The mechanism of bilirubin-photosensitized DNA strand breakage in human cells exposed to phototherapy light

Mutation Research, 112 (1983) 397-406 Elsevier

397

MTR 03786

The mechanism of bilirubin-photosensitized D N A strand breakage in human cells exposed to phototherapy light Barry S. Rosenstein a, Jonathan M. Ducore b and Scott W. Cummings c a Radiation Biology Section, Department of Radiology, b Department of Pediatrics, and ¢ Department of Biochemistry, The University of Texas Health Science Center at Dallas, 5323 Harry Hines Blvd., Dallas, Texas 75235 (U.S.A.)

(Received 29 November 1982) (Revision received 29 June 1983) (Accepted 22 July 1983)

Summary Exposure of normal human fibroblasts to visible light (420-490 nm) in the presence of exogenously added 1-100 # g / m l bilirubin enhanced the level of DNA strand breakage compared with cells irradiated in the absence of added bilirubin. Treatment of cells in the dark with an irradiated bilirubin solution also induced DNA strand breaks. However, strand breakage was not detected in cells treated with an irradiated bilirubin solution that had been incubated with catalase (H202 : H202 oxidoreductase EC 1.11.1.6). Examination of irradiated bilirubin solutions demonstrated the presence of hydrogen peroxide although, apparently, not at concentrations sufficient to account for the level of DNA strand breakage detected. Hence, irradiation of bilirubin results in the generation of hydrogen peroxide and possibly other peroxides that can cause DNA damage.

Exposure of normal human fibroblasts to visible light produced by lamps commonly used in the phototherapy treatment of neonatal hyperbilirubinemia has been found to induce DNA strand breakage (Bradley et al., 1978; Speck and Rosenkranz, 1978; Rosenstein and Ducore, 1983, 1984). However, irradiation of cells in the presence of bilirubin enhances the level of strand breaks 30-40-fold (Rosenstein and Ducore, 1984). Therefore, bilirubin can act as a photosensitizing agent causing an increase in the level of DNA damage in cells exposed to phototherapy light. This finding is of interest, because it has been estimated that from 2.5% Abbreviations: DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate

0167-8817/83/$03.00 © 1983 Elsevier Science Publishers B.V.

398 (Albrecht and Roney, 1974) to 10% of newborn infants (Speck and Rosenkranz, 1979) are exposed to high fluences of visible light used for treatment of neonatal hyperbilirubinemia (Cremer et al., 1958; Lucey et al., 1968). Hence, side effects of a carcinogenic or genetic nature may result from the DNA damage induced by phototherapy. The purpose of the work presented in this paper was to identify the photoproduct(s) generated through irradiation of bilirubin that causes DNA damage and to determine the fluence and concentration dependence of this effect. Materials and methods

Cell lines and culture conditions Normal human fibroblasts were derived from skin biopsies (Goldstein et al., 1976) and were the gift of Dr. M. Brown and Dr. J. Goldstein (Department of Biophysics and Molecular Genetics, the University of Texas Health Science Center at Dallas, Dallas, Texas). The cells were grown in Dulbecco's modified Eagle's medium (Grand Island Biological Co., Grand Island, NY) supplemented with 10% fetal calf serum (Flow Laboratories, Rockville, MD), L-glutamine at 400 # g / m l , penicillin at 100 u n i t s / m l and streptomycin at 100/~g/ml (Grand Island Biological Co.). Cultures were incubated at 37°C in a 7.5% CO 2 humidified atmosphere and the medium changed two times per week. Under these conditions the cell number doubled every 1-3 days. Labeling and irradiation conditions Human fibroblasts were plated in 25 cm 2 tissue culture flasks (Corning Glass Works, Corning, NY) at a density of 2 × 104 cells/cm 2 and either methyl[3H]thymidine was added to a final concentration of 0.05/~Ci/ml (20 C i / m m o l e , New England Nuclear, Boston, MA) or 2114C]thymidine was added to a final concentration of 0.04 # C i / m l (40 mCi/mmole). The cells were incubated for 48 h, the medium replaced with fresh non-radioactive medium and the cultures grown an additional 24 h to insure that the label was incorporated into parental DNA at the time of irradiation. The cells were then washed three times with phosphate-buffered saline (PBS, 8.0 g NaC1, 0.2 g KCI, 1.15 g N a 2 H P O 4 and 0.2 g K H 2 P O 4 in 1 1 distilled H 2 0 ). The 14C-labeled cells were covered with 5 ml of PBS or PBS containing 1, 10 or 100 ~tg/ml bilirubin (Sigma Chemical Co., St. Louis, MO; stock solution: 10 m g / m l in dimethyl sulfoxide, DMSO) and after 20 min exposed to 0 - 5 0 k J / m 2 of light from 8 Westinghouse F 2 0 T 1 2 / B B special blue lamps at a fluence rate of 16.7 W / m 2. Essentially, the entire output of these lamps is between 420 and 490 nm (Sisson et al., 1972). The fluence rate was measured with a dosimeter composed of a Beckman 12055 photocell and a Simpson 260 digital multimeter. The dosimeter was calibrated against a quartz windowed Eppley thermopile and microammeter (Keithly Model 150B) in comparison with a standard lamp (U.S. Bureau of Standards). For all irradiations, the light was filtered through 8-ram lead glass to eliminate wavelengths shorter than 350 nm and the flasks were placed on ice-water baths at I°C. For experiments in which ceils were treated with

399

irradiated bilirubin, 5 ml of 1130/~g/ml bilirubin in a 25 cm2 flask was exposed to 0, 10, 25 or 50 k J / m 2 of special blue light and the irradiated bilirubin solutions were then incubated with cells at 1°C for 10, 25 or 50 min. The 3H-labeled cells, which acted as internal standards, were exposed to 300 rad of 6°Co ~,-rays, from an AECL theratron-80 at a dose rate of 170 rad/min. Dosimetry was performed with a Victoreen air ionization chamber.

Catalase treatment 100 # g / m l bilirubin in PBS was exposed to 50 k J / m 2 of special blue light. The irradiated bilirubin solutions or 32 #M hydrogen peroxide (Malinckrodt, Inc., Paris, KY) were incubated at 37°C for 30 min with 0 or 10 # g / m l catalase (10000-25 000 units/mg protein; Sigma). Solutions were cooled to 1°C and the cultures were treated for 50 rain at this temperature. Elution conditions and calculations of DNA strand breaks After completion of the irradiations the PBS was replaced with PBS containing 0.2 m g / m l disodium EDTA and the cells were gently scraped off the surface of the flask with a rubber policeman. 106 of the 14C- and 106 of the 3H-labeled cells were lysed together on a 25 mm, 2 micron pore size polycarbonate filter (Nucleopore Corp., Pleasonton, CA) with a 2% solution of sodium dodecyl sulfate (SDS, Gallard-Schlesinger, Carleplace, NY), 0.1 M glycine and 0.02 M EDTA, pH 10. The lysis solution was allowed to flow through the filter by gravity. 2 ml of lysis solution containing 0.5 m g / m l proteinase-K (Scientific Products, Dallas, TX) was then placed on the filter followed by the elution solution of tetrapropylammonium hydroxide (RSA Corp., Ardsley, NY), 0.02 M EDTA (acid form) and 0.1% SDS, pH 12.1. The elution solution was pumped at 0.035-0.045 m l / m i n for 15 h. Five fractions were collected at 3-h intervals. Upon completion of the elution, fractions were made isovolumetric with water when necessary and 10 ml of Aquassure (New England Nuclear) was added. Filters were processed as previously described (Kohn et al., 1981). All fractions were counted in a Packard tricarb liquid scintillation counter. The frequency of breaks induced in the DNA was calculated from the following equation: Break frequency per dalton = 8.1 × 10-10

Btreatment - Bunirradiated g30o rad y-rays -- gunirradiated

where B equals the logarithm of the fraction of DNA retained on the filter after 0 h of elution minus the logarithm of the fraction DNA retained on the filter after 15 h of elution. Previous measurements (Kohn et al., 1976) have shown that a "t-ray dose of 300 rad induces 8.1 × 10 -1° single strand breaks per dalton of DNA. The specified variable, ntreatment, refers to cultures treated either with special blue light, bilirubin, bilirubin plus light, irradiated bilirubin (+ catalase) or hydrogen peroxide ( + catalase).

Measurement of hydrogen peroxide The concentration of hydrogen peroxide in the irradiated bilirubin solutions was

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determined by measurement of the oxygen released upon treatment of these solutions with catalase. The breakdown or dismutation of hydrogen peroxide by catalase yields 0.5 mole of oxygen and 1.0 mole of water per mole of hydrogen peroxide. An increase in the mV potential of an oxygen-sensitive electrode corresponds to an increase in the oxygen concentration within the electrode chamber. This response or change in oxygen concentration was quantified using a Clark-type electrode at 25°C (Prough and Ziegler, 1977). Calibration of the electrode response was performed by measuring the loss of oxygen in the presence of beef heart transport particles and known amounts of N A D H (Green and Ziegler, 1963). Results

Irradiation of cells with special blue light Exposure of normal human cells at I°C to 200 k J / m e of light from a special blue lamp caused the DNA from these cells to elute more rapidly from filters compared with DNA from unirradiated cells (Fig. 1) indicating the induction of DNA strand breaks (5 b r e a k s / 1 0 a° daltons). The cells were irradiated at 1°C to prevent both repair of induced breaks and formation of enzymatically induced breaks through excision repair. It is unlikely that this DNA breakage was caused by the formation of medium photoproducts (Wang et al., 1974) because the cells were washed three times with PBS and irradiated in PBS. However, to test this possibility, unlabeled cells were irradiated in PBS and the irradiated PBS was then incubated with labeled cells. As shown in Fig. 1, no detectable D N A strand breaks were induced. 1.0

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Fig. 1. Elution profiles of D N A from cells exposed to either special blue light, y-rays or irradiated PBS. Fig. 2. Enhancement of D N A damage of bilirubin. Elution profiles of D N A from cells treated with 1. 10 or 100 t~g/ml bilirubin and exposed to 20 k J / m 2 of special blue light.

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Effect of bilirubin concentration and special blue light fluence on strand break induction Cells were exposed to special blue light in the presence of 1, 10 or 100/~g/ml bilirubin. In each case the frequency of strand breaks was increased compared with cells irradiated in the absence of added bilirubin (Fig. 2) with strand breakage following a linear response as a function of the logarithm of bilirubin concentration (Fig. 3). In addition, the elution profile of DNA from cells allowed to incubate for 30 min at 37°C after exposure to 300 k J / m 2 of special blue light or treatment with 100 # g / m l bilirubin followed by irradiation with 15 k J / m 2 of special blue light (Fig. 4) was nearly identical to the elution profile of DNA from unirradiated cells. This demonstrates that the induced strand breaks can be repaired and thatthe DNA breakage was not the result of a general lysis of cells during the irradiation. Cells were also incubated with 1/~g/ml bilirubin at 37°C for 30 min, washed three times with PBS and exposed to 20 k J / m 2 of special blue light. The frequency of strand breaks remained elevated indicating uptake or association of bilirubin by these cells although the level of breakage (2 breaks/101° daltons) was lower than in cells irradiated in the presence of 1/~g/ml bilirubin (3 breaks/101° daltons). Cells were exposed to different fluences of light in the presence of 100/~g/ml bilirubin and the elution profiles shown in Fig. 5. DNA breakage follows a linear fluence response (Fig. 6). It should be pointed out that all fluences indicated represent the fluence to the bilirubin and not the cells. Because bilirubin absorbs strongly in the blue (Sisson, 1976), the transmittance of light to cells is low for high 1.0 I

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bilirubin concentrations. For the three concentrations used in these experiments, 100, 10 and 1 #g/ml, the per cent transmittances were 14, 80 and 97, respectively. However, irradiation of bilirubin causes its photodegradation (Lightner, 1977) resulting in higher levels of transmittance with increasing fluence (Fig. 7). After exposure to 50 k J / m 2 of special blue light the per cent transmittances through 100, 10 and 1 / ~ g / m l bilirubin solutions were 30, 98 and 100, respectively.

Treatment of cells with irradiated bilirubin 100 # g / m l bilirubin was exposed to 50 k J / m 2 of special blue light and used to treat cells in the dark at I ° C for 10, 25 or 50 min. D N A damage was induced by the irradiated-bilirubin solutions (Fig. 8) demonstrating that the D N A damage was caused by a stable photoproduct. Because the level of D N A strand breakage reached a maximum after a 25-50-min incubation at I°C, all further experiments involving irradiated-bilirubin utilized a 50-min incubation. Cells were also treated with 100 # g / m l bilirubin that was irradiated with either 0, 10, 25 or 50 k J / m 2 of special blue light (Fig. 9) and strand break induction was found to follow a linear fluence response (Fig. 10). 100 # g / m l bilirubin was exposed to 50 k J / m 2 of special blue light and incubated with 0 or 10 # g / m l catalase at 37°C for 30 min. Catalase is a hydroperoxidase and causes the destruction of the peroxide bond (Schonbaum and Chance, 1976). Cells were incubated at I ° C with the catalase-treated irradiated-bilirubin solutions and additional cultures were incubated with hydrogen peroxide or catalase-treated 1.0

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hydrogen peroxide. Incubation of the irradiated-bilirubin with catalase completely eliminated the DNA-damaging activity of this solution (Fig. 11) demonstrating that hydrogen peroxide and/or other peroxides induced the DNA strand breakage. To test the possibility that catalase caused the elimination of strand breakage through a non-specific effect unrelated to its activity as a hydroperoxidase, cells were exposed to catalase-treated bleomycin (a drug that induces DNA strand breaks through a mechanism that does not involve hydrogen peroxide generation; Suzuki et al., 1970). Catalase treatment of bleomycin had no effect on its ability to induce DNA strand breaks (data not shown). Cells were also covered with PBS or a 1% DMSO solution (the bilirubin solvent) in PBS and exposed to 50 k J / m 2 of special blue light. The level of DNA strand breaks induced (1 break/101° daltons) was approximately the same for both treatments indicating that the strand breakage caused by irradiated bilirubin was not due to irradiation of DMSO. In addition, no hydrogen peroxide was detected in either of these two solutions.

Measurement of hydrogen peroxide The concentration of hydrogen peroxide in bilirubin solutions exposed to different fluences of special blue light was determined by treating the solutions with catalase and measuring the evolution of oxygen using a sensitive oxygen electrode. 1.0 40

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Fig. 11. Effect of catalase on irradiated bilirubin and hydrogen peroxide. Elution profiles from cells treated with 100 ~ g / m l bilirubin that had been exposed to 50 k J / m 2 of special blue light and incubated at 37°C for 30 min with 0 (zx) or 10/~g/ml catalase (,L). Cells treated with 32/LM hydrogen peroxide that had been incubated at 37°C for 30 min with 0 ( O ) or 10/~g/ml catalase (e). Fig. 12. Measurement of hydrogen peroxide in irradiated bilirubin solutions. 100 # g / m l bilirubin in PBS was exposed to 0, 5, 10, 15, 20, 30, 40 or 50 k J / m 2. of special blue light. The concentration of hydrogen peroxide in these solutions was determined as described in Materials and Methods.

405

Although other peroxides act as substrates for catalas¢, only hydrogen peroxide yields oxygen upon treatment with this enzyme (Schonbaum and Chance, 1976). As shown in Fig. 12, the generation of hydrogen peroxide followed a linear fluence response up to 50 k J / m 2. However, it appears that the strand breakage was not caused by hydrogen peroxide alone, because an irradiated-bilirubin solution containing 32 #M hydrogen peroxide induced 19 breaks/101° daltons whereas a 32-#M hydrogen peroxide solution (concentration determined using the oxygen electrode) only produced 10 breaks/101° daltons (Fig. 11). Hence, potentially some other photoproduct(s) was responsible for part of the DNA damage. This other photoproduct(s) must also be a peroxide, however, because treatment of the irradiated bilirubin with catalase completely eliminated its DNA-damaging activity (Fig. 11).

Discussion Exposure of bilirubin to visible light used in the treatment of neonatal hyperbilirubinemia causes the generation of hydrogen peroxide and possibly other peroxides that can act to damage DNA in human cells. The amount of peroxide produced is dependent upon both the fluence of light and the concentration of bilirubin. The discovery that bilirubin irradiation results in the formation of DNA-damaging photoproducts is a cause for concern because thousands of newborn infants each year receive phototherapy for treatment of hyperbilirubinemia. It would therefore be prudent to limit the use of ph0totherapy to cases in which it is clearly necessary and will be beneficial, due to the association between DNA damage and carcinogenesis and genetic alterations. However, concern over the DNA-damaging activity of irradiated bilirubin in lessened somewhat by identification of hydrogen peroxide and possibly other peroxides as the critical photoproducts because cells grown in culture generally exhibit a much lower level of catalase activity compared with cells in freshly explanted tissue (Parshad et al., 1980a). Hence, cells in vivo may contain sufficient catalase to inactivate any peroxide formed. In addition, hydrogen peroxide is not a mutagen (Bradley and Erickson, 1981), although it does induce chromosome aberrations (Parshad et al., 1980b) and sister-chromatid exchanges (Bradley et al., 1979). Clearly, additional experiments are needed to determine whether, in vivo, the phototherapy treatment for neonatal hyperbilirubinemia produces harmful side effects resulting from DNA damage.

Acknowledgements This research was supported by the American Cancer Society (IN-142), the Southwestern Medical Foundation and S.W.C. is a National Institutes of Health Trainee (T32 GM07062). In addition, we thank Miss Cathy Smith for her expert secretarial assistance. This research was presented in part at the 10th Annual Meeting of the American Society for Photobiology, Vancouver, BC, 1982. References Albrecht, R.M., and P.L. Roney (1974) Hospital survey of the use of phototherapy for neonatal hyperbilirubinemia, U.S. Bureau of Radiological Health, Food and Drug Administration.

406 Bradley, M.O., and L.C. Erickson (1981) Comparison of the effects of hydrogen peroxide and X-ray irradiation on toxicity, mutation and DNA damage/repair in mammalian cells (V79), Biochim. Biophys. Acta, 654, 135-141. Bradley, M.O., L.C. Erickson and K.W. Kohn (1978) Non-enzymatic DNA strand breaks induced in mammalian cells by fluorescent light, Biochim. Biophys. Acta, 520, 11-20. Bradley, M.O., I.C. Hsu and C.C. Harris (1979) Relationships between sister chromatid exchange and mutagenicity, toxicity and DNA damage, Nature (London), 282, 318-320. Cremer, R.J., P.W. Perryman and D.H. Richards (1958) Influence of light on the hyperbilirubinemia of infants, Lancet, 1, 1094-1097. Goldstein, J.C., M.S. Brown and N.J. Stone (1976) Genetics of the LDL receptor; Evidence that the mutations affecting binding and internalization are allelic, Cell, 12, 629-641. Green, D.E., and D.M. Ziegler (1963) Electron transport particles, in: S.P. Colowick and N.O. Kaplan (Eds.), Methods in Enzymology, Vol. 6, Academic Press, New York, pp. 416-424. Kohn, K.W., L.C. Erickson, R.A.G. Ewing and C.A. Friedman (1976) Fractionation of DNA from mammalian cells by alkaline elution, Biochemistry, 15, 4629-4637. Kohn, K.W., R.A.G. Ewig, L.C. Erickson and L.A. Zwelling (1981) Measurement of strand breaks and cross-links by alkaline elution, in: E.C. Friedberg and P.C. Hanawalt (Eds.), DNA Repair, Vot. 1, Part B, Marcel Dekker, New York, pp. 379-401. Lightner, D.A. (1977) The photoreactivity of bilirubin and related pyrroles, Photochem. Photobiol., 26, 427-436. Lucey, J.F., M. Ferriero and J. Hewitt (1968) Prevention of hyperbilirubinemia of prematurity by phototherapy, Pediat., 41, 1047-1054. Parshad, R., K.K. Sanford, G.M. Jones, R.E. Torone, H.A. Hoffman and A.H. Grier (1980a) Susceptibility to fluorescent light-induced chromatid breaks associated with DNA repair deficiency and malignant transformation in culture, Cancer Res., 40, 4415-4419. Parshad, R., W.G. Taylor, K.K. Sanford, R.F. Camalier, R. Gantt and R.E. Tarone (1980b) Fluorescent light-induced chromosome damage in human IMR-90 fibroblasts; Role of hydrogen peroxide and related free radicals, Mutation Res., 73, 115-124. Prough, R.A., and D.M. Ziegler (1977) The relative participation of liver microsomal amine oxidase and cytochrome P-450 in N-demethylation reactions, Arch. Biochem. Biophys., 180, 363-373. Rosenstein, B.S., and J.M. Ducore (1983) Induction of DNA strand breaks in normal human fibroblasts exposed to monochromatic ultraviolet and visible wavelengths in the 240-546 nm range, Photochem. Photobiol., 38, 51-55. Rosenstein, B.S., and J.M. Ducore (1984) Enhancement by bilirubin of DNA damage induced in human cells exposed to phototherapy light, Pediat. Res., in press. Schonbaum, G.R., and B. Chance (1976) Catalase, in: P.P. Boyer (Ed.), The Enyzmes, Vol. XIII, Part C, Academic Press, New York, pp. 363-408. Sisson, T.R.C. (1976) Visible light phototherapy of neonatal hyperbilirubinemia, in: K.C. Smith (Ed.). Photochemical and Photobiological Reviews, Vol. 1, Plenum, New York, pp. 241-268. Sisson, T.R.C., N. Kendall, E. Shaw and L. Kechavarz-Oliai (1972) Phototherapy of jaundice in the newborn infant, 11. Effect of various light intensities, J. Pediatr., 81, 35-38. Speck, W.T., and H.S. Rosenkranz (1976) Intracellular deoxyribonucleic acid modifying activity of phototherapy lights, Pediat. Res., 10, 553-555. Speck, W.T., and H.S. Rosenkranz (1979) Phototherapy for neonatal hyperbilirubinemia, a potential environmental hazard to newborn infants, Environ. Mutagen., 1,321-336. Suzuki, H., K. Nagai, E. Akutsu, H. Yamaki, N. Tanaka and H. Umezana (1970) On the mechanism of action of bleomycin; Strand scission of DNA caused by bleomcyin and its binding to DNA in vitro, J. Antibiotics, 23, 473-480. Wang, R., J. Stoien and F. Landa (1974) Lethal effect of near-ultraviolet irradiation on mammalian cells in culture, Nature (London), 247, 42-45.