[CANCER RESEARCH 47, 410-413, January 15, 1987]
Cytotoxicity and Mutagenicity of Low Intensity, 248 and 193 nm Excimer Laser
Radiation in Mammalian Cells1
Howard Green, James Boll, John A. Parrish, Irene E. Kochevar, and Allan R. Oseroff2
Department of Dermatology, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts 02114
ABSTRACT
The cytotoxicity of 193 and 248 nm excimer laser radiation was
compared to that produced by a germicida! lamp (predominantly 254 nm)
using Chinese hamster ovary cells (CHO), and a human diploid fibroblast
line, AG-1522A. Excimer laser radiation at 248 nm (3.5 x IO2w/m2) and
germicida! radiation (53 x 10~s w/m2) caused toxicity in both cell lines,
with the AG-1522A cells (D37= 7-8 ,1/nr) being slightly more sensitive
than the CHO cells (D37= 11 J/m2). Incident 193 nm radiation was less
cytotoxic than 248 nm to AG-1522A and CHO cells with D]7 values of
18 and 85 J/m2, respectively. The mutagenic potential of UV excimer
of the subablative doses
reach cells adjacent to
question by measuring
excimer laser radiation
193 and 248 nm radiation that would
the ablated area. We examined this
the cytotoxicity of 193 and 248 nm
to CHO3 cells and AG human fibro-
blasts using a clonogenic assay, and by quantifying the muta
genicity of 193 and 248 nm excimer laser radiation in CHO
cells with the HGPRT mutation assay.
MATERIALS
AND METHODS
radiation at 193 and 248 nm was evaluated using the hypoxanthine
guanine phosphoribosyl transfer assay system with CHO cells. Excimer
laser radiation at 248 nm induced mutation in proportion to dose (1.7 x
IIIs resistant colonies per survivor per J/m2 incident radiation) up to 14
J/m2, similar to results reported for 254 nm light. However, excimer
Cells. A subclone of the near diploid CHO cell line (AA-8, Ref. 15),
supplied by R. Reynolds (Harvard School of Public Health), was used
on the mutagenesis and cytotoxicity assays. A human diploid fibroblast
cell line (IMR-AG-1522A), passages 6-9, was employed for cytotoxicity
assays. Both cell lines were routinely maintained in Eagle's minimum
laser radiation at 193 nm did not cause mutation greater than the dark
control. The decreased cytotoxicity and mutagenicity of 193 nm radiation
may be due to the shielding of the nucleus by cytoplasmic and membrane
components or to the formation of different DNA photoproducts. These
differences between 193 and 248 nm radiation may be important in
choosing an excimer wavelength for ablation in biological systems.
essential medium supplemented with 10% fetal calf serum, 1% penicil
lin and streptomycin, 1% 1-glutamine, and 1% nonessential amino
acids (GIBCO). The respective population doubling times of the CHO
and AG cells were 14 and 24 h, and the colony forming efficiencies
were 75 and 25%, respectively. The thickness of the cytoplasmic layer
overlying the nucleus in the CHO and AG cells was estimated from
measurements of the location of intracellular focal planes using a
calibrated phase contrast microscope.
INTRODUCTION
Radiation Exposure
Using 193 or 248 nm radiation, the UV excimer laser pre
cisely cuts tissue by a process termed "ablative photodecomGermicidal. Far-UV germicidal radiation, predominantly at 254 nm,
pi >siiion." removing small volumes on the order of 0.1-1 /¿m was provided by three GE-G15T8 germicidal bulbs. The incident irradiance was measured with an IL-700 radiometer using a SCE 240
thick with each 15-20-ns pulse (1). Compared to most other
cosine-corrected detector (International Light, Newburyport, MA), and
laser sources, the pulsed UV radiation causes limited thermal,
was 0.53 w/m2. All medium was removed and cells were rinsed twice
denaturative damage to surrounding tissues. However, although
with phosphate-buffered saline immediately prior to exposure, which
much of the energy incident on the tissue is utilized by the was at 24°C,in a horizontal position with the plastic dish covers
ablation process, some photons are transmitted and scattered
removed. Sham exposures were used as controls.
into tissue surrounding the ablated zone (2, 3), where they may
Excimer Laser. Cells were irradiated with a Lambda Physik EMG103
cause genetic damage.
MSC excimer laser at 193 and 248 nm, with nominal pulse durations
The cytoxicity and mutagenicity of 254 nm radiation from
of 13 and 20 ns, respectively. Laser energy was first incident on a 5low pressure mercury (germicidal) lamps is well established (4). nirn diameter circular aperature, attenuated appropriately, and then
defocussed with a quartz lens to fully illuminate 8.5-cm diameter plastic
The cytotoxicity and mutagenicity of 248 nm radiation from
tissue culture dishes (Falcon) mounted vertically. The dishes were
the excimer laser to the surrounding viable cells can be expected
rotated at 24 rpm to enhance uniform irradiation. The energy per pulse
to be similar to that of 254 nm radiation at the same absorbed
was measured after the quartz lens with a Gentec ED-200 pyroelectric
dose, unless the higher irradiano.- from the laser produces
detector
whose output was read on an oscilloscope. The energy used
different DNA photoproducts. In contrast, the biological effects was 0.4 mJ
per pulse over an area of 57 cm2, corresponding to a fluente
of 193 nm radiation have not been well studied. DNA absorbs
of 7 x 10~2J/m2 per pulse and an irradiante of 5.4 x IO4w/m2 at 193
more strongly at 193 nm than at 254 nm, but the quantum
nm and 3.5 x IO4 w/m2 at 248 nm. A variable number of pulses were
yields of the photoproducts formed at the two wavelengths
delivered within either 15 or 60 s by adjusting the repetition rate to
differ (5). The peptide bond, many amino acid side chains,
expose the cells to a total of 4, 9, 14, 18, 25, 35, 55, 65, 75, or 85 J/
unsaturated lipids, esters, and other cellular molecules absorb
nr of 193 and 248 nm radiation. Sham exposures were used as controls.
at 193 nm (6-9). Consequently, the effects of 193 nm radiation
may differ significantly from those produced by 248 and 254 Cytotoxicity Determinations
nm light.
CHO or AG cells were trypsinized, counted, serially diluted, and
Because the excimer laser has potential clinical value as a plated on 8.5-cm2 diameter plastic tissue culture dishes (Falcon). The
surgical tool (1, 10-14), we were interested in the consequences
inocula of cells were adjusted to yield 70-100 surviving colonies per
Received 2/4/86; revised 9/2/86; accepted 10/14/86.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported in part by a grant from the Arthur O. and Gullan
M. Wellman Medical Foundation and NIH Grant AM 35092-08.
2 Present address: Department of Dermatology, New England Medical Center,
Boston, MA 02111. To whom requests for reprints should be addressed.
dish after exposure. Three dishes were used for each dose. Prior to
exposure, the dishes were allowed to stand for 4-5 h at 37°Cto permit
attachment of the single cells to the surface. The medium was then
removed and the cells were rinsed twice with phosphate buffered saline
3The abbreviations used are: CHO, Chinese hamster ovary; AG, AG-1522A;
HGPRT, hypoxanthine guanine phosphoribosyl transferase; 6-TG, 6-thioguanine.
410
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MUTAGENICITY
BY EXCIMER
and irradiated without salt solution or medium. After the irradiation,
fresh medium was added and the CHO and AC cells were incubated
for 7 and 14 days, respectively, after which the colonies were scored.
The effect of excimer laser and germicida! radiation exposure on the
clonal growth was expressed as percentage of survival relative to unirradiated, sham-exposed controls. Two to four experiments were per
formed at each wavelength, and the data expressed as an average ±
standard error of the mean.
Mutagenesis Assay
The CHO/HGPRT assay was used to evaluate the mutagenicity of
excimer laser radiation at 193 and 248 nm (16, 17). After innoculation
and attachment, CHO cells were incubated at least 8 h prior to laser
irradiation. The cells were in exponential growth, with initial cell
numbers chosen to provide a minimum of 2 x 10s survivors per dose
after radiation exposure.
The irradiated cells were allowed to express the mutant phenotype
in growth medium for 8 days (with subculture every 2 days), which is
optimum for this mutation assay (17). To prevent cell-to-cell interac
tions, the cell density during subculture was 2 x IO5 cells/100-cm2
plastic dish. Five dishes, each containing 2 x IO5 treated cells, were
then plated in 100-cm2 plastic dishes containing growth medium with
10 um 6-TG presented as the selective agent. For each experiment,
cloning efficiency was simultaneously determined using sham-exposed
and irradiated cells and growth medium without 6-TG.
RESULTS
Cytotoxicity of 254 nm (Germicida!), and 193 and 248 nm
Excimer Laser UV Radiation. The dose-dependent inhibition of
the colony forming ability of CHO and AG cells after various
radiation exposures was determined. For the CHO cells, the
survival curves for the 248-nm excimer laser and the 254-nm
germicidal lamp were similar in shape, with well-defined shoul
der regions at low light doses, and exponential declines at higher
doses (Fig. IB). The D37(dose at which 37% of the cells survive)
was also approximately equal (11 J/m2). In contrast, 193 nm
radiation was almost 8-fold less cytotoxic (D37, approximately
85 J/m2).
Similar irradiation of the human fibroblast cells (AG) gave
D37 values of 8 and 7 J/m2 for 248 and 254 nm radiation,
respectively (Fig. IB). At 193 nm, the D37 for AG cells was
approximately 18 J/m2. Thus the difference in cytotoxicity
between 248 and 193 nm excimer laser radiation was much less
for AG than for CHO cells. The AG cells were more light
sensitive at all three wavelengths. At 248 and 254 nm, the AG
D37 value is about a factor of 1.5 lower than that for the CHO
cells, and a factor of 4.7 lower at 193 nm.
Mutagenicity. To examine the relative mutagenicity of 193
and 248 nm excimer laser radiation, the induction of resistance
to 6-thioguanine in CHO cells was studied. Normal CHO cells
are sensitive to 6-TG, and resistance arises through forward
mutations in the structural gene for hypoxanthine guanine
phosphoribosyl transferase (HGPRTase) (18). In Fig. 2, the
induced mutation frequency is plotted as a function of total
incident energy. Laser radiation at 248 nm induced mutation
in proportion to dose up to 14 J/m2. From 14 to 25 J/m2 the
mutation frequency was constant at about 2.5 x 10~4 TG
resistant colonies per survivor. Excimer laser radiation at 193
nm did not induce mutations above the level of spontaneous
background frequency. Thus, as in the case of the cytotoxicity
determinations, 248 nm excimer laser radiation had much
greater effects than 193 nm radiation.
DISCUSSION
The potential for precise control over tissue removal with
minimal thermal damage to the cells surrounding the ablation
LASER RADIATION
zone makes the excimer laser attractive in many areas of
medicine and surgery (1, 10-14). In cancer therapies, the exci
mer laser might permit ablation of lesions in critical locations
with minimal damage to adjacent normal tissues. However, the
biological risks of the excimer radiation have not been well
studied, particularly at 193 nm where the ablation is most
precisely controlled (1, 10, 13, 14).
Based solely on the absorption spectrum of DNA it was
expected that 193 nm radiation would be more cytotoxic than
248 nm, since the DNA absorbance is at least twice as high at
the shorter wavelength (6,7). However, our results indicate that
193 nm is less toxic to both CHO and AG cells. Between the
two cell types, the relatively rounded CHO cells were less
affected than the flatter fibroblasts. This result may be due to
competitive absorption of 193 nm radiation by cellular constit
uents, including peptide bonds and aromatic amino acids (8),
and unsaturated carbon-carbon bonds and esters (9), thus lim
iting the photon dose reaching the nucleus. In addition to
possible intrinsic differences between the two cell lines, the
greater sensitivity of the AG cells to both 193 and 248 nm
radiation may be due to differences in the thickness of the
cytoplasmic layer overlying the nucleus. In culture, the AG cells
have an umbonate, or flattened configuration; their nuclei ap
pear covered by only about l /um Of cytoplasm. The more
rounded CHO cells appear to have at least 3 nm of cytoplasm
over the nucleus.
Since accurate measurements of cytoplasmic absorption at
193 nm are not available, the effect of the cytoplasm must be
estimated from the extinction coefficients and concentrations
of absorbing components. The peptide bond absorbs at 193 nm
(e = 3 x IO3 M~') but not at 248 nm, and the aromatic amino
acids also absorb more strongly at 193 nm (e = 0.25-3.5 x 10"
M"1 cm~') than at 248 nm (c = 0.1-2 X IO3 M"1 cm"1) (4, 8).
Assuming that the cytoplasm contains a minimum of 10%
protein by weight, the concentration is 100 mg/ml. Based on
an average molecular weight for amino acids of 120, the average
concentration of peptide bonds is about 0.8 M. If the proteins
contain 9% aromatic amino acids equally distributed among
tryptophan, tyrosine, and phenylalanine, the concentration of
these species is on the order of 0.08 M, and the average extinc
tion coefficient is 2 x 10~4 M"' cm"1 at 193 nm. Then the
absorbance (A) per micron of cytoplasmic path length is ap
proximately: (3 x IO3 x 0.8 + 2 x IO4 x 0.08) x 10~" = 0.4
M~'Thus, about 60% of the incident 193 nm radiation is absorbed
by 1 Ã-¿m
of cytoplasm. Including effects of the membrane, a
similar recent calculation estimated that 87% of the incident
radiation at 200 nm is absorbed within 1 /<m of the cell surface
(19). Based on our estimates, a difference of only 1.7 firn in
cytoplasmic thickness could account for the =4.7 times greater
cytotoxicity of 193 nm radiation to AG cells compared to CHO
cells. Because of the extranuclear absorption, the 193 nm cy
totoxicity also may be at least in part due to cytoplasmic or
plasma membrane damage, particularly in cells with large cy
toplasmic volumes (19-22).
At 248 nm, there is an estimated ratio of 1.7 between um
bonate and rounded cells in the amount of radiation reaching
the nucleus (19). Our observed factor of 1.5 in cytotoxicity
between AG and CHO cells agrees well with this estimate and
with the work of Coohill et al., who found about a factor of 1.3
greater sensitivity to 254 nm radiation between flattened and
spherical CV-1 monkey kidney epithelial cells (23).
In the mutagenesis assay, our results using 248 nm laser
radiation are similar to those obtained by others for germicidal
411
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MUTAGENICITY
BY EXCIMER
LASER RADIATION
100
100
20
3O
40
50
60
UV Exposur«
DOM J/m*
70
SO
20
30
40
SO
UV Expotur« J/m2
60
70
Fig. 1. A, Surviving fraction of CHO cells after UV radiation. B, Surviving fraction of AG-1522A cells. (O) 254-nm germicida! lamp; (A) 248-nm excimer laser;
(O) 193-nm excimer laser.
absorption length for 193 n m radiation in tissue may confine
its direct genotoxic effects within the zone of thermal damage
(11). However, it is important to note that our experiments
would not detect potentially mutagenic and carcinogenic abla
tion products which might be produced in vivo during ablation
of significant thickness of tissue.
In conclusion, our data demonstrate that: (a) the cytotoxicity
and mutagenicity of 248 nm excimer laser radiation is about
the same as that caused by germicida! 254 nm radiation despite
the Hf-ibld higher irradiance of the laser, and (¿>)193 nm
radiation appears to be much less cytotoxic and genotoxic than
248 nm excimer laser radiation. Since both wavelengths can be
used for precision ablation of tissue, the greater mutagenic
potential of 248 nm radiation must be considered in the risk
assessment.
20
30
40
50
60
UV Expotur* ( J/m2 )
TO
so
90
ACKNOWLEDGMENTS
We thank Richard Reynolds and Thomas F. Deutsch for helpful
discussions, and Coherent Radiation, Inc., for providing the excimer
laser.
Fig. 2. Frequency of 6-thioguanine (TG) resistant mutants in CHO cells after
varying doses of excimer laser radiation at 248 nm (O), and at 193 nm (•).
radiation in the CHO/HGPRT
system (24, 25). The lack of
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413
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Cytotoxicity and Mutagenicity of Low Intensity, 248 and 193 nm
Excimer Laser Radiation in Mammalian Cells
Howard Green, James Boll, John A. Parrish, et al.
Cancer Res 1987;47:410-413.
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