Photochemistry and Photobiology, 2002, 75(5): 437–447
Review
Photogenotoxicity of Mammalian Cells: A Review of the Different Assays
for In Vitro Testing¶
Jean-Roch Meunier*1, Alain Sarasin2 and Laurent Marrot1
L’Oréal Advanced Research, Life Sciences Research, Aulnay-sous-Bois, France and
1
CNRS, UPR-2169, Laboratory of Genetic Instability and Cancer, Villejuif, France
2
Received 14 December 2001; accepted 22 January 2002
ABSTRACT
During the past several years, phototoxicity has been
studied at the molecular level, and these studies have pro-
vided new insights in the field of DNA lesion character-
ization, DNA repair and cell response to ultraviolet (UV)-
induced stress. The development of new antibiotics and
antiinflammatory drugs has highlighted the necessity to
develop the assessment of phototoxicity in the safety eval-
uation of new chemical compounds. This paper aims at
reviewing the known molecular mechanisms of the cel-
lular response to UV-induced stress, the in vitro methods
that can be proposed and used to screen for toxicity of
sunlight and the photosensitization process resulting
from the activation of drugs by light. UV sources, bio-
logical systems and endpoints of interest in that partic-
ular objective are listed. Phototoxic effects span from the
cytotoxic–apoptotic effect to the induction of primary
DNA damage, DNA repair and a variety of stress genes
acting on the cell cycle and the fate of the cell. Ultimately,
it can lead to the induction of hereditary DNA modifi-
cation. A variety of assays are proposed to specifically
address all these particular consequences of UV-induced
toxicity.
INTRODUCTION
All living organisms, including humans, are exposed to the
sun or to manufactured equipment that emit light. Ultraviolet
(UV) light is a well-established mutagenic and carcinogenic
agent (1), and in the recent years, phototoxicity studies at
¶Posted on the website on 28 Jauary 2002.
*To whom correspondence should be addressed at: L’Oréal Ad-
vanced Research, Life Sciences Research, Direction of Biological
Assessment and Safety, Genotoxicity Group, 1, av. E. Schueller,
F-93601 Aulnay-sous-Bois cedex, France. Fax: 33-1-4868-9180;
e-mail: jrmeunier@recherche.loreal.com
Abbreviations: BER, base excision repair; CPD, cyclobutane pyrim-
idine dimers; NER, nucleotide excision repair; PARP, poly-ADP-
ribosyl polymerase; PCR, polymerase chain reaction; 6-4 PP, py-
rimidine (6-4) pyrimidones; ROS, reactive oxygen species; SCGE,
single-cell gel electrophoresis; SSL, solar-simulated light; UDS,
unscheduled DNA synthesis; UV, ultraviolet.
q 2002 American Society for Photobiology 0031-8655/01 $5.0010.00
437
the molecular level have provided new insight in the field
of DNA lesion characterization, DNA repair, gene expres-
sion and cell cycle control.
Apart from the direct effect of UV radiation, the expo-
sures to UV light in the presence of chemical compounds
found in drugs or cosmetic ingredients can reveal an inter-
action between light absorbency and chemicals through en-
ergy transfer or chemical modifications. These interactions
can produce deleterious, cytotoxic or genotoxic effects.
Thus, some chemical compounds are well-known photosen-
sitizers: they can be of endogenous (such as porphyrin and
flavonoids) or of exogenous origins (fluoroquinolone class
of antimicrobial agents and nonsteroidal antiinflammatory
drugs) (2).
This has highlighted the necessity to harmonize and eval-
uate the strategy used to assess the phototoxic and, in par-
ticular, the photogenotoxic potential of a chemical com-
pound. Up to now, photomutagenicity testing is only re-
quired for sunscreen development.
For this purpose, different symposia, meetings and work-
shops have been organized since 1993, by the European
Center for the Validation of Alternative Methods in 1993 (3)
and 1999 (4) in Europe, in 1996 by the Food and Drug
Administration (5) and in 1999 (International Workshop of
Genotoxicity Test Procedures, IWGTP, Washington) (6), to
address this question and evaluate the existing in vitro meth-
odology. No regulatory requirement has been specified, but
a set of recommendations and guidances have been issued
(4–6). Until now, the photocytotoxicity test 3T3 neutral red
uptake (NRU) is the only in vitro method that has been val-
idated so far by European regulatory affairs (7–9). Because
this test is devoted to the evaluation of cytotoxic effect, it
represents a very preliminary step, and there is still a neces-
sity for the development of mechanistically based methods.
With this background, the fundamental research done for
years by numerous teams to understand the molecular mech-
anisms underlying the cellular response to UV-induced ge-
notoxic stress permitted one to set up various in vitro tests
for the assessment of (1) photoprotection strategies; and (2)
ingredient phototoxicity.
Obviously, the best cell system for in vitro photobiology
studies is the target cells of the skin, mostly normal human
438 Jean-Roch Meunier et al.
Figure 1. Biological endpoints for phototoxicity testing and mea-
suring photogenotoxic treatment on mammalian cells.
keratinocytes or eventually normal human melanocytes (10).
Normal skin irradiated ex vivo or reconstructed skin systems
should be even better, but more complicated, to manipulate
on a large scale. They certainly enable topical treatment in
conditions closer to those in real life; moreover, they do
constitute an alternative to animal testing for chemicals that
would be applied on the skin. But the biological endpoints,
which can be evaluated on these reconstructed organs, are
mostly cytotoxicity, cytokine release at the population level
and in situ immunochemical detection of various antigens
(11). There is still a need for developing investigative meth-
ods on these models at the single-cell level. For technical
easiness, most experiments have been, therefore, carried out
using fibroblasts or transformed cells.
A variety of light sources have been used in the literature
of photobiology. This diversity in the used UV sources and
in the dosimetry measurements renders the comparison of
results between laboratories difficult. Thus, one of the goals
of the workshops and meetings cited earlier was also to har-
monize the UV sources and to issue a recommendation on
this specific point.
Most of the studies were done with UVC light, which is
effectively screened by the ozone layer and does not reach
the earth’s surface but is easy to use in laboratory environ-
ment. Studies done with UVB or UVA tubes demonstrated
the specificity of the cellular response with regard to these
particular wavelengths. In an attempt to mimic the solar UV
spectrum, solar simulators are used, enabling the assessment
of synergistic effects of UVB and UVA on cell systems. In
all the recommendations and guidances issued so far, the
solar simulator is the recommended UV source. Moreover,
a detailed knowledge of the exact spectrum of the used
lamps is necessary to allow comparison of biological effects
and an extrapolation from in vitro testing assays with the in
vivo ‘‘real life.’’ Because these apparatus are usually very
expensive, the minimal requirement is for information on the
spectral output by determining the UVB–UVA ratio of the
source used.
This review details some of the consequences of UV ir-
radiation of living cells in order to describe most biological
assays used to screen for photogenotoxicity of light, drugs
or a combination of both (Fig. 1). It should be useful for
scientists involved in developing new assays or in testing
phototoxicity effects of various xenobiotics (Table 1).
PHOTOINDUCED CYTOTOXICITY AND
APOPTOSIS
Cytotoxicity
The photocytotoxic effect of UV light should be distin-
guished from its photogenotoxic effect. Photocytotoxicity is
the measurement of acute effect of UV stress, resulting in
life or death of a cell. Photocytotoxicity can be the result of
either necrosis or apoptosis induction. There are some spe-
cific methodologies to unequivocally demonstrate the occur-
rence of an apoptotic response, which are listed hereafter.
In contrast, the aims of photogenotoxicity studies are to
evaluate the ‘‘genetic health’’ of the surviving cells. It has
to be understood that photogenotoxicity studies should be
performed in the frame of a very well-defined cytotoxic ef-
fect of the UV stress employed. Thus, an accurate evaluation
of the photocytotoxic effect of the treatment (UV light alone
or photoactivation studies of chemical compounds) is an ab-
solute prerequisite for photogenotoxicity studies. To be bi-
ologically relevant, the genotoxicity endpoints evaluated
through a particular methodology should only be observed
in cells treated by conditions leading to no or moderate tox-
icity (survival $ 50% of the untreated controls).
There are many different methods to assess cytotoxicity.
The neutral red uptake method is currently used as a pho-
totoxicity measurement and has been validated by European
regulatory agencies as an in vitro test for phototoxicity (7,8).
This method allows an evaluation of the photoinduced cy-
totoxicity and thus should be regarded only as a primary
step.
Cytotoxicity can also be measured by the counting of cells
for several days after treatment, by assessment of the mito-
chondrial activity (3-[4,5-dimethylthiazol-2-yl]-2,5-diphen-
yltetrazolium bromide test), by the determination of the mi-
totic index (as a measurement of the dividing capacity of
the cell such as that used in the chromosomal aberration
test), by cloning efficiencies and so on.
There is, up to now, no consensus on the choice of a
preferred method for cytotoxicity measurement. Obviously,
the criterion of a reduction of 50% of cell survival would
not biologically signify the same toxicity in terms of UV
dose and concentration of compounds, depending on the
method used. This explains why usually at least two differ-
ent cytotoxicity endpoints must be used in photogenotoxicity
studies (such as cell count and mitotic index for the photo-
chromosomal aberration test, for example).
Apoptosis is an important component of the cytotoxic ef-
fect of a treatment, and the occurrence of apoptosis is a
valuable information in understanding the mechanism of
phototoxicity of a particular compound because it involves
specific pathways and cellular responses.
Apoptosis
Background and mechanisms. Cells respond to toxic stimuli
in one of two ways: by activation of the cellular stress re-
sponse or by undergoing cell death. Apoptosis is a form of
cellular suicide, with distinct morphological features, which
 Photochemistry and Photobiology, 2002, 75(5) 439

Table 1. In vitro screening assays for photogenotoxicity. State of the various described photogenotoxicity assays in terms of whether they
are used in routine screening or still in research and development stage (R&D)

Adaptability for routine screeing*† Assays

still in
Biological endpoints and assays Unknown lesions Known lesions Regulatory position R&D state

I. Cytotoxicity
Cell toxicity R1/E1 R1/E1 3T3 NRU validated (7)
Apoptosis R12/E12 R12/E12
II. Primary DNA damage
Immunodetection R2/E12 R+/E+2 Mechanistic approaches: sensitivity 1
Comet assay R1/E1 R1/E1 to be determined with a set
Comet assay + repair enzymes R12/E12 R12/E12 of chemical compounds
UDS R2/E2 R2/E2 of the same class
III. Cell response
Stress genes R12/E12 R12/E12 1
Cell cycle R2/E1 R2/E1 1
IV. Hereditary consequences
Point mutagenesis R1/E2 R1/E2 1/2‡
Chromosomal damage R1/E12 R1/E12 1/2‡

*R 5 reliability and E 5 easiness.

†R1: reliable; R12: reliability depending on the method used and damage targeted (immunodetection); R2: questionable reliability; E1:
easy to perform; E12: easiness depending on the assay used and damage targeted (immunodetection); E2: not so easy or time consuming
or needs expert skills.
‡1/2: Depending on the assay.

occurs under both physiological and pathological circum-
stances (12). These features include membrane blebbing, cy-
toplasmic shrinkage, nuclear condensation, nuclear fragmen-
tation, destruction of apoptotic cells into apoptotic bodies
and, finally, phagocytosis of these apoptotic bodies. Apopto-
sis is associated with activation of caspase proteases that
target over 100 cellular substrates. Recent developments
have identified the mitochondrion as central to their activa-
tion (13). During apoptosis, cytochrome c is released from
the mitochondria into the cytosol, where it interacts with the
cytosolic protein Apaf-1, leading to sequential activation of
pro-caspases-9 and -3 (14–16). In fact, mitochondrial chang-
es are one of the earliest processes in the apoptotic cascade.
One of the fundamental features of this type of cell death
is the recognition and phagocytosis of apoptotic cells by
macrophages or neighboring cells. However, the mechanism
of plasma membrane alterations that promote the recognition
of apoptotic cells under in vivo conditions is poorly studied.
The loss of plasma membrane phospholipid asymmetry, be-
cause of the externalization of phosphatidylserine, on the cell
surface is one of the most well-reported recognition signals
for phagocytic uptake (17). This process allowed the devel-
opment of a specific assay for the quantification of apoptosis.
Photodermatologists are well aware of the presence of
sunburn cells in the epidermis exposed to sun. These cells
express the classical morphology and behavior of apoptotic
cells. It was quickly demonstrated that UVC and UVB in-
duce apoptosis, which is considered as the best way to elim-
inate UV-damaged keratinocytes and therefore to protect the
exposed skin from cancer (18). Although UVC irradiation
has been shown to induce apoptosis via expression of the
Fas ligand and ligation of its receptor, Fas, it is believed that
p53 stabilization plays a major role in this induction. The
apoptosis of human T-helper cells mediated through the Fas
system seems to be involved in the efficiency of the UV
phototherapy (19), but UVA alone does not induce apoptosis
at relevant physiological doses. UVB and UVC induce ap-
optosis mostly by a p53-dependent pathway. Indeed, the lev-
el of apoptosis is proportional to the blockage of transcrip-
tion by the presence of unrepaired DNA lesions in tran-
scribed strands, as demonstrated by the high level of apo-
ptosis induced in DNA repair–deficient cells even by a low
dose of UV (20). Depending upon the cell types, the apo-
ptosis is more or less easy to be recorded. The choice of the
cell system is very important in this test because the level
of spontaneous or induced apoptosis is very variable accord-
ing to the cell type. Fibroblasts are very difficult to apoptose,
whereas blood cells or transformed cells exhibit a high level
of apoptosis. Similarly, cells isolated from nucleotide exci-
sion repair (NER)–deficient patients are about five times
more sensitive to the UV-induced apoptosis at low UV dos-
es. Tumoral HeLa cells are now being widely used in several
tests to screen for apoptosis induction after various treat-
ments.
Assays for apoptosis detection. Numerous assays have
been proposed for the characterization and quantification of
the apoptosis process (Fig. 2). The easiest assay is the mor-
phological study of the cells labeled by various types of
fluorescent probes. The early apoptosis process can be rec-
ognized by microscopy, showing distinct peripheral chro-
mosome condensation against the intact nuclear membrane.
Then, chromatin fragmentation and cell blebbing, with a still
intact cytoplasmic membrane, occur. Fluorescent markers,
such as the Hoechst 33342, allow one to follow living cells
and to detect apoptotic cells by fluorescent microscopy. This
assay can be standardized and automatized for a rapid
screening. The number of studied cells is, however, relative-
ly limited, making this assay not very quantitative. The sec-
440 Jean-Roch Meunier et al.
Figure 2. In vitro assays for quantifying the apoptosis level in pho-
totoxicity testing.
ond most popular determination of the apoptosis level is the
quantification of the number of cells that appeared in the
sub-G1 peak of the cell cycle profile. After labeling with
ethidium bromide, suspended cells are analyzed through
flow cytometry. Apoptotic cells appear in the window be-
tween cell debris and the classical G1 phase. This assay is
easy to be carried out, even in large-scale screening protocol,
and is considered one of the most specific.
DNA fragmentation can be recorded by many ways: gel
electrophoresis, loss of labeled genomic DNA, in situ label-
ing of DNA nicks (by the TUNEL assay) and comet assay.
These assays can be used for quantifying apoptotic efficien-
cy, but they have to be correlated with other assays because
they mainly measure the production of single-stranded
breaks in cell DNA, which are observed not only during the
apoptotic response. Thus, the TUNEL assay could also react
to the induction of strand-breaks by the DNA repair ma-
chinery. Moreover, if the well-known ‘‘DNA ladder’’ pic-
ture of apoptosis can easily be observed in circulating cells
(lymphocytes and derived–cell lines), it is usually less ob-
vious in other cell types such as skin keratinocytes or dermal
fibroblasts. Some polymerase chain reaction (PCR)–based
methods have been developed in an attempt to increase the
sensitivity of detection of the DNA ladder formation (Clon-
tech, Palo Alto, CA), but the DNA ladder endpoint can be
considered to be highly cell-type–dependent and thus some-
times quite insensitive. Modifications of cell membranes as
a result of activation of phospholipid-scramblase or inhibi-
tion of aminophospholipid-translocase (or both) cause the
externalization of phosphatidylserine during apoptosis. This
process is thought to be a general marker of apoptosis. This
event can be followed by the detection of annexin-V–fluo-
rescein isothiocyanate binding to the phosphatidylserine res-
idues that are externalized and can be quantified by flow
cytometry and thus can be used in large-scale screening as-
says, but its specificity also varies according to the cell
types.
Finally, protein modifications can also be followed during
the apoptotic pathway. Specific cleavages of the poly-ADP-
ribosyl polymerase (PARP) or of numerous caspases occur
during apoptosis. PARP cleavage has been described as a
prerequisite for the DNA fragmentation process in the apo-
ptotic cell (21) and can be followed up by the Western blot
analysis (22). These death proteases are part of a proteolytic
cascade activating proenzymes into active proteases. Serial
activation of these various caspases can be followed in cell
extracts to quantify the level of apoptosis, and several
ELISA-based immunoassays have been developed, enabling
a rapid and quantitative detection of caspase activation. Cas-
pase 3 activation is a particularly interesting endpoint be-
cause of its control role as caspase effector responsible for
the execution of the cell death program in the cell (23,24)
(Fig. 2). Several colorimetric immunoassays are now avail-
able to quantitate the activity of caspase 3 in cell lysate (25).
These methods are usually highly reliable and can be used
in large-scale screening assays (high throughput) for apopto-
sis determination.
Research conducted in the last years in the field of apo-
ptosis has lead to the identification of different molecular
events, organized in a sequential way, committing the cell
in the apoptotic pathway. These molecular endpoints have
been translated into methods enabling a rapid and quantita-
tive measurement of the apoptosis in a cell population (Fig.
2). But in order to assess a reliable and specific apoptotic
effect of the UV light or of the combination of UV light
with a photoreactive chemical compound, one should con-
firm the results by using at least two different endpoints, for
example, an early one (such as the annexin V externaliza-
tion, or the caspase 3 activation) and a late one (such as the
sub-G1 determination).
QUANTIFICATION OF GENOTOXICITY
Photolesions
Bulky lesions. UVB and UVC irradiations generate, in cel-
lular DNA, bipyrimidic photoproducts, which are mainly
cis–syn cyclobutane pyrimidine dimers (CPD) and pyrimi-
dine (6-4) pyrimidones (6-4 PP) at a ratio varying from 4:1
to 10:1, according to various studies and at various wave-
lengths (26). The 6-4 PP have been found to undergo an
efficient photoconversion into Dewar valence isomers upon
exposure to UVB light. Other types of DNA damage, such
as strand breaks or single-base modifications, have been de-
tected but at high nonphysiological doses (26). UVA irra-
diation can hardly produce direct photolesion on the DNA,
especially at the longest wavelengths, where photons are
poorly or not absorbed. Even if the induction of CPD by
UVA has been reported (27,28), this question is still a matter
of debate. Moreover, some photoactivable compounds can
also produce bulky adducts. For example, psoralen and pso-
ralen derivatives can intercalate into DNA and upon UVA
irradiation give rise to monoadducts or cross-links between
two thymine residues (29). Finally, reactive compounds pro-
duced during lipid peroxidation could also reach nuclear

DNA and form adducts (30). However, the significance of
such damage for photomutagenesis in vivo needs to be fur-
ther clarified.
Oxidized bases. UVA, and even visible light, can produce
oxidative DNA damage via the production of reactive oxy-
gen species (ROS). Oxidation of DNA by ROS produces a
large variety of lesions that have been extensively studied in
(oligo)nucleotides (31). In the case of the oxidative stress
induced by UV from sunlight, mainly 8-oxoguanine as well
as oxidized pyrimidines such as thymine glycol or uracil-
derivatives have been described in cellular systems (32–35).
Strand breaks. Endogenous photosensitizers (such as mel-
anin, porphyrin, riboflavin) and exogenous drugs (antibiot-
ics, antiinflammatory drugs. . . ) produce ROS when exposed
to UVA. Some of these ROS, such as hydroxy-radicals or
singlet oxygen, induce DNA single-strand breaks and alkali-
labile sites (36). These DNA damages, albeit certainly effi-
ciently repaired, could be a source of genetic instability. On
the other hand, double-strand breaks, which are known to
trigger a high rate of recombination, are only induced by
strong photosensitizers and have never been clearly detected
with UV from sunlight alone.
DNA Repair
Nucleotide excision repair. Bulky DNA adducts, including
UV-induced photoproducts, are recognized by the versatile
nucleotide excision repair (NER) pathway able to detect
DNA structure abnormalities and to repair them. During the
course of repair, a 26–30 bases–long oligonucleotide con-
taining the lesion is removed, and the gap is then filled in
by unscheduled DNA synthesis (UDS) carried out by DNA
polymerase d or e in the presence of PCNA and various
replication factors. Bulky DNA lesions inhibit RNA poly-
merase II giving rise to a signal that results in a preferential
repair of lesions located on transcribed strands. This tran-
scription-coupled repair is vital for cell survival after UV
irradiation. Its impairment gives rise to serious DNA-repair–
deficient diseases associating cancer development and neu-
rological disorders (37).
Base excision repair. Modified bases (such as oxidized
pyrimidines or purines) or abasic sites are repaired by base
excision repair (BER). Basically, the damaged base is rec-
ognized and removed by a specific DNA glycosylase, giving
rise to an abasic site that is further recognized and eliminated
by several classes of Ap-endonucleases. The repair of 8-
oxoguanine was particularly well studied, even in human,
where the specific repair enzyme hOGG was described (38).
The small gap is then filled in by either the b polymerase
(small patch BER) or by the same polymerases as in the
NER (long patch BER). Very recently, a transcription-cou-
pled repair for oxidized bases such as thymine glycols and
8-oxoguanine has been shown (39).
BER and NER pathways are error-free processes, in con-
trast to the translesion synthesis carried out by mutagenic
DNA polymerases.
Quantification
To characterize and quantify the lesions produced by various
UV lamps, one can use purified DNA repair enzymes, which
specifically recognize DNA lesions or classes of DNA le-
Photochemistry and Photobiology, 2002, 75(5) 441
sions usually by a DNA glycosylase activity giving rise to
nicks on purified DNA. The DNA substrates can be a plas-
mid genome (usually treated in vitro) or cellular DNA ex-
tracted from treated cells.
Specific enzymes. The phage T4 endonuclease V (or the
one from Micrococcus luteus) specifically recognizes the
CPD, whereas the Escherichia coli Fpg, NTh or Nfo proteins
cut DNA at oxidized purines, oxidized pyrimidines or abasic
sites, respectively (for a review, see Sankar and Sankar
[40]). After treatment of damaged DNA with these enzymes,
the measurement of the DNA size between two nicks allows
the quantification of DNA lesions per kilobasepair of DNA.
For example, using the T4-endonuclease V enzyme on UV-
irradiated plasmid DNA, Kuluncsics et al. (28) determined
the yield of CPD formation by UVC (1.5 3 1022 CPD/[kbp
J m2]), UVB (1.5 3 1024 CPD/[kbp J m2]), UVA (1.4 3
1027 CPD/[kbp J m2]) and solar-simulated light (SSL) (1.5
3 1026 CPD/[kbp J m2]). With UVA and SSL irradiations,
oxidized pyrimidines and purines as well as abasic sites were
detected at similar levels as CPD. It is important to note that
the absolute values concerning the production of DNA le-
sions after various wavelength irradiations vary a lot from
one laboratory to another and among the various biological
systems used. Other than the method using the bacterial Fpg
protein, 8-oxoguanine can also be measured by high-perfor-
mance liquid chromatography (HPLC) of DNase-treated
DNA, followed by electrochemical detection (41). Such a
measurement can improve the detection of ROS effects in
treated biological samples. This assay is still difficult to be
performed on a large scale, and measurements of 8-oxo-
guanine are still controversial and subject to artifacts. A
more quantitative and specific determination of DNA lesion
can be performed using HPLC followed by electrospray ion-
ization tandem mass spectrometry (LC-MS/MS) of purified
irradiated DNA (42). This allows one to measure all classes
of UV-induced photoproducts after UVC or UVB irradia-
tion. However, this technique is used by sophisticated lab-
oratories, and we do not yet know if this could be applied
in the near future for screening assays.
In terms of photoscreening, plasmid DNA or genomic
DNA can be used to quantify the protection level on known
DNA lesions (such as CPD, for example) or to detect new
classes of DNA damage produced by given photoactivable
chemicals. This can be realized by using the Fpg, Nfo or
Nth enzymes, which recognize numerous classes of DNA
damage, whereas the T4-endonuclease V can only be utilized
for quantifying CPD.
The E. coli NER system can be reproduced in vitro using
the UvrA, UvrB and UvrC purified proteins, which are able
to recognize bulky DNA lesions and to produce single-strand
gaps in damaged DNA. This assay has been successfully
used to study the respective repair kinetics of CPD and 6-4
DNA lesions after UV irradiation (43). However, the avail-
able enzymes of commercial preparation are relatively dif-
ficult to use in a quantitative way.
Antibodies. A popular test involves the use of polyclonal
or monoclonal antibodies able to recognize different types
of bulky lesions such as UV-induced photoproducts, chem-
ical carcinogen–induced lesions and some oxidized bases
(44). The use of antibodies against CPD or 6-4 PP (45,46)
is possible in a screening assay on genomic DNA or for in
442 Jean-Roch Meunier et al.
situ immunohistochemistry to follow the presence of lesions
on irradiated native skin or reconstructed skin in vitro (47).
This assay can be ‘‘roughly quantified,’’ although its cali-
bration in terms of number of lesions per unit of DNA is
still difficult. It is, however, a relatively easy technique for
measuring a protection index by a given molecule on UV-
irradiated skin. Antibodies against 8-oxoguanine, as a mea-
sure of oxidized purines, do exist, but their specificity and
possible use on tissue slides are still not reliable enough.
Comet assay. Sensitive detection of DNA damage is the
goal of a variety of techniques used in environmental toxi-
cology. Because the effects of environmental genotoxics,
such as UV light, are often tissue- and cell-type specific, it
is of importance to rely on techniques that can detect DNA
damage in the individual target cell.
The comet assay, also called single-cell gel electropho-
resis (SCGE), is a powerful method devoted to the assess-
ment of primary DNA damage (but not their biological ef-
fects such as mutations or clastogenesis). Although other
techniques, such as immunological detection of strand
breaks (48) or the alkaline elution, exist for detecting DNA
damage, the comet assay presents the advantage of being
relatively easy, reliable and fast enough to perform.
Analysis of DNA damage by SCGE was first described in
1984 by Ostling and Johanson (49). They demonstrated that
DNA of cells embedded in a low-melting agarose microgel
on microscope slides migrates in an electrophoretic field in
a pattern determined by the extent of DNA damage. Because
the migrating DNA resembles comet tails, the assay has been
termed as comet assay. The technique first described with
the use of a neutral electrophoresis buffer that allows only
the detection of double-strand breaks has then evolved (50)
with the use of alkaline electrophoresis conditions that per-
mit the detection of single-strand DNA breaks, alkali-labile
sites as well as double-strand breaks. The comet assay pro-
tocols have been discussed and harmonized during the In-
ternational Workshop on Genotoxicity Test Procedures in
1999 (for a review, see Tice et al. [51]).
Thanks to image analysis, the comet assay allows quan-
titative measurement of DNA strand-breaks induction, either
caused by the genotoxin itself or by the activity of DNA
repair enzymes removing the damage. Several software
packages are available in the market. They enable the cal-
culation of the tail moment, a measure of the extent of dam-
age, taking into account the length of the comet tail and the
amount of DNA found both in the tail and in the nucleus
(52). Statistical analysis, accurate cytotoxicity data and ki-
netic studies are necessary to interpret the biological impact
of the observed comet results (51).
This assay can be combined with the use of specific an-
tibodies to detect the nature of the damage, its localization
and the number of specific DNA damage such as pyrimidine
dimers (53) or with DNA repair enzymes such as endonu-
clease III or Fpg enzymes (54).
The comet assay is widely used today both in in vivo and
in vitro studies for the evaluation of the genotoxic potential
of a variety of DNA-damaging agents such as chemical com-
pounds, ionizing radiation and UV radiation. In the field of
phototoxicology, the comet assay has been used to demon-
strate the genotoxic potential of UVC, UVB or UVA light
(55) as well as SSL on normal human melanocytes in culture
(10). The comet assay enables the evaluation of photopro-
tective effects of sunscreen or antioxidants (56,57), or the
photogenotoxic potential of chemical compounds when ir-
radiated with UV light, such as the antibacterial fluoroquin-
olone Lomefloxacin (58). This can be performed on every
cell type, and thus is a particularly interesting endpoint in a
strategy of in vitro photogenotoxicity testing.
Unscheduled DNA synthesis. Quantification of NER by
UDS, i.e. by incorporation of labeled DNA precursors during
resynthesis, corresponds to an indirect measure of the pres-
ence of DNA lesions in an NER-proficient cell. This ap-
proach has been used to study the repair of photolesions such
as pyrimidine dimers (59). The level of UDS is proportional
to the UV dose until a plateau indicating a steady-state level
of repair is reached (which is around 15 J/m2 for UVC and
500 J/m2 for UVB). This assay allows one to measure repair,
and therefore the amount of damage even for unknown
bulky adducts. It does, however, not easily detect DNA le-
sions repaired by BER, such as oxidized bases because in
this case, the patch size is too small to give rise to detectable
labeling. UDS analysis can be used in automatic and large-
scale assays using labeled or fluorescent detection probes.
Positive results with UDS indicate the presence of DNA le-
sions recognized and repaired by NER and therefore allow
the quantification of initial genotoxicity. This does not, how-
ever, permit one to determine if the repair step is complete
and faithful. For a rapid and early test in a screening pro-
cedure, UDS is one of the most reliable in vitro assays, but
in some cases (particularly with chemical compounds) it can
be relatively insensitive, especially for DNA damage re-
paired through BER mechanisms (small patch of nucleotide
removed).
Reporter gene. Bulky DNA adducts as well as some ox-
idized bases strongly inhibit RNA transcription because of a
blockage of the RNA polymerase II elongation. Plasmid
DNA expressing a reporter gene (chloramphenicol acetyl
transferase, luciferase or Green Fluorescent Protein) can be
used as a genetic marker of DNA repair after transfection in
mammalian cells. Damaged plasmids will not express the
reporter gene because of the inhibition of transcription, but
during the course of repair, the gene will become expressed
again as a result of an efficient repair. This assay is relatively
easy to be performed in a medium-scale range, and gene
expression, i.e. repair efficiency, can be quantified by auto-
matic procedures. However, this assay necessitates treating
the plasmid in vitro as naked DNA, which exclude all bio-
logical reactions produced inside the cells. Damaged plas-
mids need to be transfected in the host cell, this necessitating
the use of a control undamaged plasmid as a marker for
DNA transfection efficiency. Most experiments on a large-
scale basis have been done with human lymphocytes, but in
theory all mammalian cells can be used for this assay, de-
pending upon their transfection efficiency (for a review, see
Benhamou and Sarasin [60]).
GLOBAL CELLULAR RESPONSE TO THE UV
STRESS
Gene expression
UV irradiation of mammalian cells produces damage at the
level of proteins, DNA and lipids, according to the wave-

length utilized, but many of its deleterious effects have gene
expression as endpoints.
Among the major responses to UV irradiation, the p53
pathway is probably the most documented one (Fig. 1). The
p53 protein accumulates in a dose-dependent manner in
mammalian cells after exposure to various genotoxic agents
(61). In the first hours after treatment, p53 is stabilized by a
posttranslational process (probably linked to phosphoryla-
tion, acetylation or both) independent of its transcription lev-
el (62). This then leads to the transactivation of p53-respon-
sive genes such as those involved in cell cycle regulation,
apoptosis, DNA repair or replication. A relatively easy meth-
od to detect genotoxic stress consists in the analysis of p53
stabilization and accumulation by Western blotting or im-
munohistochemistry techniques using one of the numerous
commercially available antibodies. This can be performed
easily on cell extracts or in vitro cultured cells, as well as
on skin biopsies (63). A comparable type of assay can be
performed by following the kinetics of some expressed p53-
responsive genes that are triggered upon p53 activation; P21,
Gadd45, PCNA and Bax genes are classically used as pos-
itive markers of UV damage either at the mRNA level or
with specific antibodies directed toward the proteins. UVC
and UVB induce this p53 pathway, whereas the impact of
UVA is less clear and seems to depend on cell type.
A major consequence of p53 stabilization is a cell cycle
arrest via the induction of the p21 pathway, to allow time
for DNA repair before replication. The cell cycle arrest oc-
curs mostly at the G1/S checkpoint after UV irradiation, al-
though some G2 arrest has also been reported. The mea-
surement of G1-arrested cells immediately after treatment by
a genotoxic agent can be considered as a qualitative analysis
of the consequences of DNA damage induction, and the ki-
netics of normal cell cycle recovery can be taken as a quan-
titative analysis of DNA repair. This kind of analysis can be
performed automatically by monitoring the number of cells
in each cell cycle compartment. The choice of the utilized
cells is critical for this type of assay because one should
choose cells that are very active in cycling.
Photoinduced mutagenesis
The production of bipyrimidic lesions by UVC or UVB ir-
radiation induces a spectrum of mutations characteristic of
these wavelengths. Results of experiments with cells from
different organisms, ranging from bacteria to human, clearly
show that UVC and UVB radiation give rise mainly to base
pair–substitutions, targeted at pyrimidine–pyrimidine se-
quences, and mostly to GC-to-AT transitions (64,65). A high
level of tandem CC-to-TT transitions are found only in UV-
irradiated samples (around 10–20% in DNA repair–profi-
cient systems), whereas this number is less than 1023 in mu-
tation spectra produced by genotoxic agents different from
UV. Interestingly enough, DNA repair–deficient cells (es-
sentially xeroderma pigmentosum cells) exhibit a very high
level of CC-to-TT tandem mutations (about 60–70%) for still
not fully understood reasons. This result is found in tumor
suppressor genes isolated from xeroderma pigmentosum
(XP) skin cancers, such as p53, PTCH and p16 (65–67).
Mutation spectra of these genes are characterized by muta-
tional hot spots where the same type of mutation is found at
Photochemistry and Photobiology, 2002, 75(5) 443
the same position in tumors isolated from different individ-
uals. This type of hot spot can be used as a marker for
screening the deleterious effects of UV exposure and UV
mutagenesis. Because the largest mutation database has been
constructed with the p53 gene, we used this gene to screen
for hot spots demonstrating UV exposure. This assay is
much easier with the UV-induced mutations because one can
look for the presence of the double mutations CC to TT,
which renders the assay easier and more specific. A test us-
ing the mutant allele–specific PCR has been developed for
looking at the appearance of CC-to-TT mutations at the co-
don 247/248 hot spot of the p53 gene, with a sensitivity as
low as 1026. By using this assay, we could show that normal
skin biopsy from an exposed body site could easily be dif-
ferentiated from unexposed skin from the same individual
(68). Usual sun exposure induced detectable CC-to-TT tan-
dem mutation in a normal skin biopsy. This assay, when
done in a very controlled manner, is relatively easy to be
carried out, and it is possible to use it as a screening test for
antimutagenic protocols from either in vivo or ex vivo skin
biopsies, or from reconstructed skin. It is obvious that the
mutation induction represents the first step in the carcino-
genic process, and therefore monitoring the presence of mu-
tations can be considered as a direct way to measure this
initial process.
The mutagenicity of the UVA wavelength has not been
studied as well as for UVB and UVC. Episomal shuttle vec-
tors stably replicating inside the cell nucleus have been used
in order that the total response of the cells such as production
of free radicals is obtained after UVA treatment. UVA-in-
duced mutation spectra show an absence of the tandem CC-
to-TT mutations, a majority of C-to-T transitions, but not
always located at bipyrimidic sequences, and a high level of
mutations at A–T base pairs compared with the UVC- and
UVB–induced mutations (69,70). Although this high level
of mutations at A–T base pairs could be considered as some
type of molecular fingerprint of UVA, the lack of obvious
hot spots, with the small numbers of studied cases, does not
allow to propose a screening assay specific for UVA muta-
genesis yet.
On the other hand, UVA-induced 8-methoxypsoralen mu-
tagenesis has also been studied in the framework of psora-
len1UVA therapy (71).
The point mutagenesis assay on the V79 cell, called the
hgprt assay, has been adapted in a photo-hgprt assay and
developed in some laboratories to perform photomutagen-
icity screening of compounds (72).
Photoclastogenicity
As a result of DNA repair failure, a primary DNA damage
induced by UV light may be converted into a hereditary
damage after cellular DNA replication, which can be a point
mutation, a deletion or a chromosomal damage. The impact
of UV-induced point mutagenesis is well documented, as
described previously. These mutations represent a major
event in nonmelanoma skin cancers but are still a matter of
debate in their role in the development of the very aggressive
melanoma, at least for the p53 gene. Point mutagenesis
should, however, not hinder the importance of other damage
and in particular chromosomal damage. These can be divid-
444 Jean-Roch Meunier et al.
ed into clastogenic events (resulting in the breakage of the
chromosome) and aneugenic events (resulting in a complete
loss of a chromosome). Such events are sources of genetic
instability, which could ultimately lead to the development
of cancer. Data generated so far with known photosensitizing
compounds show a good correlation between in vitro mam-
malian photoclastogenic and photocarcinogenesis potential
in animals (73).
In general, chromosomal damage is assessed through the
chromosomal aberration test (74) or through the in vitro mi-
cronucleus test. Photoinduced chromosomal damage is then
evaluated either through the photoaberration test (usually on
CHO cell line) or through the photomicronucleus test on
various cell types (immortalized CHO cells, V79 and normal
human skin cells). Because the photochromosomal aberra-
tion test (although considered as a regulatory test) is partic-
ularly difficult to perform and sensitive to the effect of cy-
totoxicity, the photomicronucleus test has been proposed as
a feasible alternative. The in vitro micronucleus test, in the
process of international validation, is by far more adapted
for screening purpose: it is easier and faster to carry out.
Moreover, the use of the micronucleus test renders photo-
induced clastogenic studies on human normal skin cells fea-
sible (studies which are very difficult with the chromosomal
aberration test because of the two-fold increase in the num-
ber of chromosomes to be analyzed, as compared with CHO
cells). Finally, the micronucleus test is recognized as the
only one being able to detect both clastogenic and aneugenic
events (75). These two kinds of events can then be distin-
guished using molecular gene probing technologies, such as
fluorescence in situ hybridization (76). A concordance of
80–95% has been found between these two tests.
Thus, the photomicronucleus test has been recommended
in the strategy for photochemical genotoxicity assessment in
the IWGTP workshop held in Washington in 1999 (6). UV
light (UVB, UVA as well as SSL) has been shown to induce
chromosomal damage in the micronucleus test (77,78). A
variety of photosensitizing compounds have been evaluated
in the photomicronucleus test on V79 cells (79) or on cir-
culating lymphocytes.
The manual scoring of the slides under a microscope is,
today, the major limitation for an extensive use of the pho-
tomicronucleus test for screening purpose. Several attempts
have been made to ease the reading of the results, either
through image analysis system or through flow cytometry.
The development of a reliable automatized system for scor-
ing would enable the routine use of photomicronucleus test
in screening programs for photoinduced genotoxicity of
chemical compounds (78).
CONCLUDING REMARKS
Among the toxicological sciences, phototoxicology appears
to be a quite new and emerging field, which is illustrated by
the relative lack of a definite regulatory framework for test-
ing as compared with other toxicological endpoints such as
chemical-induced mutagenesis. But phototoxicology is,
above all, quite unique among the toxicological fields be-
cause (1) the biological hazard of its etiologic agent, UV
light, is well characterized; and (2) the target organ, the skin,
is unique, quite well known and modeled in vitro through
the human reconstructed skin models. These characteristics
enable the development of in vitro tests on human skin cells
or on human reconstructed skin models, which can be con-
sidered as predictive for the photoprotective or phototoxic
effect of chemical compounds in vivo. Thus, the develop-
ment of in vitro methodology as an alternative to animal
testing is particularly advanced and relevant in the field of
phototoxicology.
Photobiological research conducted in numerous labora-
tories worldwide has permitted researchers to unravel, at the
cellular and molecular levels, the biological mechanisms of
photoinduced hazards. This fundamental research work en-
ables the determination of biological endpoints, which can
be used to set up in vitro tests. This review has aimed at
describing these various endpoints, from cytotoxicity and ap-
optosis to photomutagenesis and chromosomal damage,
through the assessment of primary DNA damage and cellular
response to UV-induced DNA lesions. For all these partic-
ular biological endpoints, specific in vitro approaches can be
used to set up a strategy for testing of photoinduced toxicity
and genotoxicity.
This review does not aim to propose a definite and stan-
dard strategy for testing but attempts to demonstrate the
strength of the mechanistically based in vitro methods on
mammalian cells for phototoxicology assessment. In this
context, one has to also consider other existing in vitro meth-
ods, which can be quicker and easier to perform than tests
on mammalian cells in culture and could be used in a pre-
screening step in a funnel-shaped strategy. Among these
steps, some use a bacterial system (Photo-Ames test), but
the extreme sensitivity of bacteria to UV light makes the use
of UV doses irrelevant when compared with human expo-
sure. The yeast Saccharomyces cerevisiae can also be used
as a quick and very informative model for photocytotoxicity
and photogenototoxicity assessment (80,81).
Finally, the use of the reconstructed skin models from the
second generation, including melanocytes, Langherans cells
(82) or cells defective for DNA repair (83), will enable the
development of new in vitro models that are always more
predictive and informative of the phototoxic potential of UV
light and of photosensitizing xenobiotics or more useful to
quantify the photoprotective effects of sunscreens.
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