Photosensitive cataractogens, chlorpromazine and methoxypsoralen, cause DNA repair synthesis in lens epithelial cells.

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1 Number 7 Reports 687 From the Department of Ophthalmology and the Clinical Laboratoiy, Beilinson Medical Center, Sackler School of Medicine, Tel-Aviv University, Petah Tikva, Israel. Submitted for publication Dec. 19, Reprint requests: R. S. Manor, Department of Ophthalmology, Beilinson Hospital, Petah Tikva, Israel. Key words: cell-mediated immunity, malignant melanoma, migration-inhibition factor, extracts of choroidal malignant melanoma-associated antigens REFERENCES 1. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.: Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193:265, Tripodi, D. S., Lyons, S., and Davies, D.: Separation of peripheral leukocytes by Ficoll density gradient centrifiigation, Transplantation 11:487, David, J. R. S., Ah-Ashari, H. S., Lawrence, H. S., and Thomas, L.: Delayed hypersensitivity in vitro. I. The specificity of inhibition of cell migration by antigens. J. Immunol. 92:244, Kuritzky, A., Livni, E., Munitz, H., et al.: Cell mediated immunity to human myelin basic proteins in schizophrenic patients, J. Neurol. Sci. 30:369, Rajapakse, D. A., and Glynn, L. E.: Macrophage migration inhibition test using guinea pig macrophages and human lymphocytes, Nature 226:857, Char, D. H., Hollinshead, A., Cogan, D. C, Ballintine, E. J., Hogan, M. J., and Herberman, R. B.: Cutaneous delayed hypersensitivity reactions to soluble melanoma antigen in patients with ocular malignant melanoma, N. Engl. J. Med. 291:274, Char, D. H.: Inhibition of leukocyte migration with melanoma-associated antigens in choroidal tumors, INVEST. OPHTHALMOL. VISUAL SCI. 16:176, Char, D. H., Jerome, L., McCoy, J. L., and Herberman, R. B.: Cell-mediated immunity to melanoma associated antigens in patients with ocular malignant melanoma, Am. J. Ophthalmol. 79:812, Char, D. H.: Immunologic aspects and management of malignant intraocular pigmented neoplasma. In Peyman, G. A., Apple, D. J., and Sanders, D. R., editors: Intraocular Tumors, New York, 1977, Appleton-Century-Crofts. Photosensitive cataractogens, chlorpromazine and methoxypsoralen, cause DNA repair synthesis in lens epithelial cells. JULE G. JOSE AND K. LEMONE YIELDING. Autoradiographic techniques showed that photoactivation of methoxypsoralen or chlorpromazine caused diffuse nuclear labeling of lens epithelial cells by thymidine. Chlorj)romazine and light also induced thymidine incorporation into lymphocytes, whereas light or drug alone did not cause unscheduled DNA synthesis. Certain photosensitizing agents have been implicated in the production of cataracts in guinea pigs, mice, and humans. 1 " 3 It has been suggested that photo-induced protein coagulation accounts for the opacification. Of course few, if any, drugs show absolute specificity for a specific cellular component, and many photosensitizers affect DNA as well as protein. 4 Several cataractogenic agents such as x-ray, ultraviolet (UV) radiation, and alkylating agents affect DNA as their major biological target, suggesting the importance of examining lens DNA as a potential site of action of cataractogenic photosensitizers. We report here that each of two cataractogenic photosensitizers, methoxypsoralen (8-MOP) and chlorpromazine, in combination with near-uv radiation induce unscheduled DNA synthesis ("repair synthesis") in lens epithelial cells. This was observed autoradiographically and is presumptive evidence for repair of damage to DNA. 5 Photosensitized damage to DNA was substantiated by the additional finding that combined treatment with the cataractogens and near-uv radiation provoked repair synthesis in human peripheral lymphocytes treated with hydroxyurea. These results therefore suggest alteration of epithelial cell DNA as an additional or alternate mechanism by which such compounds can produce lens opacities. Methods Incubation and irradiation of lenses. Male Osborne-Mendel rats approximately 175 to 200 gm in weight were anesthetized with chloroform, and the lenses were removed. The lenses were then placed in 4 ml of Kinoshita's medium as described by von Sallmann and Grimes," to which the photosensitizer was added. After being incubated with the photosensitizing drug for Vi hr at 37 C, the lenses were irradiated cell side up for time periods of 2 to 30 min with a Raymaster lamp (tube type B, Black Raymaster; George W. Gates & Co., Inc., Franklin Square, N. Y.) with its energy peak near 360 nm and practically no radiation shorter than 300 nm, filtered to remove all but a trace of visible light (manufacturer's specifications). This lamp was calibrated with a Yellow Springs Instrument Co. (Yellow Springs, Ohio) meter which recorded a flux of 1.1 x 10 3 ergs/cm sec at 13 cm, the working distance which was used throughout. A piece of plate glass 0.5 cm thick was placed between the lens and the light source as a means of eliminating extraneous far- UV radiation. Two types of control lenses were always maintained. One group was kept in the dark after a V /78/ $00.50/ Assoc. for Res. in Vis. and Ophthal., Inc.

2 688 Invest. Optilhalmol, Visual Sci. July 1978 Reports 2 * % Ik Fig. 1. Auto radiograms of flat-mounted epithelial cells from rat lenses incubated in vitro with HTdR and stained with Schiffs reagent, a, Lens incubated with 8-MOP (5 X lo^m) and irradiated 20 min with near-uv. b, Example of an 8-MOP-treated lens which was kept in the dark, c, Diffuse nuclear incorporation of 3HTdR following exposure to chlorpromazine (3 x 1O~4M) and 10 min near-uv light, d, Chlorpromazine-treated lens kept in the dark, e, Control rat lens epithelium irradiated with near-uv light 10 min but not treated with any drug. 8-MOP was prepared as a stock solution of 1 mg/ml in ethanol and diluted into the incubation medium to give a final ethanol concentration of 1%. Controls also contained 1% ethanol. 3 hr incubation with the drug. The second control group was exposed to near-uv radiation following l A hr incubation in medium lacking the drug. Immediately after each of these three treatments, the lenses were placed in fresh medium containing 5 /u,ci/ml tritiated thymidine (3HTdR, specific activity 40 /xci/mm; New England Nuclear, Boston, Mass.). After 2 hr, they were rinsed in phosphate buffer and fixed in Carney's solution, and epithelial cell flat mounts were prepared. 7 The flat mounts were dipped in Kodak NTB-2 emulsion and refrigerated. After 2 weeks' exposure, they were developed with D-19 and fixed with Kodak rapid fixer. Lymphocyte assay. This assay was a slight modification of the procedure developed by Evans

3 Number 7 Reports 689 and Norman 8 and Gaudin et al. 9 to study repair synthesis following UV irradiation. Whole blood was collected from human donors and passed through a column containing saline-washed nylon fibers. The blood was then mixed with an equal volume of dextran in normal saline (2 gm of dextran per 40 ml of saline). The blood was allowed to settle for 1 hr, and then the upper layer containing the lymphocytes was removed by aspiration. Equal aliquots of this suspension, each containing 5 x 10 6 cells, were placed in disposable plastic tubes, centrifuged, and resuspended in Spinner's minimal essential medium containing chlorpromazine at 0, 3 X 10~ 6, or 3 X 1(T 5 M. After V 2 hr of incubation, the cells were irradiated with near-uv light for various periods of time and stirred at 3 min intervals to promote even irradiation. The cells were then centrifuged, resuspended in 2 ml of medium containing 5 /u-ci/ml of 3 HTdR plus 2 X 10~ 3 M hydroxyurea, and incubated at 37 C with shaking. Two hours later, 1 ml was removed, and the DNA was collected on Nucleopore filters (Nuclepore Corp., Pleasanton, Calif.) placed in vials containing Aquasol and counted on a Beckman scintillation counter (Beckman Instruments, Inc., Palo Alto, Calif.). For each tube, the number of cells per milliliter was determined by means of a Coulter cell counter (Coulter Electronics, Inc., and the data were expressed as counts per minute (cpm) per 10 6 cells. Results. Fig. 1, a, shows the diffuse nuclear labeling typical of DNA repair synthesis, 5 which was induced in lens cells by 8-MOP and near-uv in combination. Such labeling did not occur if the drug-exposed lenses were kept in the dark (Fig. 1, b). Similar results were observed following chlorpromazine plus near-uv radiation as is demonstrated in the irradiated cells in Fig. 1, c, which show diffuse nuclear labeling vs. those kept in the dark (Fig. 1, d) which show no label. At the doses used (1.32 to 19.8 x 10 s ergs/cm) near-uv radiation alone did not induce 3 HTdR incorporation into the lens cells (Fig. 1, e). The tests with chlorpromazine were carried out over several concentrations (1.5 to 6 x 10~ 4 ) at irradiation times from 2 to 30 min. Discernible labeling was present even in those cells irradiated for as little as 2 min. These results show that the cataractogenic photosensitizers 8-MOP and chlorpromazine induce unscheduled DNA labeling in lens epithelial cells when photosensitized. In other cell types, such labeling has been found to characterize DNA repair synthesis 5 and we presume that it has a similar meaning in lens epithelial cells. It seems especially unlikely that such labeling is from replicative synthesis, since virtually all cells were Minutes of Irradiation Fig. 2. Thymidine incorporation in lymphocytes induced by chlorpromazine in combination with near-uv light. Open circles, 3 x 10" 5 M chlorpromazine; closed circles, 3 X 10~ 6 M chlorpromazine; X, no drug. Error bars indicate ± 1 standard deviation from the mean. For details see text. labeled following the combined insults of drug and irradiation. Although 8-MOP had been shown previously to induce repair synthesis in bacterial and mammalian cells, l0 ' '' chlorpromazine had not been studied. Therefore this drug was tested further with a standard lymphocyte assay for DNA repair synthesis. 8 ' 9 This assay also provided quantitative information regarding the relationship between thymidine incorporation and drug or light exposure, which was difficult to obtain by autoradiography of the lens epithelium. These findings are given in Fig. 2. Each point represents the mean of the counts determined in four experiments. Background counts (zero time in light in the presence or absence of drug) varied somewhat from experiment to experiment (from 90 to 180 cpm/10 6 cells). Therefore the zero value was substracted from the corresponding value obtained after radiation before the mean value was determined for each point given on the graph. The graph in Fig. 2 shows that chlorpromazine in combination with near-uv light induced repair synthesis in lymphocytes. The amount of incorporation was dependent upon both light exposure and drug concentration until toxic levels of the drug were achieved.

4 690 Reports Invest. Ophthalmol. Visual Sci. July 1978 Thus the ability of the combination of chlorpromazine and light to provoke unscheduled DNA labeling in the lens epithelium was correlated with the production of DNA repair synthesis in lymphocytes. Discussion. The results presented in this report demonstrate that both 8-MOP and chlorpromazine, if photoactivated, can induce unscheduled (repair) synthesis of DNA in lens epithelial cells. 8-MOP has previously been shown to have this capacity in both bacterial and mammalian cells " 8-MOP is reported to intercalate between the bases of the DNA backbone and, when photolyzed, to crosslink the strands covalently. These lesions can be repaired in Escherichia coli cells by a process involving recombination and excision repair. 10 Photolysis of chlorpromazine within intact cells may also be presumed to cause DNA damage as evidenced by resulting DNA repair synthesis. Although the nature of the lesion(s) is unknown, the related compound methylene blue has been found to photo-oxidize guanine bases of DNA. 12 Perhaps chlorpromazine acts in a similar manner. Chlorpromazine has also been shown to cause mutations in Salmonella tester strains, but only when photo-excited. 13 The compound is less mutagenic for strains having proficient DNA repair systems, suggesting that at least some of the lesions produced by chlorpromazine are also repaired in Salmonella. We believe that the unscheduled DNA labeling observed in these experiments represents synthesis and that such labeling reflects damage to DNA. The primary reason for our contention is that such labeling has been correlated firmly with repair synthesis in other cell lines, 5 and autoradiography is now used routinely to test for DNA-damaging capacity. Furthermore, we previously have reported 14 that such unscheduled labeling occurs following far-uv irradiation, a classic experimental tool used to damage DNA. The possibility that the observed labeling resulted from stimulation of S-phase synthesis appears ruled out by the presence of labeling in virtually 100% of cells. The possibility may be raised that terminal addition to DNA was stimulated by light and the photosensitizing drugs. Although this posibility has not been ruled out directly, it seems quite unlikely in view of the fact that neither UV nor 8-MOP stimulates such activity in other cell systems. Also, terminal deoxynucleotidyl transferase has been demonstrated to exist in only two types of cells, those of the thymus and the bone marrow. 15 ' l6 Such terminal addition, however, would still reflect a provoked change in the normal genetic complement of the lens cell. The significance of DNA damage and repair in the lens is yet to be established. However, our findings that the photosensitive cataractogens chlorpromazine and 8-MOP in combination with near-uv can produce unscheduled DNA (presumably repair) synthesis in the lens suggests one mechanism by which they could produce cataracts. In the case of a continual insult to the DNA, the accumulation of unrepaired lesions could lead to erroneous cell differentiation, altered metabolism, and anomalous crystallin production and contribute ultimately to opacification. The capacity of such compounds to interact with mammalian cell DNA suggests that caution should be employed in their use. For example, patients might be protected from some of their deleterious ocular effects by the use of proper filtering spectacles while they are using such drugs. The phenothiazine derivatives are known to accumulate in the lens and other tissues, 2 which could present a long-term risk for such patients. These findings call into serious question the wisdom of increasing the amount of near-uv light in lamps designed for human use as proposed by Ott, 17 especially since chlorpromazine and 8-MOP are but two of many known man-made or naturally occurring photosensitizers which might act in a similar manner. 18 These results also suggest that tests for potential drug toxicity might include measurements of drug- and drug-light-provoked DNA repair synthesis, as indicators of DNA damage, particularly in instances where such drugs concentrate appreciably in such light-exposed tissues as the lens. From the Laboratory of Molecular Biology, University of Alabama in Birmingham, Birmingham, Ala. This work was supported by NIH postdoctoral fellowship EYO to Jule G. Jose and NIH grant CA These experiments were reported in the Ph.D. dissertation of Jule Jose, University of Alabama at Birmingham Submitted for publication Nov. 4, Reprint requests: Dr: Jule G. Jose, School of Optometry, University of California, Berkeley, Calif Key words: cataract, photosensitizer, DNA repair, methoxsalen, chlorpromazine REFERENCES 1. Cloud, T. M., Hakim, R., and Griffen, A.: Photosensitization of the eye with methoxsalen. II. Chronic effects, Arch. Ophthalmol. 66:689, Howard, R. O., McDonald, C. J., Dunn, B., and Creasey, W.: Experimental chlorpromazine cataracts, INVEST. OPHTHALMOL. 8:413, Setogawa, T., Tamai, A., Matsuura, H., and Ogura, C: Lens changes associated with long-term psychotropica therapy, Yonago Acta Med. 19:103, 1975.

5 Number 7 Reports Eisenstark, A.: Mutagenic and lethal effects of visible and near-ultraviolet light on bacterial cells, Adv. Genet. 16:167, Rasmussen, R. E., and Painter, R. B.: Radiationstimulated DNA synthesis in cultured mammalian cells, J. Cell Biol. 29:11, von Sallmann, L., and Grimes, P.: Effects of isoproterenol on cell division in cultured rat lenses, IN- VEST. OPHTHALMOL. 13:210, Howard, A.: Whole mounts of rabbit lens epithelium for cytological study, Stain Technol. 27:313, Evans, R. G., and Norman, A.: Unscheduled incorporation of thymidine in ultraviolet-irradiated human lymphocytes, Radiat. Res. 36:287, Gaudin, D., Gregg, R. S., and Yielding, K. L.: Inhibition of DNA repair by cocarcinogens, Biochem. Biophys. Res. Commun. 48:945, Cole, R. S., Levitan, D., and Sinden, R. R.: Removal of psoralen interstrand cross-links from DNA of Escherichia coli: mechanism and genetic control, J. Mol. Biol. 103:39, Baden, H. P., Parrington, J. M., Delanty, J. D. A. and Pathak, M. A.: DNA synthesis in nonnal and xeroderma pigmentosum fibroblasts following treatment with 8-methoxypsoralen and long wave UV light, Biochim. Biophys. Acta 262:247, Simon, M. I., and Van Vunakis, H.: The dye sensitized photo-oxidation of purine and pyrimidine derivatives, Arch. Biochem. Biophys. 105:197, MacPhee, D. G., and Imray, F. P.: Mutagenesis by photoactivation of chlorpromazine, a tranquilizer of the phenothiazine group, Aust. J. Biol. Sci. 27:231, Jose, J. G., and Yielding, K. L.: Unscheduled DNA synthesis in lens epithelium following UV irradiation, Exp. Eye Res. 24:113, Baltimore, D.: Is terminal deoxynucleotidyl transferase a somatic mutagen in lymphocytes? Nature 248:409, Bollum, F. J., Chang, L., Tsiapalis, C. M., and Dorson, J. W.: Nucleotide polymerizing enzymes from calf thymus gland, Methods Enzymol. 29:70, Ott, J.: The effect of light and its control, World Hospitals; THopital danes le Monde 3:277, Pathak, M. A.: Basic aspects of cutaneous photosensitization. In Urbach, F., editor: The Biologic Effects of Ultraviolet Radiation, New York, 1969, Pergamon Press, pp Ocular penetration of 5-fluorocytosine. JULIA A. WALSH, DAVID A. HAFT, MICHAEL H. MILLER, MURIEL R. LORAN, AND ALAN H. FRIEDMAN. Ocular penetration of 5-fluorocytosine (5FC) was studied in uninfected rabbits after subconjunctival and oral administration. With oral administration, 5FC achieved therapeutic levels in both the vitreous and aqueous humors. The use of a phannacokinetic model permitted, objective comparison of kinetic events within the eye chambers and the serum. The rates of entry and elimination in the vitreous were found to be slower than those in the aqueous, but the mean concentration over 24 hr was the same. The therapeutic levels achieved in the aqueous after subconjunctival administration were of shorter duration, and no detectable levels occurred in the vitreous. Oral administration is clearly therapeutically superior to subconjunctival administration in this model. In the past decade there has been an increase in the clinical recognition of fungal endophthalmitis. ' 2 Amphotericin B, the antifungal agent commonly used in therapy, is associated with significant toxicity, and the ocular penetration of this drug is poor. 3 Another antifungal agent, 5-fluorocytosine (5FC) is considerably less toxic. 4 Unlike amphotericin B, 5FC is readily absorbed from the gastrointestinal tract, and following a standard oral dose (37.5 mg/kg) serum levels of between 50 and 100 /Ag/ml are obtained. 4 Although in vivo selection of resistant mutants may limit its clinical usefulness, 5FC has been used effectively against pathogenic fungi including several of the organisms associated with fungal endophthalmitis. 4 Most pretreatment isolates of Candida spp. and Cryptococcus neoformans,' for example, are sensitive to less than 5 /u,g/ml. 4 5FC penetrates well into tissue compartments, including cerebrospinal fluid where concentrations in the presence of inflammation average 74% of the simultaneous serum concentrations. 4 Since the blood-aqueous and blood-cerebrospinal fluid barriers may be similar, 5 therapeutic intraocular concentrations following oral administration might be possible. In order to characterize the ocular pharmacokinetics of 5FC, drug concentrations in the serum, aqueous, and vitreous humors were determined in uninfected rabbits following both oral and subconjunctival administration. Materials and methods Ocular concentration after oral administration of 5FC. Twenty-three New Zealand white rabbits weighing approximately 3 kg were used. The animals were anesthetized with ketamine, and a pediatric feeding tube was inserted through the m.outh into the stomach. The contents of a 250 mg capsule (1.5 gm/m 2 ) of 5FC (Hoffman-LaRoche, Inc., Nutley, N. J.) diluted in 10 cc of water was administered. The animals were sacrificed atregular intervals with 100 mg of intravenous pentobarbital, and a terminal blood specimen for drug assay, was obtained. The eyes were immediately enucleated, and samples were taken. Approxi /78/ $00.40/ Assoc. for Res. in Vis. and Ophthal., Inc.