Vol. 90, No. 2, 1979 September
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
27, 1979
Pages 460-465
PHOTODYNAMIC MUTAGENICITY Nachman Gruener*
MAMMALIAN CELLS
IN
and Michael
P. Lockwood
Tulane University School of Public Health and Tropical Department of Environmental Health New Orleans, Louisiana 70112 Received
August
Medicine Sciences
lo,1979 SUMMARY
Photoactivation of bound rose bengal in the presence of oxygen causes mutations in Chinese hamster embryo cells. Visible light by itself can also cause a slight increase in mutation frequency. Both reactions are amplified by deuterium oxide. Singlet oxygen reactive compounds, B-carotene and 1,3diphenylisobenzofuran diminish the toxic and mutagenic effects. 12-0tetradecanoyl-phorbol-13-acetate, a potent tumor promoter, increases the number of mutants induced by the photodynamic action. The enhancement of mutagenesis by deuterium oxide and its reduction by specific singlet oxygen antagonists suggest that this active oxygen species is the direct mutagen. Active hydroxyl
species
radicals
and are glet
oxygen
toxic
oxygen
are
(lO2)
absorbtion
of light,
C30,).
This
oxygen
(102)
and is
energy
then
transfer
and ground
state
1 3
*To whom correspondence
systems
anions
biological
processes
in vivo
or in vitro
spin
to the
inversion
by ground
reaction
results
sensitizer
(1,2),
(3,4).
Sin-
relatively
state in the
is
excited
short-lived
relatively triplet formation
by the singlet
long-lived molecular
oxygen
of singlet
(Sen).
Sen A>
'Sen
Sen p>
3 Sen > Sen + '0
Sen + 302 __
should
(Sen)
to the
quenched
and
of many photosensitized
a photosensitizer
transformed
1Sen undergoes is
in normal
superoxide
shown to be the mediator
In such reactions
3 Sen, which
species,
oxygen,
to be formed
biological
has been
(5).
(ISen).
known
to various
oxidations
state
such as singlet
2
be addressed.
PBS, phosphate buffered saline (pH, 7.4); DPBF, 1,3-diphenylisoAbbreviations: benzofuran; TPA, 12-O-tetradecanoyl-phorbol-13-acetate; DMEM, Dulbecco's Modified fetal calf serum (56OC, 30 min.); Minimum Essential Medium; IFBS, inactivated Sen, photosensitizer. 0006-291X/79/180460-06$01.00/0 Copyright All rights
@ I979
by Academic Press, Inc. in anyform reserved.
of reproduction
460
BIOCHEMICAL
Vol. 90, No. 2, 1979
The sensitizer ground
state
The excited
is a photoreactive exceeds 102 is
material
22 Kcal/mole., then
AND BIOPHYSICAL
the
available
for
whose
AE between
AE between
reactions
RESEARCH COMMUNICATIONS
with
the triplet
oxygen's
'02
suitable
and
and 302 states.
oxidizable
mole-
cules. In the induce
following
mutations
paper
we will
in mammalian
present
evidence
that
singlet
oxygen
can
cells.
MATERIALS
AND METHODS
Chemicals. Chemicals and cell culture reagents were purchased as follows : Sensitox II, (the photoreactive rose bengal immobilized on polystyrene beads) from Hydron Laboratories (New Brunswick, N.J.); B-carotene, deuterium oxide (D20), and ouabain from Sigma Chemicals (St. Louis, MO.). 1,3-diphenylisobenzofuran from Aldrich (Milwaukee, Wis.); and all cell culture media and reagents from GIBCO (Grand Island, N.Y.). 12-O-tetradecanoyl-phorbol-13acetate was kindly supplied by R.K. Boutwell. Cell Culture. Chinese hamster embryonic lung cells (V79) were cultured in DMEM (5) supplemented with 10% IFBS. Cells were maintained at 37OC in a 10% CO2 atmosphere. Subcultures were performed using 0.25% trypsin in 0.01% ethylened-iaminetetraacetic acid. Ouabain resistant mutants were isolated from V79 Mutagenesis Assays. cells which had been exposed to the bound rose bengal in the presence of light. Briefly, 3X106 V79 cells were plated in 60 mm plastic petri dishes (Falcon) 16 h prior to treatment, in 4 ml DMEM plus 10% IFBS. Cells were washed and exposed to 100 mgfplate of the bound rose bengal suspended in 2 ml PBS at The light source was a 15 watt 24OC in the presence and absence of light. fluorescent lamp (Buchler Instruments) approximately 20 cm above the plates, yielding 150 ft-candles at the cell s,urface. The light caused no increase in temperature under these conditions. After exposure, the plates were washed three times with PBS. Each plate was then trypsinized, counted and replated in 10 plates with 3X105 cells per plate in 4 ml DMEM plus 10% IFBS. One hundred cells were also seeded in each of 4 plates per point for determination of cytotoxic effect. Forty-eight hours after treatment, ouabain (final concentration, 1mM) was added to plates being assayed for mutagenicity. Seven days after plating, cytotoxicity was determined by cloning efficiency in those plates previously seeded with 100 cells. Cells were fixed in methanol and stained with Giemsa. Plates being assayed for ouabain-resistant mutants were Ouabain resistant mutants isolated fixed and stained 14 days after treatment. at random and grown for one month without ouabain were found to be resistant to different concentrations up to 1mM. B-carotene, DPBF and TPA stock solutions were prepared in acetone immediately before testing. Acetone levels did not exceed 0.02% per plate. When the effects of D20 were examined, it was substituted for the H20 in PBS. RESULTS AND DISCUSSION Data presented induced
a slight
in increase
Fig.
la shows that in the
number
461
illumination of mutants
of V79 cells above
in PBS
the background.
The
Vol. 90, No. 2, 1979
BIOCHEMICAL
AND BlOPHYSlCAL
TIME
RESEARCH COMMUNICATIONS
hn)
Fig. 1. Time course of photodynamic mutagenicity. Assays were performed as described under Materials and Methods for the following conditions: D20 in the dark (A A), PBS in light (0 n ), D20 in light (0 a), Sen in light (00): Sen and D20 in light (0 4). Open symbols indicate plating efficiency and closed symbols indicate mutation frequency. aMutation frequency is expressed as number of mutants per lo6 surviving cells.
exchanged presence
of Hz0 with of light.
Others
have also
H20 medium
toxic
lethality
D20 in the dark
is
somehow
found
leads
that
Light which
Previous chemicals
extended
to an increase
mechanism.
such as flavins
studies
may excite
shown that
To test
a bound
photosensitizer.
the
dye is
and is
unequivocally Control
not
eluted
recovered
easily
from
oxygen
is
omitted
(9,lO).
(Table
for
the
experiments
(data
beads
medium after
However,
462
in cells
in the
compounds
oxygen
1
to produce
02.
photosensitive
The possibility
that
lo2
DNA damage could be 1 O2 is the mutagen we used not presented)
to the medium,
in the presence 1).
involved
of soluble
that
from the
can be demonstrated
or light
irradiation
light
of mammalian
O2 is
state
the hypothesis
the
or mutagenicity
that
by ground
responsible
to visible
frequency 1
in the
mutagenic.
photosensitive
in bacteria
are
but not
of exposure
intracellular
can be scavenged
or both
toxic
of D20 suggests
can cause mutagenicity
implied.
periods
and mutagenicity
in the mutation
effect
have
or a dye derivative
cells
cell
The enhancement
(7,S).
that
D2 0 enhanced
cannot
incubation. of the
indicated penetrate
the
No toxicity
dye when either
in the presence
of all
three
BIOCHEMICAL
Vol. 90, No. 2, 1979
Table
1.
The
enhancement
AND BIOPHYSICAL
and
inhibition in
Treatment
Plating
V79
of
RESEARCH COMMUNICATIONS
photodynamic
mutagenicity
cells.
efficiency
Number per
(%I
of
106 cells plated
mutants
per
106 surviving cells
94
0.3
0.3
92
0.6
0.6
81
1.3
1.4
29
4.0
12.8
72
0.3
1.3
64
1.3
2.4
5mM DPBFb
80
1.3
1.6
Sen + 5mM DPBFb
38
2.6
6.5
Control
(full
medium)a
Sena Sen
(N2 atmosphere)b
Senb 0.1
mM B-caroteneb
Sen + 0.1
mM fi-caroteneb
Senb + TPA (0.1
ug/ml)c
28
8.0
26.6
Senb
pg/ml)c
24
10.0
39.1
+ TPA (0.5
The experimental conditions are described under Materials aThese treatments were done in the dark. bCells were exposed to these compounds for 30 min. in the %A was added immediately after exposure and was present of the experiment. factors,
02 and light),
(Sen,
a pronounced
increased
with
exposure
chemical
species
is
produced
outside
by light
and depends
on the
presence
hypothesis causes
that
'02
which
the mutagenicity,
lifetime
of
toxicity
and mutagenicity
a specific
'02
is
IO2 scavenger
and mutagenicity
been
shown to increase
enhanced
while
not
(Fig.
being
'02 mutagenicity
lb).
It
the
H20 with
by a factor
(12) effects. mutation itself
effect
feasible
a mutagen
in V79 cells.
463
is
To support
(Fig.
cancer
induced (14).
that
formation
In this
which a
catalyzed the
medium,
(ll), the
and as expected,
1 02 trapper
TPA, a potent frequency
D20.
increased
and DPBF an
found
2 ps in H20 medium
of 10 (ll),
impressively
is
of light. remainder
to assume
and its
of only
Methods.
presence for the
of Sen and oxygen.
we exchanged
are
is
cell,
has a lifetime
increased
city
cells,
time
mutagenic
and
(13)
lb).
S-carotene,
inhibit
promoter
by mutagens
the
the
toxi-
has recently in mammalian
As shown in Table
1, TPA also
Vol. 90, No. 2, 1979
A major
question
studies
in bacterial
not
carcinogens
all
cyclic
produced
(15) are
skin
(18).
has been
may result recently These
further
It
with
whereas, U.V.
study: (16)
that
that
indicate
accumulation
that
cancer the role
Early
(19)
compared
work
by U.V.
of singlet
might
oxygen
that
compounds
sensitizer
alone
gave
noted
skin
tumors
that
controls.
in human skin
Bodaness
if
poly-
showed
to untreated
of porphyrins
caused
most
many carcinogenic
nor
(18).
Recent
shown that
of photosensitive
Epstein mice
a carcinogen?
have
that
illumination
irradiation,
'02
(17).
solutions
neither
RESEARCH COMMUNICATIONS
systems
activity
carcinogenesis skin
Is
was found
in B-carotene-treated
in photodynamic
studies
warrants
mutagens.
After
suggested
suggested
this
and mammalian
injected
tumors,
slower
from
AND BIOPHYSICAL
show photodynamic
of mice
effect
developed It
evolves
hydrocarbons
illumination
this
BIOCHEMICAL
with
and Chan (20) be mediated
by
aging have 1 02.
in carcinogenesis
investigation.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Boss, N., Saran, M., Lengfelder, E., Michel, C., Fuchs, F., and Frenzel, C. (1978) Photochem. Photobiol. 28, 629-638. Foote, C.S. (1976) in Free Radicals in Biology, Pryor, N.A., Ed., pp. 85133, Academic Press, New York. Ito, T. (1978) Photochem. Photobiol. 28, 493-508. Rahimtula, A.D., Hawco, F.J., and O'Brien, P.J. (1978) Photochem. Photobiol. 28, 811-815. Smith, J.D., Freeman, G., Vogt, M., and Dulbecco, R. (1960) Virology 12, 185 196. Spikes, J.D. (1977) in The Science of Photobiology, Smith, K.C., Ed., pp. 87-112, Plenum Press, New York and London. Burki, H.J., and Lam, C.K. (1978) Mutation Res. 54, 373-377. Jacobson, E.D., Krell, K., Dempsey, M.J., Lugo, M.H., Ellingson, O., and Hench, C.W. (1978) Mutation Res. 51, 61-75. Imray, P.F., and Macphee, D.G. (1975) Mutation Res. 27, 299-306. Gutter, B., Speck, W.T., and Rosenkranz, H.S. (1977) Mutation Res. 44, 177-182. Merkel, P.B., and Kearns, D.R. (1972) J. Am. Chem. Sot. 94, 7244-7253. Foote, C-S., and Denny, R.W. (1968) J. Am. Chem. Sot. 90, 6233-6235. Matheson, I.B.C., and Lee, J. (1970) Chem. Phys. Lett. 7, 475-476. Lancas, G.R., Baxter, C.S., and Christian, R.T. (1978) J. Toxicology and Environmental Health 4, 31-36. McCann, J., Choi, E., Yamasaki, E., and Ames, B.N. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 5135-5139. Hsie, A.W., Couch, B.D., San Sebastian, J.R., Sun W.N.C., Riddle, J.C., Brimes, P.A., Machanoff, R., Forber, N-L., Kenny, R.T., and Hsie, M.H. (1978) Proc. Am. Assoc. Cancer Res. 19, 93. Malling, H.V., and Chu, E.H.Y. (1970) Cancer Res. 30, 1236-1240.
464
Vol. 90, No. 2, 1979
18. 19. 20.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Santamaria, L., and Prino, G. (1964) in Research Progress in Organic, Biological and Medicinal Chemistry, Gallo, U. and Santamaria, L., Ed., pp. 260-336, Soueta Editoriale Farmacentica, Milan. Epstein, J.H. (1977) Photochem. and Photobiol. 16, 117-119. Bodaness, R.S., and Chan, P.C. (1979) Biochem. and Biophys. Res. Commun. 87, 1116-1123.
465
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
27, 1979
Pages 460-465
PHOTODYNAMIC MUTAGENICITY Nachman Gruener*
MAMMALIAN CELLS
IN
and Michael
P. Lockwood
Tulane University School of Public Health and Tropical Department of Environmental Health New Orleans, Louisiana 70112 Received
August
Medicine Sciences
lo,1979 SUMMARY
Photoactivation of bound rose bengal in the presence of oxygen causes mutations in Chinese hamster embryo cells. Visible light by itself can also cause a slight increase in mutation frequency. Both reactions are amplified by deuterium oxide. Singlet oxygen reactive compounds, B-carotene and 1,3diphenylisobenzofuran diminish the toxic and mutagenic effects. 12-0tetradecanoyl-phorbol-13-acetate, a potent tumor promoter, increases the number of mutants induced by the photodynamic action. The enhancement of mutagenesis by deuterium oxide and its reduction by specific singlet oxygen antagonists suggest that this active oxygen species is the direct mutagen. Active hydroxyl
species
radicals
and are glet
oxygen
toxic
oxygen
are
(lO2)
absorbtion
of light,
C30,).
This
oxygen
(102)
and is
energy
then
transfer
and ground
state
1 3
*To whom correspondence
systems
anions
biological
processes
in vivo
or in vitro
spin
to the
inversion
by ground
reaction
results
sensitizer
(1,2),
(3,4).
Sin-
relatively
state in the
is
excited
short-lived
relatively triplet formation
by the singlet
long-lived molecular
oxygen
of singlet
(Sen).
Sen A>
'Sen
Sen p>
3 Sen > Sen + '0
Sen + 302 __
should
(Sen)
to the
quenched
and
of many photosensitized
a photosensitizer
transformed
1Sen undergoes is
in normal
superoxide
shown to be the mediator
In such reactions
3 Sen, which
species,
oxygen,
to be formed
biological
has been
(5).
(ISen).
known
to various
oxidations
state
such as singlet
2
be addressed.
PBS, phosphate buffered saline (pH, 7.4); DPBF, 1,3-diphenylisoAbbreviations: benzofuran; TPA, 12-O-tetradecanoyl-phorbol-13-acetate; DMEM, Dulbecco's Modified fetal calf serum (56OC, 30 min.); Minimum Essential Medium; IFBS, inactivated Sen, photosensitizer. 0006-291X/79/180460-06$01.00/0 Copyright All rights
@ I979
by Academic Press, Inc. in anyform reserved.
of reproduction
460
BIOCHEMICAL
Vol. 90, No. 2, 1979
The sensitizer ground
state
The excited
is a photoreactive exceeds 102 is
material
22 Kcal/mole., then
AND BIOPHYSICAL
the
available
for
whose
AE between
AE between
reactions
RESEARCH COMMUNICATIONS
with
the triplet
oxygen's
'02
suitable
and
and 302 states.
oxidizable
mole-
cules. In the induce
following
mutations
paper
we will
in mammalian
present
evidence
that
singlet
oxygen
can
cells.
MATERIALS
AND METHODS
Chemicals. Chemicals and cell culture reagents were purchased as follows : Sensitox II, (the photoreactive rose bengal immobilized on polystyrene beads) from Hydron Laboratories (New Brunswick, N.J.); B-carotene, deuterium oxide (D20), and ouabain from Sigma Chemicals (St. Louis, MO.). 1,3-diphenylisobenzofuran from Aldrich (Milwaukee, Wis.); and all cell culture media and reagents from GIBCO (Grand Island, N.Y.). 12-O-tetradecanoyl-phorbol-13acetate was kindly supplied by R.K. Boutwell. Cell Culture. Chinese hamster embryonic lung cells (V79) were cultured in DMEM (5) supplemented with 10% IFBS. Cells were maintained at 37OC in a 10% CO2 atmosphere. Subcultures were performed using 0.25% trypsin in 0.01% ethylened-iaminetetraacetic acid. Ouabain resistant mutants were isolated from V79 Mutagenesis Assays. cells which had been exposed to the bound rose bengal in the presence of light. Briefly, 3X106 V79 cells were plated in 60 mm plastic petri dishes (Falcon) 16 h prior to treatment, in 4 ml DMEM plus 10% IFBS. Cells were washed and exposed to 100 mgfplate of the bound rose bengal suspended in 2 ml PBS at The light source was a 15 watt 24OC in the presence and absence of light. fluorescent lamp (Buchler Instruments) approximately 20 cm above the plates, yielding 150 ft-candles at the cell s,urface. The light caused no increase in temperature under these conditions. After exposure, the plates were washed three times with PBS. Each plate was then trypsinized, counted and replated in 10 plates with 3X105 cells per plate in 4 ml DMEM plus 10% IFBS. One hundred cells were also seeded in each of 4 plates per point for determination of cytotoxic effect. Forty-eight hours after treatment, ouabain (final concentration, 1mM) was added to plates being assayed for mutagenicity. Seven days after plating, cytotoxicity was determined by cloning efficiency in those plates previously seeded with 100 cells. Cells were fixed in methanol and stained with Giemsa. Plates being assayed for ouabain-resistant mutants were Ouabain resistant mutants isolated fixed and stained 14 days after treatment. at random and grown for one month without ouabain were found to be resistant to different concentrations up to 1mM. B-carotene, DPBF and TPA stock solutions were prepared in acetone immediately before testing. Acetone levels did not exceed 0.02% per plate. When the effects of D20 were examined, it was substituted for the H20 in PBS. RESULTS AND DISCUSSION Data presented induced
a slight
in increase
Fig.
la shows that in the
number
461
illumination of mutants
of V79 cells above
in PBS
the background.
The
Vol. 90, No. 2, 1979
BIOCHEMICAL
AND BlOPHYSlCAL
TIME
RESEARCH COMMUNICATIONS
hn)
Fig. 1. Time course of photodynamic mutagenicity. Assays were performed as described under Materials and Methods for the following conditions: D20 in the dark (A A), PBS in light (0 n ), D20 in light (0 a), Sen in light (00): Sen and D20 in light (0 4). Open symbols indicate plating efficiency and closed symbols indicate mutation frequency. aMutation frequency is expressed as number of mutants per lo6 surviving cells.
exchanged presence
of Hz0 with of light.
Others
have also
H20 medium
toxic
lethality
D20 in the dark
is
somehow
found
leads
that
Light which
Previous chemicals
extended
to an increase
mechanism.
such as flavins
studies
may excite
shown that
To test
a bound
photosensitizer.
the
dye is
and is
unequivocally Control
not
eluted
recovered
easily
from
oxygen
is
omitted
(9,lO).
(Table
for
the
experiments
(data
beads
medium after
However,
462
in cells
in the
compounds
oxygen
1
to produce
02.
photosensitive
The possibility
that
lo2
DNA damage could be 1 O2 is the mutagen we used not presented)
to the medium,
in the presence 1).
involved
of soluble
that
from the
can be demonstrated
or light
irradiation
light
of mammalian
O2 is
state
the hypothesis
the
or mutagenicity
that
by ground
responsible
to visible
frequency 1
in the
mutagenic.
photosensitive
in bacteria
are
but not
of exposure
intracellular
can be scavenged
or both
toxic
of D20 suggests
can cause mutagenicity
implied.
periods
and mutagenicity
in the mutation
effect
have
or a dye derivative
cells
cell
The enhancement
(7,S).
that
D2 0 enhanced
cannot
incubation. of the
indicated penetrate
the
No toxicity
dye when either
in the presence
of all
three
BIOCHEMICAL
Vol. 90, No. 2, 1979
Table
1.
The
enhancement
AND BIOPHYSICAL
and
inhibition in
Treatment
Plating
V79
of
RESEARCH COMMUNICATIONS
photodynamic
mutagenicity
cells.
efficiency
Number per
(%I
of
106 cells plated
mutants
per
106 surviving cells
94
0.3
0.3
92
0.6
0.6
81
1.3
1.4
29
4.0
12.8
72
0.3
1.3
64
1.3
2.4
5mM DPBFb
80
1.3
1.6
Sen + 5mM DPBFb
38
2.6
6.5
Control
(full
medium)a
Sena Sen
(N2 atmosphere)b
Senb 0.1
mM B-caroteneb
Sen + 0.1
mM fi-caroteneb
Senb + TPA (0.1
ug/ml)c
28
8.0
26.6
Senb
pg/ml)c
24
10.0
39.1
+ TPA (0.5
The experimental conditions are described under Materials aThese treatments were done in the dark. bCells were exposed to these compounds for 30 min. in the %A was added immediately after exposure and was present of the experiment. factors,
02 and light),
(Sen,
a pronounced
increased
with
exposure
chemical
species
is
produced
outside
by light
and depends
on the
presence
hypothesis causes
that
'02
which
the mutagenicity,
lifetime
of
toxicity
and mutagenicity
a specific
'02
is
IO2 scavenger
and mutagenicity
been
shown to increase
enhanced
while
not
(Fig.
being
'02 mutagenicity
lb).
It
the
H20 with
by a factor
(12) effects. mutation itself
effect
feasible
a mutagen
in V79 cells.
463
is
To support
(Fig.
cancer
induced (14).
that
formation
In this
which a
catalyzed the
medium,
(ll), the
and as expected,
1 02 trapper
TPA, a potent frequency
D20.
increased
and DPBF an
found
2 ps in H20 medium
of 10 (ll),
impressively
is
of light. remainder
to assume
and its
of only
Methods.
presence for the
of Sen and oxygen.
we exchanged
are
is
cell,
has a lifetime
increased
city
cells,
time
mutagenic
and
(13)
lb).
S-carotene,
inhibit
promoter
by mutagens
the
the
toxi-
has recently in mammalian
As shown in Table
1, TPA also
Vol. 90, No. 2, 1979
A major
question
studies
in bacterial
not
carcinogens
all
cyclic
produced
(15) are
skin
(18).
has been
may result recently These
further
It
with
whereas, U.V.
study: (16)
that
that
indicate
accumulation
that
cancer the role
Early
(19)
compared
work
by U.V.
of singlet
might
oxygen
that
compounds
sensitizer
alone
gave
noted
skin
tumors
that
controls.
in human skin
Bodaness
if
poly-
showed
to untreated
of porphyrins
caused
most
many carcinogenic
nor
(18).
Recent
shown that
of photosensitive
Epstein mice
a carcinogen?
have
that
illumination
irradiation,
'02
(17).
solutions
neither
RESEARCH COMMUNICATIONS
systems
activity
carcinogenesis skin
Is
was found
in B-carotene-treated
in photodynamic
studies
warrants
mutagens.
After
suggested
suggested
this
and mammalian
injected
tumors,
slower
from
AND BIOPHYSICAL
show photodynamic
of mice
effect
developed It
evolves
hydrocarbons
illumination
this
BIOCHEMICAL
with
and Chan (20) be mediated
by
aging have 1 02.
in carcinogenesis
investigation.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
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