- Email: [email protected]
Photorepair of ultraviolet radiation-induced pyrimidine dimers in corneal DNA
Mutation Research, 194 (1988) 49-55 DNA Repair Reports Elsevier
49
MTR 06284
Photorepair of ultraviolet radiation-induced pyrimidine dimers in corneal D N A R o n a l d D . L e y , L e e A . A p p l e g a t e a n d S t e v e n E. F r e e m a n Photocarcinogenesis Program, Division of Biomedical Research, Lovelace Medical Foundation, 2425 Ridgecrest Drive, S.E., Albuquerque, N M 87108 (U.S.A.) (Received 19 June 1987) (Revision received 18 January 1988) (Accepted 22 January 1988)
Keywords: Pyrimidine dimers; Cornea; Photorepair; Marsupial; Nucleases; Monodelphis domestica; Micrococcus luteus
Summary The induction and photorepair of pyrimidine dimers in D N A have been measured in the ultravioletirradiated, corneal epithelium of the marsupial, Monodelphis dornestica, using damage-specific nucleases from Micrococcus luteus in conjunction with agarose gel electrophoresis. We observed that FS-40 sunlamps (280-400 nm) induced 7.2 _+ 1.0 × 10 -5 pyrimidine dimers per kilobase (kb) of D N A per J / m 2. Following 100 J / m 2, 50% and > 90% of the dimers were photorepaired during a 10- and 30-min exposure to photoreactivating light (320-400 nm), respectively. In addition, - 70% and - 60% of the dimers induced by 300 and 500 J / m 2, respectively, were repaired by a 60-min exposure to photoreactivating light. The capacity of the corneal epithelium of M. domestica to photorepair pyrimidine dimers identifies this animal as a potentially useful model with which to determine whether pyrimidine dimers are involved in pathological changes of the irradiated eye.
Ultraviolet radiation (UVR)-induced photodamage of the eye has been observed in both humans and experimental animals. Photokeratitis, acute corneal injury, occurs in individuals who use arc welders (National Institute for Occupational Safety and Health, 1972) and from intensive exposure to U V R from sunlight reflected off surfaces such as snow or sand (Diffey, 1982). Ultraviolet radiation-induced corneal damage has also been
Correspondence: Dr. Ronald D. Ley, Division of Biomedical Research, Lovelace Medical Foundation, 2425 Ridgecrest Drive, S.E., Albuquerque, NM 87108 (U.S.A.).
extensively studied in primates and rabbits (Pitts, 1974; Pitts and Tredici, 1971). Ultraviolet radiation induces a number of lesions in DNA, the most prevalent being a cyclobutane-type dimer between adjacent pyrimidines on the same D N A strand (Patrick and Rahn, 1976). Pyrimidine dimers can be split in situ by a light-dependent repair process called photoreactivation (PR). This repair pathway requires the presence of the PR enzyme and exposure to longwavelength visible radiation in the range of 320-500 nm (Cook, 1970). Photoreactivation has been reported to occur in human (Sutherland et al., 1980; D'Ambrosio et al., 1981; Eggset et al., 1983) and marsupial (Ley, 1984) skin, but is much
0167-8817/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
50 less evident in mice (Ley, et al., 1978). The specificity of the PR repair pathway for pyrimidine dimers (Setlow and Setlow, 1963) has been used to identify the lesion as a major cause of lethal and mutagenic (Harm, 1976), tumorigenic (Hart et al., 1977), and transformational (Sutherland et al., 1980) events. We have previously used post-(UV-B) exposure to UV-A (320-400 nm, photoreactivating light, PRL) to identify pyrimidine dimers as the major photoproduct involved in UVR-induced pathological changes in the skin of a South American opossum Monodelphis domestica (Ley, 1985; Ley and Applegate, 1985). In addition, we have observed the induction of corneal opacification and neovascularization (unpublished observations) and tumors (Applegate et al., 1985) of the cornea during long-term, chronic irradiations of M. domestica with UV-B. Thus, M. domestica m a y provide us with a valuable model with which to determine whether UV-induced pyrimidine dimers in D N A are involved in pathological changes of the irradiated eye. It should be noted that individuals who suffer from the autosomal recessive disease xeroderma pigmentosum express an enhanced susceptibility to corneal abnormalities which include corneal clouding and vascularization and neoplasms possibly induced by U V R (Kraemer et al., 1987). Before initiating studies to determine whether post-(UV-B) exposure to P R L suppresses the induction of photodamage of the cornea of M. domestica, we wished to determine: (1) whether measurable PR of pyrimidine dimers occurs in the cornea of M. domestica; and, (2) if present, the kinetics of PR after various doses of UV-B. The results of that study are reported herein. Materials and methods
Experimental animals We raised the gray, short-tailed opossums (South American opossums) to approximately 4 - 5 - m o n t h s of age under relatively simple husbandry methods adapted in part from F a d e m et al. 1982). Pertinent exceptions to these husbandry methods were that the opossums had free access to water and fox food, reproduction diet (Milk Specialties, New Holstein, Wisconsin) and that their diet was supplemented 3 times per
week with cabbage and various fruits for roughage along with 3 - 4 g of a wet food mixture composed of 425 g of Ken-L-ratio dog food, 650 g of found fox food, 12 g of powdered milk, 6 g of bran, and - 625 ml of water. The animals were housed under red fluorescent lights (General Electric F40R) to avoid exposure to photoreactivating wavelengths of light. The rooms were maintained at 2 4 ° C with the humidity at - 40%.
Exposure conditions For animal irradiations, the opossums were anesthetized by open-drop inhalation of methoxyfluorane (Metofane, Pitman-Moore Inc., Washington Crossing, N J) in a closed chamber system. Whiskers were removed from the eye and facial area before irradiations in which the eyes were held open and exposed to a Westinghouse FS-40 sunlamp at doses of 100, 300 or 500 J / m 2. The sunlamp emits wavelengths between 280 and 400 n m with a peak emission at 313 nm and with relative emissions of 0.04, 0.27, 0.69, 1.0 and 0.09 at 280, 290, 300, 313 and 360 nm, respectively (Grube et al., 1977). Dose rate from the FS-40 sunlamp was 6.2 W / m 2 between 280 and 400 nm as determined with an Optronic Model 742 spect r o r a d i o m e t e r (Optronics Laboratories, Inc., Orlando, FL). This scanning spectroradiometer measures the spectral emissions at 1-nm intervals and the emitted energies are summed over the range of 280-400 nm. Eyes which received photoreactivation were taped around the eye, held open, and exposed immediately after UVB exposure to a BLB lamp (Sylvania) filtered with 3 m m of window glass for 5, 10, 15, 30 or 60 min. Animals were anesthetized during the PR treatment and the eyes irrigated with phosphate-buffered saline (0.01 M PO4; 0.15 M NaC1; p H 7.2) (PBS) to prevent drying. The filtered BLB lamps emit wavelengths between 320 and 400 n m with a dose rate of 10 W / m 2 as measured with the spectroradiometer. Enucleations After irradiations and respective photoreactivation treatments, opossums were killed by methoxyfluorane inhalation and their eyes immediately enucleated and placed in PBS on ice. Enucleations were routinely carried out under yellow safelights
51 (General Electric F40GO) and subdued lighting conditions. The samples were then subjected to heat-cold shock by submersion for 30 sec in a 5 5 ° C circulating water bath (Lauda K - 2 / R , Brinkman Instruments Inc., Westbury, NY) followed immediately by 3 min in 0 ° C distilled water. After treatment, the corneal epithelium was easily separated from the eye by mild scraping with a scalpel (feather blade) into 0.25 ml of A1 buffer (50 m M Tris, p H 8; 10 m M EDTA; 0.04 M NaC1). DNA preparation The corneal epithelial cells were lysed by adding 25 /~1 of a 10% ( w / v ) solution of sodium dodecyl sulfate in A1 buffer and incubating at 3 7 ° C for 30 min. To the samples, 25 /~1 of proteinase K (10 m g / m l in A1 buffer) was added and incubation continued for 30 min at 37 o C. Redistilled phenol (0.25 ml) (Amresco) was added, the samples were rocked slowly for 30 min, and then centrifuged at 1300 g for 5 min at room temperature. The upper aqueous phase was gently removed with a wide bore pasteur pipette, 0.25 ml of sec-butanol added, and the samples mixed by inversion. The samples were centrifuged at 1300 g for 5 min at room temperature and then the lower aqueous phase removed and mixed with 0.5 ml of chilled absolute ethanol. The D N A was allowed to precipitate at - 7 0 ° C for 1-2 h. The samples were centrifuged at 16000 g for 15 min and the D N A pellets resuspended in 35 /~1 of a solution containing 30 mM Tris, pH, 8.0, 40 m M NaC1, 1 m M disodium EDTA. Aliquots of D N A (40-60 ng) were then treated with enough of the pyrimidine-dimer specific enzyme from Micrococcus luteus (UV endonuclease) (Carrier and Setlow, 1970) to give stoichiometric cleavage at dimer sites while parallel samples were not treated with UV endonuclease. Following incubation with UV endonuclease, an alkaline stop mix [25% ( v / v ) glycerol, 0.125% ( w / v ) bromocresol green and 0.5 N NaOH] was added to all samples and aliquots of the D N A mixtures (20-45 ng) were loaded into wells of 0.4% agarose gels (Sigma Type II agarose in 50 m M NaCI, 4 m M disodium EDTA). Molecular weight markers (bacteriophage D N A from T4, T7 and a HindIII digest of lambda) were added to at least one of the
wells on each gel and electrophoresis was carried out for 2 h at 40 V in 2 mM disodium E D T A and 30 mM N a O H using a BioRad minigel apparatus. The gels were neutralized, stained with ethidium bromide, destained in water and photographed on a UV transilluminator (Haakebuchler UVT) through a 23A Wratten Filter using Polaroid type 55 positive-negative film. The D N A was quantitated by comparison with the fluorescence of calf-thymus D N A standards following electrophoresis o n agarose gels. The level of background breaks in the D N A as a result of extraction was consistent for all samples. Gel scans The D N A distribution in each lane was determined by scanning the photographic negative with a GS300 scanning densitometer (Hoefer Scientific Instruments). The voltage output of the densitometer is digitized with an 8-bit analog-todigital ( A / D ) converter and stored using the GS350 Data System (Hoefer Scientific Instruments) on disc using an IBM P C / X T . The digitized gels scans were then transferred to a VAX microcomputer (Digital Equipment Corporation) for pyrimidine dimer analysis. Pyrimidine dimer analysis The method of analyzing photographic negatives to determine D N A concentration in an alkaline agarose gel and to determine the number of pyrimidine dimers in D N A has been described (Sutherland et al., 1984; Freeman et al., 1986; Freeman and Thompson, in press). We have adapted this method of calculation to the results from densitometric tracings of photographic negatives of D N A gels using the Hoefer densitometer. In brief, gel fluorescence [p(x)] is the DNA fluorescence at migration distance (x) down a lane and can be described by the equation (Pulleyblank et al., 1977; Sutherland et al., 1984):
Here 7 is the slope of the linear portion of a plot of OD versus the logarithm of film exposure, I 0 and I are the intensity of incident light from the densitometer probe and intensity of light trans-
52
mitted through the negative, respectively. In practice, film exposure times are within the linear portion of the O D versus logarithm of film exposure curve. For Polaroid Type 55 film 7 = 0.73. A parameter proportional to the intensity of the D N A fluorescence [p(x)] from the gel is measured as a voltage potential from the densitometer tracing which in turn is digitized with the 8-bit A / D converter. The digitized voltage potential (V) can be related to the optical density (OD) of the film by the equation: V = kOD
(2)
which can be rearranged to give:
OD=V/k
(3)
In these expressions, k is a constant that is influenced by the gain setting on the densitometer, find V is generally expressed in millivolts. In practice for comparison of D N A from lane to lane on a given gel, the gain setting is kept the same. The densitometer response has been determined to be linear over the O D range exhibited by D N A concentrations. Each electrophoresed lane is scanned using the densitometer and the values of V / k stored in digital form. It follows from Beers law that:
I o / I = 10 °D
ESS kb
1 Lr,( + endo)
1 Ln( - endo)
(7)
The reciprocal number average molecular length is given by:
fxm~ p ( x ) dx L'71
=
m,o
L(x)
fx£To(x) d x
(8)
(4)
Since from Eqn. 3, O D = Vk, we can now combine Eqns. 1 and 3 to arrive at:
p ( X ) = [loV/k] 1"37- -
1
(5)
Thus, p ( x ) is derived from V / k values obtained by digitizing a densitometer scan. A set of molecular length standards is included in each gel in order to determine the coefficients of an analytical expression that describes D N A molecular length ( L ) as a function of migration distance (x):
L(x)
infinite length and x 0 is the migration distance of a D N A molecule of zero length. A comprehensive discussion of Eqn. 6 is provided in Freeman et al. (1986). The constants c, x ~ and x 0 are determined for each gel through regression analysis of migration distances exhibited by the set of molecular length standards. The L ( x ) model thus derived is used as the standard curve for subsequent analysis of unknown samples. For the specific case of UVR-induced pyrimidine dimers, single strand breaks can be induced quantitatively by the dimer-specific endonuclease from Micrococcus luteus (Setlow et al., 1975). We can compute the number of UV-endonuclease sensitive sites (ESS) per 1000 bases (kb) as the difference between reciprocal number average molecular lengths ( 1 / L n ) of irradiated D N A with a without endonuclease treatment; that is
c
c
X -- X 0
X 0 -- Xoe
(6)
where c is an empirically derived constant, x ~ is the migration distance of a D N A molecule of
Results We first determined the extent of D N A damage and photorepair in opossum corneal epithelium exposed in situ to ultraviolet radiation from an FS-40 sunlamp (290-320 nm). The eyes of the opossum were irradiated with U V R in situ and the corneal cells were either removed immediately or exposed to P R L for varying periods of time. The D N A was then extracted from the corneal epithelial cells, treated with UV endonuclease, and subjected to electrophoresis in 0.4% alkaline agarose gels. After neutralization and staining, the gels were photographed and the negatives scanned as described above. Fig. 1 shows traces of a negative in an experiment where the corneal epithelial cells of an opossum eye exposed to 300 J / m 2
53
!!1
ILl O Z LU
I
'
I
. 10
'
'
'
!
O
ILl nO ..I I.I. ...I U.I
O , 0
r
~
I 10
. . . .
MIGRATION
, 0
DISTANCE
,
,
(mrn)
Fig. 1. Profiles of corneal, epithelial D N A after alkaline agarose gel electrophoresis. Corneal D N A was isolated from eyes of M. domestica that had been exposed in situ to 300 J / m 2 from a FS-40 sunlamp and either held in the dark or treated for 1 h PRL. Panel A: U V R + d a r k 1 h+UV-endonuclease; Panel B: U V R + d a r k 1 h: Panel C: U V R + I h P R L + U V endonuclease; Panel D: U V R + I h PRL.
without (traces A and B) and with (traces C and D PRL treatment. DNAs in traces A and C were treated with UV endonuclease whereas those in traces B and D were not. Comparison of traces A and B (Fig. 1), which is D N A from corneal epithelium cells of an eye irradiated with U V R without exposure to PRL, yields 0.038 ESS (dimers)/kb (see equation above), whereas D N A from cells exposed to 60 min of PRL after UVR exposure (Fig. 1, traces C and D) was observed to contain 0.013 ESS/kb. Thus the PR treatment has monomerized 66% of the UVRinduced pyrimidine dimers. To determine the kinetics of photorepair, we exposed opossum eyes to 100 J / m 2 from the FS-40 sunlamp and then exposed the eyes to PRL for various times. Fig. 2 shows the kinetic analysis for 0, 15, 30 or 60 min PRL. The average number of sites induced by 100 J / m 2 from the FS-40 is 0.023 E S S / k b while the average number following 60 min of PRL is 0.002 ESS/kb. Therefore, approximately 92% of the dimers are repaired by 60 min of PRL. Only 10 and 30 min of PRL was required to repair - 5 0 % and > 90% of the dimers, respectively. When an exposed eye was held in the dark for 30 rain or exposed to 30 min PRL prior to U V R and then held in the dark for 30 min
the number of pyrimidine dimers observed in both cases was 0.018/kb. These values fall well within the error estimate for the number of pyrimidine dimers induced by 100 J / m 2 (see above) and suggests that (1) pre-PRL does not reduce the number of pyrimidine dimers induced by subsequent irradiation; (2) pre-PRL does not stimulate repair of dimers during a 30-min repair period post UVR; and, (3) that dark repair does not contribute significantly to dimer reduction in 30 min after irradiation. We also determined the dose response for pyrimidine dimer induction in the opossum cornea by irradiation of the eye with UVR from the FS-40 sunlamp (0 to 1.0 × 103 J / m 2) (Fig. 3). We estimate the number of E S S / k b produced per dose of U V R ( E S S / k b per J / m 2) by linear regression analysis. The slope of the line which yields that estimate is 7.2 _+ 1.0 × 10 -5. In parallel experiments, we determined the extent of photorepair by following U V R exposures of 300 and 500 J / m 2 with 60 min of PRL. Approximately 70% and 60% of the dimers induced by 300 and 500
5,0 g" C) x
w I-
\
2.0 \ I'-
laJ
LO
d z 0 t~ z w
0
I
I
I
I
0
5
I0
15
:50 P R L (min)
60
Fig. 2. The number of E S S / k b in D N A from UVR-irradiated corneas followed by varying exposures to PRL. Eyes of M. domestica were exposed in situ to 100 J / m 2 from an FS-40 sunlamp and the eyes were enucleated either immediately or after 5, 10, 15, 30 or 60 min of PRL exposure. The number of pyrimidine dimers were determined in the D N A using alkaline agarose gel electrophoresis and expressed as endonuclease sensitive sites per 1000 bases (kb). Each point is the average of 2-5 independent Expts. + SD.
54
O
io
x v
w F--
t--
8
6
z
co < w
z O
2
8.-
z LO
~ 0
I
I
I
I
I
2
4
6
8
I0
genetic d a m a g e as skin epithelial cells. The lower level of D N A d a m a g e in the skin epithelium of M. d o m e s t i c a presumably reflects attenuation of the incident U V R by the stratum corneum. We also report here that exposure to P R L after U V B resulted in m o n o m e r i z a t i o n of the UVB-induced dimers. O u r previous studies have shown that chronic exposure of the eye of M . d o m e s t i c a to U V R resulted in the occurrence of corneal tumors (Applegate et al., 1985) and opacification and n e o v a s c u l a r i z a t i o n ( u n p u b l i s h e d observation). Thus, the capacity to photorepair pyrimidine dimers in corneal D N A of M . d o m e s t i c a m a y provide us with a useful model with which to determine whether pyrimidine dimers are involved in these, and other, U V R - i n d u c e d , pathological changes of the irradiated cornea.
DOSE (d/m 2x10 -2) Fig. 3. The n u m b e r of E S S / k b in D N A from corneas exposed in situ to increasing doses of U V R from the FS-40 sunlamp. Eyes of M. domestica were exposed in situ to U V R from an FS-40 sunlamp and the eyes enucleated immediately (O) or after 1 h PRL (©).
J / m 2, respectively, were repaired by the 60 min exposure to P R L (Fig. 3). Discussion The data presented herein show that U V R - i n duced pyrimidine dimers can be quantitatively measured in D N A of the corneal epithelium of M. d o m e s t i c a following exposure to relatively low doses of UVB. Dimers ( 0 . 0 2 3 / k b of D N A ) were readily measurable after 100 J / m 2. In comparison, a single dose of - 4 0 0 J / m 2 from the FS-40 sunlamps would be required to induce a barely perceptible reddening (erythema) of the skin of M. d o m e s t i c a (Ley, 1985) and a similar level of dimers in epidermal D N A (Applegate and Ley, 1987). Using the alkaline agarose gel electrophoresis m e t h o d we have previously shown that a dose of 400 J / m 2 of U V B from a fluorescent sunlamp is required to induce similar dimer yields in the skin epithelium of the o p o s s u m as 100 J / m 2 induces in corneal epithelium (Freeman et al., 1988). These induction data show that for equal exposures to U V R from the FS-40 sunlamp the corneal epithelium would suffer from four times as m u c h
Acknowledgements We wish to acknowledge the excellent technical assistance provided b y Sharon L. R y a n during the course of this study and helpful suggestions from Dr. K.D. Ley concerning preparation of the manuscript. This study was funded by the Lovelace Medical F o u n d a t i o n . References Applegate, L.A., and R.D. Ley (1987) Excision repair of pyrimidine dimers in marsupial cells, Photochem. Photobiol., 45, 241-245. Applegate, L.A., T.D. Stuart and R.D. Ley (1985) Ultraviolet radiation (UVR)-induced histopathological changes in the skin and eyes of Monodelphis domestica, Photochem. Photobiol., 41S, 93. Carrier, W.L., and R.B. Setlow (1970) An endonuclease from Micrococcus luteus which has activity toward ultravioletirradiated deoxyribonucleic acid: Purification and properties, J. Bacteriol., 102, 178-186. Cook, J.S. (1970) Photoreactivation in animal cells, in: A.C. Giese (Ed.), Photophysiology, Vol. 5, Academic Press, New York, pp. 191-223. D'Ambrosio, S.M, J.W. Whestone, L. Slazinski and E. Lowney (1981) Photorepair of pyrimidine dimers in human skin in vivo, Photochem. Photobiol., 34, 461-464. Diffey, B.L. (1982) Ultraviolet Radiation in Medicine, Adam Hilger, Bristol. Eggsett, G., G. Volden and H. Krokan (1983) UV-induced DNA damage and its repair in human skin in vivo studies by sensitive immunohistochemical methods. Carcinogenesis, 4, 745-750.
55 Fadem, B.H., G.L. Trupin, E. Maliniak and V. Hayssen (1982) Care and breeding of the gray, short-tailed opossum (Monodelphis domestica), Lab. Anim. Sci. 32, 405-409. Freeman, S.E., and B.D. Thompson (1988) Evaluation of densitometry data using interactive computer graphics: Application to DNA agarose gels, Int. J. of Biomed. Computing, in press. Freeman, S.E., A.D. Blackett, D.C. Monteleone, R.B. Setlow, B.M. Sutherland and J.C. Sutherland (1986) Quantitation of radiation-, chemical-, or enzyme-induced single strand breaks in nonradioactive DNA by alkaline gel electrophoresis: Application to pyrimidine dimers, Anal. Biochem. 158, 119-129. Freeman, S.E., L.A. Applegate and R.D. Ley (1988) Excision repair of UVR-induced pyrimidine dimers in corneal epithelium, Photochem. Photobiol., 47, 159-163. Grube, D.D., R.D. Ley and R.J.M. Fry (1977) Photosensitizing effects of 8-methoxypsoralen on the skin of hairless mice, II. Strain and spectral differences of tumorigenesis, Photochem. Photobiol., 25, 269-276. Harm, H. (1976) Repair of UV-irradiated biological systems Photoreactivation, in: S.E. Wang (Ed.), Photochemistry and Photobiology of Nucleic Acids, Vol. II, Academic Press, New York, pp. 219-263. Hart, R.W., R.B. Setlow and A.D. Woodhead (1977) Evidence that pyrimidine dimers in DNA can give rise to tumors, Proc. Natl. Acad. Sci. (U.S.A.), 75, 5574-5578. Kraemer, K.H., M.M. Lee and J. Scotto (1987) Xeroderma pigmentosum: cutaneous, ocular, and neurologic abnormalities in 830 published cases, Arch. Dermatol., 123, 241-250. Ley, R.D. (1984) Photorepair of pyrimidine dimers in the epidermis of the of the marsupial Monodelphis domestica, Photochem. Photobiol., 40, 141-143. Ley, R.D. (1985) Photoreactivation of UV-induced pyrimidine dimers and erythema in the marsupial Monodelphis domestica, Proc. Natl. Acad. Sci. (U.S.A.), 82, 2409-2411. Ley, R.D., and L.A. Applegate (1985) Ultraviolet radiation-induced histopathologic changes in the skin of the marsupial
Monodelphis domestica, II. Quantitative studies of the photoreactivation of induced hyperplasia and sunburn cell formation, J. Invest. Dermatol., 85, 365-367. Ley, R.D., B.A. Sedita and D.D. Grnbe (1978) Absence of photoreactivation of pyrimidine dimers in the epidermis of hairless mice following exposure to ultraviolet light, Photochem. Photobiol., 27, 483-485. National Institute for Occupational Safety and Health (1972) Criteria for a recommended standard on occupational exposure to ultraviolet radiation, U.S. Department of Health, Education and Welfare. Patrick, M.H., and R.O. Rahn (1976) Photochemistry of DNA and polynucleotides: Photoproducts, in: S.Y. Wang (Ed.). Photochemistry and Photobiology of Nucleic Acids, Vol. II, Academic Press, New York, pp. 83-87. Pitts, D.G. (1974) The Human Ultraviolet Action Spectrum, Am. J. Optom. Physiol. Optics, 51, 946-960. Pitts, D.G., and T.J. Tredici (1971) The effects of ultraviolet on the eye, Am. Indust. Hyg. Assoc. J., 32, 235-246. Pulleyblank, D.E., M. Shure and J. Vinograd (1977) The quantitation of fluorescence by photography, Nuc. Acids Res., 4, 1409-1418. Setlow, J.K., and R.B. Setlow (1963) Nature of the photoreactivable ultraviolet lesion in DNA, Nature (London), 197, 560-562. Setlow, R.B., W.L. Carrier and J. Stewart (1975) Endonuclease sensitive sites in uv-irradiated DNA, Biophys. J., 15, 194a. Sutherland, B.M., J.S. Cimino, N. Delihas, A.G. Shih and R.P. Oliver (1980a) Ultraviolet light-induced transformation of human cells to anchorage-independent growth, Cancer Res., 40, 1934-1939. Sutherland, B.M, L.C. Harber and I.E. Kochevar (1980b) Pyrimidine dimer formation and repair in human skin, Cancer Res., 40, 3181-3185. Sutherland, J.C., D.C. Monteleone, J. Trunk and G. Ciarrochi (1984) Two-dimensional, computer controlled film scanner: Quantitation of fluorescence from ethidium bromide-stained DNA gels, Anal. Biochem, 139, 390-399.
49
MTR 06284
Photorepair of ultraviolet radiation-induced pyrimidine dimers in corneal D N A R o n a l d D . L e y , L e e A . A p p l e g a t e a n d S t e v e n E. F r e e m a n Photocarcinogenesis Program, Division of Biomedical Research, Lovelace Medical Foundation, 2425 Ridgecrest Drive, S.E., Albuquerque, N M 87108 (U.S.A.) (Received 19 June 1987) (Revision received 18 January 1988) (Accepted 22 January 1988)
Keywords: Pyrimidine dimers; Cornea; Photorepair; Marsupial; Nucleases; Monodelphis domestica; Micrococcus luteus
Summary The induction and photorepair of pyrimidine dimers in D N A have been measured in the ultravioletirradiated, corneal epithelium of the marsupial, Monodelphis dornestica, using damage-specific nucleases from Micrococcus luteus in conjunction with agarose gel electrophoresis. We observed that FS-40 sunlamps (280-400 nm) induced 7.2 _+ 1.0 × 10 -5 pyrimidine dimers per kilobase (kb) of D N A per J / m 2. Following 100 J / m 2, 50% and > 90% of the dimers were photorepaired during a 10- and 30-min exposure to photoreactivating light (320-400 nm), respectively. In addition, - 70% and - 60% of the dimers induced by 300 and 500 J / m 2, respectively, were repaired by a 60-min exposure to photoreactivating light. The capacity of the corneal epithelium of M. domestica to photorepair pyrimidine dimers identifies this animal as a potentially useful model with which to determine whether pyrimidine dimers are involved in pathological changes of the irradiated eye.
Ultraviolet radiation (UVR)-induced photodamage of the eye has been observed in both humans and experimental animals. Photokeratitis, acute corneal injury, occurs in individuals who use arc welders (National Institute for Occupational Safety and Health, 1972) and from intensive exposure to U V R from sunlight reflected off surfaces such as snow or sand (Diffey, 1982). Ultraviolet radiation-induced corneal damage has also been
Correspondence: Dr. Ronald D. Ley, Division of Biomedical Research, Lovelace Medical Foundation, 2425 Ridgecrest Drive, S.E., Albuquerque, NM 87108 (U.S.A.).
extensively studied in primates and rabbits (Pitts, 1974; Pitts and Tredici, 1971). Ultraviolet radiation induces a number of lesions in DNA, the most prevalent being a cyclobutane-type dimer between adjacent pyrimidines on the same D N A strand (Patrick and Rahn, 1976). Pyrimidine dimers can be split in situ by a light-dependent repair process called photoreactivation (PR). This repair pathway requires the presence of the PR enzyme and exposure to longwavelength visible radiation in the range of 320-500 nm (Cook, 1970). Photoreactivation has been reported to occur in human (Sutherland et al., 1980; D'Ambrosio et al., 1981; Eggset et al., 1983) and marsupial (Ley, 1984) skin, but is much
0167-8817/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
50 less evident in mice (Ley, et al., 1978). The specificity of the PR repair pathway for pyrimidine dimers (Setlow and Setlow, 1963) has been used to identify the lesion as a major cause of lethal and mutagenic (Harm, 1976), tumorigenic (Hart et al., 1977), and transformational (Sutherland et al., 1980) events. We have previously used post-(UV-B) exposure to UV-A (320-400 nm, photoreactivating light, PRL) to identify pyrimidine dimers as the major photoproduct involved in UVR-induced pathological changes in the skin of a South American opossum Monodelphis domestica (Ley, 1985; Ley and Applegate, 1985). In addition, we have observed the induction of corneal opacification and neovascularization (unpublished observations) and tumors (Applegate et al., 1985) of the cornea during long-term, chronic irradiations of M. domestica with UV-B. Thus, M. domestica m a y provide us with a valuable model with which to determine whether UV-induced pyrimidine dimers in D N A are involved in pathological changes of the irradiated eye. It should be noted that individuals who suffer from the autosomal recessive disease xeroderma pigmentosum express an enhanced susceptibility to corneal abnormalities which include corneal clouding and vascularization and neoplasms possibly induced by U V R (Kraemer et al., 1987). Before initiating studies to determine whether post-(UV-B) exposure to P R L suppresses the induction of photodamage of the cornea of M. domestica, we wished to determine: (1) whether measurable PR of pyrimidine dimers occurs in the cornea of M. domestica; and, (2) if present, the kinetics of PR after various doses of UV-B. The results of that study are reported herein. Materials and methods
Experimental animals We raised the gray, short-tailed opossums (South American opossums) to approximately 4 - 5 - m o n t h s of age under relatively simple husbandry methods adapted in part from F a d e m et al. 1982). Pertinent exceptions to these husbandry methods were that the opossums had free access to water and fox food, reproduction diet (Milk Specialties, New Holstein, Wisconsin) and that their diet was supplemented 3 times per
week with cabbage and various fruits for roughage along with 3 - 4 g of a wet food mixture composed of 425 g of Ken-L-ratio dog food, 650 g of found fox food, 12 g of powdered milk, 6 g of bran, and - 625 ml of water. The animals were housed under red fluorescent lights (General Electric F40R) to avoid exposure to photoreactivating wavelengths of light. The rooms were maintained at 2 4 ° C with the humidity at - 40%.
Exposure conditions For animal irradiations, the opossums were anesthetized by open-drop inhalation of methoxyfluorane (Metofane, Pitman-Moore Inc., Washington Crossing, N J) in a closed chamber system. Whiskers were removed from the eye and facial area before irradiations in which the eyes were held open and exposed to a Westinghouse FS-40 sunlamp at doses of 100, 300 or 500 J / m 2. The sunlamp emits wavelengths between 280 and 400 n m with a peak emission at 313 nm and with relative emissions of 0.04, 0.27, 0.69, 1.0 and 0.09 at 280, 290, 300, 313 and 360 nm, respectively (Grube et al., 1977). Dose rate from the FS-40 sunlamp was 6.2 W / m 2 between 280 and 400 nm as determined with an Optronic Model 742 spect r o r a d i o m e t e r (Optronics Laboratories, Inc., Orlando, FL). This scanning spectroradiometer measures the spectral emissions at 1-nm intervals and the emitted energies are summed over the range of 280-400 nm. Eyes which received photoreactivation were taped around the eye, held open, and exposed immediately after UVB exposure to a BLB lamp (Sylvania) filtered with 3 m m of window glass for 5, 10, 15, 30 or 60 min. Animals were anesthetized during the PR treatment and the eyes irrigated with phosphate-buffered saline (0.01 M PO4; 0.15 M NaC1; p H 7.2) (PBS) to prevent drying. The filtered BLB lamps emit wavelengths between 320 and 400 n m with a dose rate of 10 W / m 2 as measured with the spectroradiometer. Enucleations After irradiations and respective photoreactivation treatments, opossums were killed by methoxyfluorane inhalation and their eyes immediately enucleated and placed in PBS on ice. Enucleations were routinely carried out under yellow safelights
51 (General Electric F40GO) and subdued lighting conditions. The samples were then subjected to heat-cold shock by submersion for 30 sec in a 5 5 ° C circulating water bath (Lauda K - 2 / R , Brinkman Instruments Inc., Westbury, NY) followed immediately by 3 min in 0 ° C distilled water. After treatment, the corneal epithelium was easily separated from the eye by mild scraping with a scalpel (feather blade) into 0.25 ml of A1 buffer (50 m M Tris, p H 8; 10 m M EDTA; 0.04 M NaC1). DNA preparation The corneal epithelial cells were lysed by adding 25 /~1 of a 10% ( w / v ) solution of sodium dodecyl sulfate in A1 buffer and incubating at 3 7 ° C for 30 min. To the samples, 25 /~1 of proteinase K (10 m g / m l in A1 buffer) was added and incubation continued for 30 min at 37 o C. Redistilled phenol (0.25 ml) (Amresco) was added, the samples were rocked slowly for 30 min, and then centrifuged at 1300 g for 5 min at room temperature. The upper aqueous phase was gently removed with a wide bore pasteur pipette, 0.25 ml of sec-butanol added, and the samples mixed by inversion. The samples were centrifuged at 1300 g for 5 min at room temperature and then the lower aqueous phase removed and mixed with 0.5 ml of chilled absolute ethanol. The D N A was allowed to precipitate at - 7 0 ° C for 1-2 h. The samples were centrifuged at 16000 g for 15 min and the D N A pellets resuspended in 35 /~1 of a solution containing 30 mM Tris, pH, 8.0, 40 m M NaC1, 1 m M disodium EDTA. Aliquots of D N A (40-60 ng) were then treated with enough of the pyrimidine-dimer specific enzyme from Micrococcus luteus (UV endonuclease) (Carrier and Setlow, 1970) to give stoichiometric cleavage at dimer sites while parallel samples were not treated with UV endonuclease. Following incubation with UV endonuclease, an alkaline stop mix [25% ( v / v ) glycerol, 0.125% ( w / v ) bromocresol green and 0.5 N NaOH] was added to all samples and aliquots of the D N A mixtures (20-45 ng) were loaded into wells of 0.4% agarose gels (Sigma Type II agarose in 50 m M NaCI, 4 m M disodium EDTA). Molecular weight markers (bacteriophage D N A from T4, T7 and a HindIII digest of lambda) were added to at least one of the
wells on each gel and electrophoresis was carried out for 2 h at 40 V in 2 mM disodium E D T A and 30 mM N a O H using a BioRad minigel apparatus. The gels were neutralized, stained with ethidium bromide, destained in water and photographed on a UV transilluminator (Haakebuchler UVT) through a 23A Wratten Filter using Polaroid type 55 positive-negative film. The D N A was quantitated by comparison with the fluorescence of calf-thymus D N A standards following electrophoresis o n agarose gels. The level of background breaks in the D N A as a result of extraction was consistent for all samples. Gel scans The D N A distribution in each lane was determined by scanning the photographic negative with a GS300 scanning densitometer (Hoefer Scientific Instruments). The voltage output of the densitometer is digitized with an 8-bit analog-todigital ( A / D ) converter and stored using the GS350 Data System (Hoefer Scientific Instruments) on disc using an IBM P C / X T . The digitized gels scans were then transferred to a VAX microcomputer (Digital Equipment Corporation) for pyrimidine dimer analysis. Pyrimidine dimer analysis The method of analyzing photographic negatives to determine D N A concentration in an alkaline agarose gel and to determine the number of pyrimidine dimers in D N A has been described (Sutherland et al., 1984; Freeman et al., 1986; Freeman and Thompson, in press). We have adapted this method of calculation to the results from densitometric tracings of photographic negatives of D N A gels using the Hoefer densitometer. In brief, gel fluorescence [p(x)] is the DNA fluorescence at migration distance (x) down a lane and can be described by the equation (Pulleyblank et al., 1977; Sutherland et al., 1984):
Here 7 is the slope of the linear portion of a plot of OD versus the logarithm of film exposure, I 0 and I are the intensity of incident light from the densitometer probe and intensity of light trans-
52
mitted through the negative, respectively. In practice, film exposure times are within the linear portion of the O D versus logarithm of film exposure curve. For Polaroid Type 55 film 7 = 0.73. A parameter proportional to the intensity of the D N A fluorescence [p(x)] from the gel is measured as a voltage potential from the densitometer tracing which in turn is digitized with the 8-bit A / D converter. The digitized voltage potential (V) can be related to the optical density (OD) of the film by the equation: V = kOD
(2)
which can be rearranged to give:
OD=V/k
(3)
In these expressions, k is a constant that is influenced by the gain setting on the densitometer, find V is generally expressed in millivolts. In practice for comparison of D N A from lane to lane on a given gel, the gain setting is kept the same. The densitometer response has been determined to be linear over the O D range exhibited by D N A concentrations. Each electrophoresed lane is scanned using the densitometer and the values of V / k stored in digital form. It follows from Beers law that:
I o / I = 10 °D
ESS kb
1 Lr,( + endo)
1 Ln( - endo)
(7)
The reciprocal number average molecular length is given by:
fxm~ p ( x ) dx L'71
=
m,o
L(x)
fx£To(x) d x
(8)
(4)
Since from Eqn. 3, O D = Vk, we can now combine Eqns. 1 and 3 to arrive at:
p ( X ) = [loV/k] 1"37- -
1
(5)
Thus, p ( x ) is derived from V / k values obtained by digitizing a densitometer scan. A set of molecular length standards is included in each gel in order to determine the coefficients of an analytical expression that describes D N A molecular length ( L ) as a function of migration distance (x):
L(x)
infinite length and x 0 is the migration distance of a D N A molecule of zero length. A comprehensive discussion of Eqn. 6 is provided in Freeman et al. (1986). The constants c, x ~ and x 0 are determined for each gel through regression analysis of migration distances exhibited by the set of molecular length standards. The L ( x ) model thus derived is used as the standard curve for subsequent analysis of unknown samples. For the specific case of UVR-induced pyrimidine dimers, single strand breaks can be induced quantitatively by the dimer-specific endonuclease from Micrococcus luteus (Setlow et al., 1975). We can compute the number of UV-endonuclease sensitive sites (ESS) per 1000 bases (kb) as the difference between reciprocal number average molecular lengths ( 1 / L n ) of irradiated D N A with a without endonuclease treatment; that is
c
c
X -- X 0
X 0 -- Xoe
(6)
where c is an empirically derived constant, x ~ is the migration distance of a D N A molecule of
Results We first determined the extent of D N A damage and photorepair in opossum corneal epithelium exposed in situ to ultraviolet radiation from an FS-40 sunlamp (290-320 nm). The eyes of the opossum were irradiated with U V R in situ and the corneal cells were either removed immediately or exposed to P R L for varying periods of time. The D N A was then extracted from the corneal epithelial cells, treated with UV endonuclease, and subjected to electrophoresis in 0.4% alkaline agarose gels. After neutralization and staining, the gels were photographed and the negatives scanned as described above. Fig. 1 shows traces of a negative in an experiment where the corneal epithelial cells of an opossum eye exposed to 300 J / m 2
53
!!1
ILl O Z LU
I
'
I
. 10
'
'
'
!
O
ILl nO ..I I.I. ...I U.I
O , 0
r
~
I 10
. . . .
MIGRATION
, 0
DISTANCE
,
,
(mrn)
Fig. 1. Profiles of corneal, epithelial D N A after alkaline agarose gel electrophoresis. Corneal D N A was isolated from eyes of M. domestica that had been exposed in situ to 300 J / m 2 from a FS-40 sunlamp and either held in the dark or treated for 1 h PRL. Panel A: U V R + d a r k 1 h+UV-endonuclease; Panel B: U V R + d a r k 1 h: Panel C: U V R + I h P R L + U V endonuclease; Panel D: U V R + I h PRL.
without (traces A and B) and with (traces C and D PRL treatment. DNAs in traces A and C were treated with UV endonuclease whereas those in traces B and D were not. Comparison of traces A and B (Fig. 1), which is D N A from corneal epithelium cells of an eye irradiated with U V R without exposure to PRL, yields 0.038 ESS (dimers)/kb (see equation above), whereas D N A from cells exposed to 60 min of PRL after UVR exposure (Fig. 1, traces C and D) was observed to contain 0.013 ESS/kb. Thus the PR treatment has monomerized 66% of the UVRinduced pyrimidine dimers. To determine the kinetics of photorepair, we exposed opossum eyes to 100 J / m 2 from the FS-40 sunlamp and then exposed the eyes to PRL for various times. Fig. 2 shows the kinetic analysis for 0, 15, 30 or 60 min PRL. The average number of sites induced by 100 J / m 2 from the FS-40 is 0.023 E S S / k b while the average number following 60 min of PRL is 0.002 ESS/kb. Therefore, approximately 92% of the dimers are repaired by 60 min of PRL. Only 10 and 30 min of PRL was required to repair - 5 0 % and > 90% of the dimers, respectively. When an exposed eye was held in the dark for 30 rain or exposed to 30 min PRL prior to U V R and then held in the dark for 30 min
the number of pyrimidine dimers observed in both cases was 0.018/kb. These values fall well within the error estimate for the number of pyrimidine dimers induced by 100 J / m 2 (see above) and suggests that (1) pre-PRL does not reduce the number of pyrimidine dimers induced by subsequent irradiation; (2) pre-PRL does not stimulate repair of dimers during a 30-min repair period post UVR; and, (3) that dark repair does not contribute significantly to dimer reduction in 30 min after irradiation. We also determined the dose response for pyrimidine dimer induction in the opossum cornea by irradiation of the eye with UVR from the FS-40 sunlamp (0 to 1.0 × 103 J / m 2) (Fig. 3). We estimate the number of E S S / k b produced per dose of U V R ( E S S / k b per J / m 2) by linear regression analysis. The slope of the line which yields that estimate is 7.2 _+ 1.0 × 10 -5. In parallel experiments, we determined the extent of photorepair by following U V R exposures of 300 and 500 J / m 2 with 60 min of PRL. Approximately 70% and 60% of the dimers induced by 300 and 500
5,0 g" C) x
w I-
\
2.0 \ I'-
laJ
LO
d z 0 t~ z w
0
I
I
I
I
0
5
I0
15
:50 P R L (min)
60
Fig. 2. The number of E S S / k b in D N A from UVR-irradiated corneas followed by varying exposures to PRL. Eyes of M. domestica were exposed in situ to 100 J / m 2 from an FS-40 sunlamp and the eyes were enucleated either immediately or after 5, 10, 15, 30 or 60 min of PRL exposure. The number of pyrimidine dimers were determined in the D N A using alkaline agarose gel electrophoresis and expressed as endonuclease sensitive sites per 1000 bases (kb). Each point is the average of 2-5 independent Expts. + SD.
54
O
io
x v
w F--
t--
8
6
z
co < w
z O
2
8.-
z LO
~ 0
I
I
I
I
I
2
4
6
8
I0
genetic d a m a g e as skin epithelial cells. The lower level of D N A d a m a g e in the skin epithelium of M. d o m e s t i c a presumably reflects attenuation of the incident U V R by the stratum corneum. We also report here that exposure to P R L after U V B resulted in m o n o m e r i z a t i o n of the UVB-induced dimers. O u r previous studies have shown that chronic exposure of the eye of M . d o m e s t i c a to U V R resulted in the occurrence of corneal tumors (Applegate et al., 1985) and opacification and n e o v a s c u l a r i z a t i o n ( u n p u b l i s h e d observation). Thus, the capacity to photorepair pyrimidine dimers in corneal D N A of M . d o m e s t i c a m a y provide us with a useful model with which to determine whether pyrimidine dimers are involved in these, and other, U V R - i n d u c e d , pathological changes of the irradiated cornea.
DOSE (d/m 2x10 -2) Fig. 3. The n u m b e r of E S S / k b in D N A from corneas exposed in situ to increasing doses of U V R from the FS-40 sunlamp. Eyes of M. domestica were exposed in situ to U V R from an FS-40 sunlamp and the eyes enucleated immediately (O) or after 1 h PRL (©).
J / m 2, respectively, were repaired by the 60 min exposure to P R L (Fig. 3). Discussion The data presented herein show that U V R - i n duced pyrimidine dimers can be quantitatively measured in D N A of the corneal epithelium of M. d o m e s t i c a following exposure to relatively low doses of UVB. Dimers ( 0 . 0 2 3 / k b of D N A ) were readily measurable after 100 J / m 2. In comparison, a single dose of - 4 0 0 J / m 2 from the FS-40 sunlamps would be required to induce a barely perceptible reddening (erythema) of the skin of M. d o m e s t i c a (Ley, 1985) and a similar level of dimers in epidermal D N A (Applegate and Ley, 1987). Using the alkaline agarose gel electrophoresis m e t h o d we have previously shown that a dose of 400 J / m 2 of U V B from a fluorescent sunlamp is required to induce similar dimer yields in the skin epithelium of the o p o s s u m as 100 J / m 2 induces in corneal epithelium (Freeman et al., 1988). These induction data show that for equal exposures to U V R from the FS-40 sunlamp the corneal epithelium would suffer from four times as m u c h
Acknowledgements We wish to acknowledge the excellent technical assistance provided b y Sharon L. R y a n during the course of this study and helpful suggestions from Dr. K.D. Ley concerning preparation of the manuscript. This study was funded by the Lovelace Medical F o u n d a t i o n . References Applegate, L.A., and R.D. Ley (1987) Excision repair of pyrimidine dimers in marsupial cells, Photochem. Photobiol., 45, 241-245. Applegate, L.A., T.D. Stuart and R.D. Ley (1985) Ultraviolet radiation (UVR)-induced histopathological changes in the skin and eyes of Monodelphis domestica, Photochem. Photobiol., 41S, 93. Carrier, W.L., and R.B. Setlow (1970) An endonuclease from Micrococcus luteus which has activity toward ultravioletirradiated deoxyribonucleic acid: Purification and properties, J. Bacteriol., 102, 178-186. Cook, J.S. (1970) Photoreactivation in animal cells, in: A.C. Giese (Ed.), Photophysiology, Vol. 5, Academic Press, New York, pp. 191-223. D'Ambrosio, S.M, J.W. Whestone, L. Slazinski and E. Lowney (1981) Photorepair of pyrimidine dimers in human skin in vivo, Photochem. Photobiol., 34, 461-464. Diffey, B.L. (1982) Ultraviolet Radiation in Medicine, Adam Hilger, Bristol. Eggsett, G., G. Volden and H. Krokan (1983) UV-induced DNA damage and its repair in human skin in vivo studies by sensitive immunohistochemical methods. Carcinogenesis, 4, 745-750.
55 Fadem, B.H., G.L. Trupin, E. Maliniak and V. Hayssen (1982) Care and breeding of the gray, short-tailed opossum (Monodelphis domestica), Lab. Anim. Sci. 32, 405-409. Freeman, S.E., and B.D. Thompson (1988) Evaluation of densitometry data using interactive computer graphics: Application to DNA agarose gels, Int. J. of Biomed. Computing, in press. Freeman, S.E., A.D. Blackett, D.C. Monteleone, R.B. Setlow, B.M. Sutherland and J.C. Sutherland (1986) Quantitation of radiation-, chemical-, or enzyme-induced single strand breaks in nonradioactive DNA by alkaline gel electrophoresis: Application to pyrimidine dimers, Anal. Biochem. 158, 119-129. Freeman, S.E., L.A. Applegate and R.D. Ley (1988) Excision repair of UVR-induced pyrimidine dimers in corneal epithelium, Photochem. Photobiol., 47, 159-163. Grube, D.D., R.D. Ley and R.J.M. Fry (1977) Photosensitizing effects of 8-methoxypsoralen on the skin of hairless mice, II. Strain and spectral differences of tumorigenesis, Photochem. Photobiol., 25, 269-276. Harm, H. (1976) Repair of UV-irradiated biological systems Photoreactivation, in: S.E. Wang (Ed.), Photochemistry and Photobiology of Nucleic Acids, Vol. II, Academic Press, New York, pp. 219-263. Hart, R.W., R.B. Setlow and A.D. Woodhead (1977) Evidence that pyrimidine dimers in DNA can give rise to tumors, Proc. Natl. Acad. Sci. (U.S.A.), 75, 5574-5578. Kraemer, K.H., M.M. Lee and J. Scotto (1987) Xeroderma pigmentosum: cutaneous, ocular, and neurologic abnormalities in 830 published cases, Arch. Dermatol., 123, 241-250. Ley, R.D. (1984) Photorepair of pyrimidine dimers in the epidermis of the of the marsupial Monodelphis domestica, Photochem. Photobiol., 40, 141-143. Ley, R.D. (1985) Photoreactivation of UV-induced pyrimidine dimers and erythema in the marsupial Monodelphis domestica, Proc. Natl. Acad. Sci. (U.S.A.), 82, 2409-2411. Ley, R.D., and L.A. Applegate (1985) Ultraviolet radiation-induced histopathologic changes in the skin of the marsupial
Monodelphis domestica, II. Quantitative studies of the photoreactivation of induced hyperplasia and sunburn cell formation, J. Invest. Dermatol., 85, 365-367. Ley, R.D., B.A. Sedita and D.D. Grnbe (1978) Absence of photoreactivation of pyrimidine dimers in the epidermis of hairless mice following exposure to ultraviolet light, Photochem. Photobiol., 27, 483-485. National Institute for Occupational Safety and Health (1972) Criteria for a recommended standard on occupational exposure to ultraviolet radiation, U.S. Department of Health, Education and Welfare. Patrick, M.H., and R.O. Rahn (1976) Photochemistry of DNA and polynucleotides: Photoproducts, in: S.Y. Wang (Ed.). Photochemistry and Photobiology of Nucleic Acids, Vol. II, Academic Press, New York, pp. 83-87. Pitts, D.G. (1974) The Human Ultraviolet Action Spectrum, Am. J. Optom. Physiol. Optics, 51, 946-960. Pitts, D.G., and T.J. Tredici (1971) The effects of ultraviolet on the eye, Am. Indust. Hyg. Assoc. J., 32, 235-246. Pulleyblank, D.E., M. Shure and J. Vinograd (1977) The quantitation of fluorescence by photography, Nuc. Acids Res., 4, 1409-1418. Setlow, J.K., and R.B. Setlow (1963) Nature of the photoreactivable ultraviolet lesion in DNA, Nature (London), 197, 560-562. Setlow, R.B., W.L. Carrier and J. Stewart (1975) Endonuclease sensitive sites in uv-irradiated DNA, Biophys. J., 15, 194a. Sutherland, B.M., J.S. Cimino, N. Delihas, A.G. Shih and R.P. Oliver (1980a) Ultraviolet light-induced transformation of human cells to anchorage-independent growth, Cancer Res., 40, 1934-1939. Sutherland, B.M, L.C. Harber and I.E. Kochevar (1980b) Pyrimidine dimer formation and repair in human skin, Cancer Res., 40, 3181-3185. Sutherland, J.C., D.C. Monteleone, J. Trunk and G. Ciarrochi (1984) Two-dimensional, computer controlled film scanner: Quantitation of fluorescence from ethidium bromide-stained DNA gels, Anal. Biochem, 139, 390-399.