell transformation assay. A cytotoxicity assay is carried out prior to the SHE cell transformation assay to determine the
appropriate test agent exposure, e.g. concentration. The exposure that causes 50% cytotoxicity is taken as the highest in the main study. The assay includes solvent
(VC) and positive control treatment groups in addition to test agent treatments. The SHE cells are exposed for 24 h or 7 days before being fixed, stained and
microscopically assessed for cell/colony morphology.
Why the SHE assay remains relevant
The SHE cells are metabolically competent and can activate
many chemicals to their ultimate carcinogenic form without
the addition of an exogenous activation system (2,5). As they
are embryo-derived, the SHE cell population is composed
of multiple cell types that are at different stages of the
differentiation process, thus providing a broad spectrum of
cellular targets for the transformation response (2,15–18); this
attribute is believed to contribute towards the overall sensitivity
of the assay. Under standard culture conditions (pH 7.3), .472
agents have been tested in the SHE cell transformation assay
(15,19). Only 213 (of 472) agents have in vivo rodent
carcinogenicity data available; the pH 7.3 version of the assay
classified 177 (of 213) agents as rodent carcinogens and 36 as
non-carcinogens (19). For these 213 physical and chemical
agents, the SHE assay at pH 7.3 has a sensitivity of 82% (146/
177), a specificity of 69% (25/36) and an overall concordance
with rodent bioassay of 80% (171/213) (19).
In 1987, the pH 7.3 of the Dulbecco’s Modified Eagle Medium
(DMEM) cell culture medium was reduced to pH 6.7 by LeBoeuf
and Kerckaert (13). This resulted in better SHE cell clonal growth,
improved morphological transformation performance and less
assay-to-assay variability (13,15). Lowering the culture pH had
direct effects on the SHE cell phenotype including a decrease in
levels of mRNA transcripts for gap junction protein leading to
reduced intercellular gap junction communication, changes in
protein charge, changes in protein phosphorylation at serine/
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threonine and tyrosine residues, and changes in some gene expression (4,16,19).
Under the reduced pH methodology, the SHE assay has
a sensitivity of 87%, specificity of 83% and an overall concordance with rodent carcinogenicity data of 85% (2,8,15). These
improved accuracy and performance outcomes of the SHE assay
are consistent with a database of .200 chemicals (2,8,15). In
comparison, a battery of S. typhimurium bacterial mutagenicity, in
vitro chromosome aberrations and mouse lymphoma tests
positively detected 47 of 60 carcinogens; the SHE assay alone
was sensitive to 52 of these test agents (2,8,20). Additionally, the
three-test battery had a specificity of 32% (13/40), whereas the
SHE assay had a specificity of 83% (33/40) (2,8,20). The SHE
assay was also shown to be able to detect S. typhimurium-positive
carcinogens and S. typhimurium-negative carcinogens with
a sensitivity of 100% and 78%, respectively (2,8,20).
Limitations of the assay in current practice
Although the SHE cell transformation assay is an attractive
model, several challenges have made it problematic for routine
use in risk assessment. Within the SHE cell population, 1/3
of the clonable cells are capable of undergoing morphological
transformation following carcinogen exposure (15). These
sensitive-to-morphological-transformation cells are relatively
undifferentiated and, based on their responsiveness to growth
differentiation factor, they can be divided into three populations:
an undifferentiated cell population, a committed progenitor cell
Biospectroscopy to objectively score the SHE assay
population with partial epithelial cell-like characteristics and a
committed progenitor cell population possessing mesenchymal
cell-like characteristics (15). While this composition of different
cell types gives the SHE assay the advantage of capability to
detect a wide range of carcinogens, including those with tissuespecific modes of action, it is also the cause of variation in
sensitivity to chemical transformation when different aliquots of
cells from the same culture are used (15,16,18).
The ability of an agent to induce pre-neoplastic changes is
measured by visually scoring the morphologically transformed
SHE colonies. At present, there is currently no objective
molecular marker to identify transformed SHE cells (21); thus,
a trained eye is required to score the colonies under the
microscope (Figures 1 and 2). Large sample sizes are required
in order to derive appropriate statistical analysis because of
the low frequency of induced morphological transformation
following chemical treatment (2,17).
In vitro simulation of in vivo multistage neoplastic
transformation
Neoplastic transformation is a multistage process in which cells
acquire multiple genetic alterations resulting in loss of the
organism-determined growth control of individual cells (4).
A number of in vitro cell transformation assays have been used
to explore the mechanistic basis of both in vivo and in vitro
multistage neoplastic transformation. Different in vitro assays
model different stages of the multistage carcinogenesis process
(22,23). For instance, the BALB/c 3T3 and C3H/10T½ cell
assays have been used to predict the conversion of immortal
cells to malignant cells, which is the final stage of neoplastic
transformation. In contrast, the SHE cell transformation assay
is the only in vitro assay which models multistage carcinogenesis from a normal diploid cell to a fully malignant one
(4,10,12). When treated with carcinogen, the SHE cells
undergo morphological transformation due to genetic alter-
ation. Morphologically transformed SHE cells are more susceptible to further genetic alterations compared to normal SHE
cells. Thus, further genetic changes in morphologically transformed SHE cells results in an immortal cell line. Immortal
SHE cells acquire additional genetic alterations en route to
becoming tumourigenic and eventually malignant. Each stage
of this transformation process is examined below.
Morphological transformation
The SHE cells can acquire morphological transformation
following exposure to genotoxic or non-genotoxic carcinogenic
agents (1,24). Morphologically transformed SHE cells display
a high nuclear-to-cytoplasmic ratio with criss-crossed, randomly oriented and three-dimensional colony growth characteristics (4,15,24) (Figure 2). Morphological transformation is
believed to arise from a block in the normal cellular
differentiation of stem cells within the SHE cell population
(25). It has been shown that 75% of the morphologically
transformed SHE cells exhibit reduced expression of h19
gene compared to normal SHE cells; this is believed to be
responsible for the blockade in cellular differentiation (15,25).
In addition, re-establishment of wild-type h19 in tumourigenic
SHE cells results in the suppression of tumourigenicity in the
cell population (25). The h19 gene is believed to be involved in
the regulation of cellular differentiation in embryogenesis.
Expression of h19 is low in undifferentiated embryonic stem
cells but increases upon the induction of embryonic stem cell
differentiation into embryoid bodies (25–28).
Different agents induce morphological transformation in
SHE cells by targeting different genes. Zinc has anti-apoptotic
properties and can induce morphological transformation of
normal SHE cells (29). Zinc-induced morphologically transformed SHE cells show up-regulation of anti-apoptotic bcl-2
and down-regulation of pro-apoptotic bax expression (30,31).
Studies of the anti-apoptotic properties of zinc suggest that
Fig. 2. The multistage SHE neoplasm. Normal SHE cells (A) may acquire a morphologically transformed phenotype (B) after exposure to carcinogenic test agent.
Morphologically transformed SHE cells senesce unless they acquire further genetic alteration en route to immortalisation and tumorigenicity (C). When
tumourigenic cells are injected into nude mice, cancer sites may develop (D), which through further mutation transform into malignant cells and metastasise.
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A. A. Ahmadzai et al.
inhibition of apoptosis occurs by an epigenetic mechanism and
contributes to the early stage of transformation by extending
the viability of altered cells (29,30). However, over expression
of c-myc is not observed in zinc-induced morphologically
transformed SHE cells. Benzo[a]pyrene (B[a]P) also has no
effect on c-myc expression at transforming concentrations in
SHE cells, whereas 2,4-diaminotoluene (2,4-D) induces over
expression of this gene (32). The proto-oncogene c-myc is
believed to play an important role in cell apoptosis and is
closely linked to bcl-2 (30,33–35). These contrasting observations suggest that different carcinogens mediate their transforming effects via different genes and proteins in SHE cells.
The rate of spontaneous morphological transformation in the
SHE assay (pH 6.7) approximates 0.1–0.4% (4). Morphological transformation of SHE cells can result in either a stable or
a reversible phenotype depending upon the carcinogen being
tested and different test agents transform SHE cells with
different frequencies (4). For instance, the frequency of morphological transformation caused by B[a]P is 1–3% whereas
12-O-tetradecanoylphorbol 13-acetate, a cancer promoter,
induces a level of 10% in SHE cells. However, the window
of opportunity within which morphological transformation can
occur is believed to be transient and is lost at higher passages
as the number of differentiated daughter cells (in which a block
in differentiation is impossible) continually increases, whereas
the stem cell population (in which differentiation could be
blocked) decreases in number (15).
Immortality
Morphologically transformed cells acquire immortality with
a frequency of 10–100% depending on the test agent being
used to induce morphological transformation (4,11,36). For
instance, the frequency for B[a]P-induced morphologically
transformed cells going on to establish an immortal cell line is
30% whereas the level derived from 3-methylcholanthrene
(MCA)-induced morphological transformation is 14% (11,36).
However, control SHE cells achieve immortality with a frequency
of 0–3% (11,36). Immortality is the result of further genetic
alterations in morphologically transformed SHE cells; otherwise, the cells senesce (4,37). Cellular senescence appears to be
primarily controlled by four genetic complementation groups
involving at least eight chromosomal loci, including the tp53
and rbi (19,38). Multiple biochemical factors including
mortalin, DNA methylation, ceramide, telomerase activity
and prohibitin are also considered to be pivotal in the control
of cellular senescence (19,39–41).
Although the exact nature and number of mutations required
to give rise to immortal SHE cells remains unknown, the
majority that do acquire immortalisation are aneuploid (11,42).
The karyotype of chemically induced SHE cell lines often
exhibits double non-disjunctions resulting in trisomy of
chromosome 11 and duplication of a translocated chromosome
(43). Cytogenetic studies on early-passage SHE cell lines that
were exposed to asbestos showed that six of the eight cell lines
had trisomy chromosome 11, whereas the remaining two cell
lines had other chromosomal abnormalities, including X or
Y, þ3, 8p-, -13 and t(13;21) (44). Four of the eight cell lines
had a gain of chromosome 8 (44). Endo and Hieber (45)
reported that four of six radiation-induced immortal SHE cell
lines exhibited non-random rearrangements in the long arms of
chromosome 6 (6qþ) and chromosome 3 (3qþ); however,
spontaneously transformed SHE cells did not show the same
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genetic alterations. B[a]P-induced immortal SHE cell lines
exhibit trisomy of chromosome 8, monosomy of chromosome
16, monosomy of chromosome 10 and deletion of 2p. Other
chromosomal alterations that have been reported in immortal
SHE cell lines include trisomy of chromosomes 3 and 7, and
monosomy of chromosomes 13 and 15 (4,46–50). All the
available studies to date on immortal SHE cells suggest that
different agents cause different chromosomal alterations.
Untransformed SHE cells are stimulated in response to
epidermal growth factor (EGF), to some members of fibroblast
growth factor family proteins, to certain cytokines [including
interleukin (IL)-4, IL-9 and erythropoietin] and the plateletderived growth factor (PDGF) family, while being inhibited by
other cytokines (including IL-1a and IL-1b), transforming
growth factor (TGF)-b and nerve growth factor (18). However,
immortal SHE cell lines are unresponsive to EGF, PDGF and
TGF-b1 (18). Furthermore, immortal SHE cells express
retinoblastoma (rb) protein, which is normally in its phosphorylated form (51). Such immortal SHE cells can be induced to
undergo senescence by introducing a normal copy of human
chromosome 1 into them, suggesting that some genes located
on this chromosome are involved in the regulation of cellular
senescence (4,52).
Towards acquisition of tumourigenicity
Immortal SHE cells may then go on to acquire tumourigenicforming capacity following further genetic alterations, which
mostly occur spontaneously as a result of genetic instability (4).
Transformed SHE cells cycle at twice the rate of untransformed
SHE cells and exhibit anchorage-independent growth in soft
agar (53,54). The resultant tumourigenic SHE cells express
ornithine decarboxylase at twice the levels of those found
in untransformed SHE cells and high levels of cytosolic
NAD(P)H: quinone oxidoreductase (DT diaphorase); after 24
passages, they are nearly 100% effective at inducing cancer in
newborn hamsters within 6 weeks (53).
Several proto-oncogene and tumour suppresser genes have
been identified that are believed to contribute towards
neoplastic transformation. The c-srv is a non-receptor thymidine kinase; mutations in the c-srv gene leading to increased
expression of altered forms of pp60c-src protein have been noted
in asbestos-induced tumourigenic SHE cell lines (55). Cells
that did not translate the correct pp60c-src protein failed to grow
in soft agar (55,56). In another study, 20% of SHE cell lines
transformed by 3-MCA were shown to contain an inactive tp53
gene (57), whereas the asbestos- or diesthylstilbestrol (DES)transformed SHE cell lines carried wild-type tp53 (58). Thus,
transformed cells that carry wild-type tp53 probably contain
mutations in other apoptosis-regulating genes.
Arsenic-exposed tumourigenic SHE cells over express c-myc
and c-Ha-ras oncogenes (59). Nevertheless, the SHE cells that
were treated with DES also expressed elevated levels of c-Haras oncogene mRNA transcripts at the neoplastic stage, whereas
trenbolone induced high transcriptional levels of c-myc and cHa-ras at the pre-neoplastic (immortal) stage (60). However,
asbestos, N-methylnitrosourea and aflatoxin B1 had no apparent
effect on the transcriptional levels of these oncogenes (60).
Other mutated oncogenes that have been identified in neoplastic
SHE cells include c-Nras, cph and c-fos (4,60–63).
The proliferation of normal cells is strictly controlled by cell
cycle checkpoints that have been shown to be impaired in
cancer cells (64). These checkpoints are in place to best ensure
Biospectroscopy to objectively score the SHE assay
the fidelity of the DNA sequence and, to this end, regulate the
timing of cell cycle events (64,65). Any damage in DNA
should lead to a blockade of mitosis at the G2/M-phase
checkpoint, which provides the cell enough time to repair
or activate apoptosis (64). This control is bypassed in SHE
cells that have been transformed by malachite green; these
have abrogated G2/M-phase checkpoint control resulting in
uncontrolled cell division (64).
Malignancy and metastasis in vivo
Tumourigenic SHE cells undergo further genetic alteration in
vivo to acquire a malignant state and an ability to metastasise
(Figure 2) (4). Metastasis, the spread of malignant cells from the
primary site, is a non-random multistep process that is dependent
on the properties of the tumour cells and the host response
(66,67). Acquisition of the metastatic phenotype is not well
understood. However, the general process involves the production of proteolytic enzymes by tumour cells to enable
detachment from the primary site so that they may invade the
surrounding tissues and pass through the blood stream to lodge at
distant (or secondary) sites (67). Such malignant cells exhibit
loss of cell adhesion molecules and acquire the ability to escape
from host immune surveillance (67,68). Tumour cells in vivo are
surrounded by extracellular matrix (ECM), which is mainly
composed of proteoglycans, heparan sulfate, collagen and
keratan sulfate. ECM plays a key role in providing protection
for tumour cells against chemotherapy-induced cell death (69).
Tumour cells also express increased levels of plasminogen
activators (PAs), which convert plasminogen to plasmin,
conferring the property of enhanced fibrinolytic activity (70–
72). Although PAs are involved in metastasis, increased activity
is not always correlated with malignancy (73–77).
When introduced into nude mice or neonatal hamster, transformed SHE cells give rise to tumours with high frequency
(10,12) (Figure 2). Malignant SHE cells are capable of
metastasizing as they produce ECM proteins and t-PA and
show resistance towards macrophages (4,10,77–80). Mutations
leading to activated Ras have been reported in numerous
human tumours, a process that is involved in both tumourigenicity and metastasis in rodent cells in vitro (66,81–84). In
SHE cells, activated Ha-ras oncogene-induced metastasis goes
through the Ras/Ra1GDS/Ra1 signalling pathway in vivo (66).
Fourier-transform infrared (IR) spectroscopy as an approach
to score SHE colonies
Using metabolomic profiling, thousands of molecules can be
identified and quantified within biological samples (85).
Different tools can be used for metabolomic assessment, including mass spectroscopy, nuclear magnetic resonance (86),
Fourier-transform IR (FTIR) spectroscopy, metabolite arrays
and Raman spectroscopy. These tools generate complex
datasets, which need to be reduced, using visualisation
software, to derive interpretable datasets for analysis (85).
Visualisation software packages often apply principal component analysis (PCA), hierarchical cluster analysis, Fisher
discriminant analysis, linear discriminant analysis (LDA) or
artificial neural networks (85,87,88).
Cancer cells have altered metabolic activities associated with
increased rates of lipolysis, protein turnover and glycolysis
(89–94). Of huge benefit would be a method capable of
fingerprinting the complexity of any cell (Figure 3) (95,96).
Fig. 3. Low-temperature scanning electron microscopy images of cells after
cryo-planing to expose cell interior on the cold stage of a JEOL 840 using
a Hexland CT1000 cryo-trans system. (A) Cells appear vitrified, but cellular
features evident include nucleus (Nu), cell membrane (CM; identified by
arrow), nuclear envelope (NuEnv; identified by arrow), mitochondrion (Mit;
example identified by arrow) and cytoplasm (Cy). Scale bar 5 3.0 lm. (B)
Higher magnification with cellular features evident including nucleus (Nu),
nuclear envelope (NuEnv; identified by arrow) and mitochondrion (Mit;
example identified by arrow). Scale bar 5 1.0 lm.
Such complexity is based on structural and functional characteristics and, as such, will lend itself to a particular chemical
composition. Thus, if one can fingerprint the normal complexity,
deviations from this can be associated with various pathological
states, such as morphological transformation (97). The emerging
field of biospectroscopy offers a range of technologies capable
of interrogating the signatures of cell populations down to
individual cell organelles (87). This approach has been applied
for cancer screening (98), stem cell characterisation (99) and
monitoring effects of contaminants or mixtures thereof in mammalian cells (100–103). The application of biospectroscopy tools
generates spectra containing hundreds of variables; as such,
mathematical models for exploratory (e.g. PCA) or classification
(e.g. LDA) purposes are required (104) (Figure 4). The SHE
assay has conventionally relied on subjective visual scoring of
stained foci; however, there is now evidence that FTIR
spectroscopy can be applied as an objective tool to perform
such predictions. Using FTIR in attenuated total reflection
(ATR) mode, Walsh et al. (14) and Trevisan et al. (105)
demonstrated that that SHE foci exhibited IR spectral alterations
associated with test agent treatment or morphological transformation. In ATR-FTIR spectroscopy, the IR beam undergoes
total internal reflection within a crystal (made of germanium,
zinc selenide or diamond) generating an evanescent wave that
penetrates beyond a surface of the crystal by a few microns to
interact with a sample (in this case, biological) placed in close
contact (87). The beam exiting the crystal is received by a
detector that generates a spectrum with a number of absorbance
intensity peaks based on the sample-attenuated signal, each
characteristic of a particular biomolecule. The derived spectral
range of 1800–900 cm1 has been termed the ‘biochemical-cell
fingerprint’ as it contains the fundamental vibrational modes of
structures present in biological systems. Figure 4 shows the
work flow that Walsh et al. (14) and Trevisan et al. (105)
employed; the SHE cells were treated with test agents and then
fixed with methanol on low-E reflective slides. Each SHE
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Fig. 4. Identifying the classifying biomarkers for transformed versus non-transformed SHE cells using IR spectroscopy. The SHE cells are treated with test agent,
cultured and then fixed on low-E reflective slides. Each colony on the slides is interrogated using IR spectroscopy. Multivariate computational analysis is applied to
identify key biomarkers.
colony was located on the slides, referenced and interrogated
using ATR-FTIR spectroscopy.
The applicability of this approach to the cell transformation
assay in terms of how one might manage the derived dataset
in order to extract meaningful information is illustrated in
Figures 5 and 6. The IR spectra derived from transformed
colonies exhibit significant alterations when compared
to those classed as non-transformed SHE colonies, e.g. Nnitroso-N-methylnitroguanidine (MNNG)-treated transformed
versus MNNG-treated non-transformed category spectral
points in an LDA scores plot significantly segregate with a
P-value ,10100; the same is the case for B[a]P treatment
(Figure 5, left panels). When a per-wave number t-test was
carried out for MNNG-treated or B[a]P-treated transformed
versus non-transformed colony categories, significant spectral
alterations are noted in the 1750–1200 cm1 (associated with
protein and nucleic acids) and 1205–900 cm1 (associated
with nucleic acids) regions. In addition, IR spectra derived
from a particular test agent treatment exhibit significant differences compared to those of the vehicle control (VC) category.
The derived spectra from a test agent treatment could be used
to classify that particular test agent against other treatments,
e.g. MNNG versus VC or B[a]P versus VC (Figure 6). The
potential to identify biochemical effects related to test agent
treatment or morphological transformation in SHE foci
represents an important move towards the development of
a novel risk assessment approach that can be applied to new
or unknown entities. Because of the non-destructive nature
of interrogation by FTIR spectroscopy, this novel scoring
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system can be correlated for validation purposes with visual
screening subsequent to staining.
Conclusions
The SHE cell transformation assay has recently acquired
renewed focus as a tool for chemical safety risk assessment and
the identification of potential chemical carcinogens (106).
However, despite the fact that it exhibits good correlation with
rodent bioassay data, progressing its application has been
limited primarily because of the complexity in setting up the
assay and more importantly the lack of an objective scoring
methodology. Ideally, such a scoring methodology would
allow the derivation of a phenotypic measure of SHE colonies
post-treatment with test agent that would then allow one to
assign a class label of ‘no risk’, ‘DNA-reactive transforming
agent’ or ‘non-reactive DNA transforming agent’. The
advantage of the SHE cell transformation assay is that it has
a high sensitivity and specificity for rodent carcinogens and
non-carcinogens, respectively (15). This makes it a potential
replacement for in vivo testing strategies (106). An objective
measure of morphological transformation would allow risk
assessors to make informed quantitative decisions regarding the
categorisation of a particular test agent. We propose that by
harnessing the availability of new physical sciences sensing
tools in combination with new computational approaches,
the SHE cell transformation assay has the potential to gain
prominence as a tool for chemical safety risk assessment and as
an alternative to in vivo testing.
Biospectroscopy to objectively score the SHE assay
Fig. 5. Techniques for distinguishing between non-transformed versus transformed colonies using IR spectroscopy. Left panels: LDA scores plots showing
segregation between non-transformed and transformed IR spectra for two different chemical treatments [MNNG (12.5 lg/ml) and B[a]P (5 lg/ml)]. A statistical
hypothesis test (‘two-sample t-test’) found in both cases a P-value , 10100. Right panels: plots showing [wavenumber] [log10(P-value)] for the same
chemicals as in the left panels (note that plotting the logarithm of the P-values allows for more detail than plotting the P-values themselves). The significance
threshold is marked in both plots (significance level of 5%; log10(0.05) 1.3). Significant differences between non-transformed versus transformed spectra are
observed throughout the spectral region (non-significant wavenumbers are hachured out in gray). The MNNG treatment exhibits its highest significance levels
between 1130 and 930 cm1, whereas B[a]P shows highest significance between 1620 and 1220 cm1. The gaps in the plots correspond to wavenumbers where
log10(p) 5 N or equivalently, P 5 0.
Fig. 6. Techniques for distinguishing between test agent treatments using IR spectroscopy. Left panels: LDA scores plots showing segregation between different
chemical (MNNG or B[a]P) versus their respective VC category (dimethyl sulfoxide; DMSO). A statistical hypothesis test (‘two-sample t-test’) found in both cases
a P-value , 10100. Right panels: two plots showing [wavenumber] [log10(P-value)] for the same chemicals (note that plotting the logarithm of the P-values
allows for more detail than plotting the P-values themselves). The significance threshold is marked in both plots (significance level of 5%; log10(0.05) 1.3).
Significant differences between treatment versus VC is observed throughout the spectral region (non-significant wavenumbers are hachured out in gray). The gaps in
the plots correspond to wavenumbers where log10(p) 5 N or equivalently, P 5 0.
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Funding
Unilever as ‘‘part of Unilever’s ongoing effort to develop novel
ways of delivering consumer safety’’.
Acknowledgements
Conflict of interest statement: None declared.
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