Journal o f Photochemistry and Photobiology, B: Biology, 7 (1990) 2 6 1 - 2 7 6

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I N V E S T I G A T I O N S OF A M A N G A N E S E - C O N T A I N I N G MIMIC OF S U P E R O X I D E D I S M U T A S E IN RIBOFLAVIN P H O T O T O X I C I T Y IN H U M A N C E L L S / N VITRO BERNHARD ORTEL, RICHARD WILLIAM GANGE and TAYYABA HASANt

Wellman Laborataries o f Photomedicine, Department o f Dermatology, Harvard Medical School, Massachusetts General Hospital, 50 Blossom Street, Boston, MA 02114 (U.S..a-) (Received February 20, 1990; accepted February 28, 1990)

K e y w o r d s . Riboflavin, phototoxicity, manganese desferioxamine, SOD mimic, superoxide anion.

Summary The activity and specificity of a manganese-containing low molecular weight mimic of superoxide dismutase (manganese desferioxamine (Mndf)) were investigated in riboflavin CRf) photosensitization in solution and cell culture. In addition to the very high superoxide dismutase-like activity of Mndf at micromolar concentrations, photochemical studies in solution indicated that it could quench excited singlet and triplet states at minimolar concentrations. Human erythrocytes, human lymphocytes and a human bladder carcinoma cell line were used to evaluate the potential of Mndf for i n vivo use. The efficacy and toxicity of Mndf in the protection against Rf photoxicity varied between the different cell types.

1. Introduction Superoxide dismutase (SD) is one of the enzymes which protects aerobic organisms from oxygen toxicity. SD was first described by McCord and Fridovich in 1969 [1] and a number of sensitive assays have been developed for the detection of minute quantities of this metalloprotein (for a review, see ref. 2). The significance of superoxide anion (O2-) has been demonstrated to be crucial in paraquat toxicity [3-5], reperfusion injury in organ transplantation [6, 7] and hyperoxygenation pulmonary toxicity in animals [8, 9]. O2- can induce single-strand breaks in DNA [10] and its involvement has been implied in tumor promotion [11, 12]. It may also play a role in cutaneous photosensitization [13, 14]. *Author to whom correspondence should be addressed.

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262 In order to clarify the role of 0 2 - in biological systems, SD has been used for selective dismutation [12-16]. A [email protected] problem with the use of SD is its high molecular weight (greater than 30 kilodalton) which limits its diffusibility into cells. I n v i v o delivery is limited by rapid clearance and potential antigenicity, although this problem may be circumvented by the use of polyethylenegiycol-coated enzyme. Another approach to dismutate O2- in biological systems involves the development of SD mimics which are low molecular weight chelators with complexed metal atoms exhibiting SD activity. The advantage of using small molecules with SD activity is the potentially easier access of these mimics into ceils and cell organelles. One such compound, copper(II) complexed into diisopropyisalicylate (Cudips), was originally developed for the treatment of rheumatoid arthritis [ 17]. Cudips has SD activity in solution. Its effectiveness in 0 2 - dismutation in biological systems is, however, controversial. Athar et al. [18] have reported it to be protective against oxidative damage i n v i v o . However, Darr et al. [19] have demonstrated that it does not show SD activity in the presence of protein, probably owing to the disintegration of the complex caused by copper-protein chelation. More recently a manganese(IV)-containing compound has been reported to be a very efficient mimic of SD [19]. This compound, manganese desferioxamine (Mnd0, possesses high SD activity. A concentration of about 1 p2¢I Mndf mimics the activity of 1 U SD in the standard assay. Unlike Cudips, Mndf is not inactivated by the presence of protein [19]. In cultured cells protection from 02--mediated paraquat toxicity has been demonstrated in algae [16] and in Chinese hamster ovary cells [15]. Because of the role oxygen radicals frequently play in cutaneous phototoxicity [14], Mndf was considered to be potentially effective in preventing this undesirable side effect of clinical photodynamic therapy with hematoporphyrin derivative (HPD) [18, 20]. As a prelude to final testing i n v i v o we have investigated the quenching potential of Mndf in photoseusitized reactions of riboflavin (Rf) (traditionally considered to be an efficient generator of 0 2 - ) in solution and three cell culture systems. Since the protective effect of Mndf has been demonstrated in one eukaryotic cell culture system [15], we explored three different human cell types in order to investigate the protective effect of Mndf at different leveis of cellular activity and structure. We investigated the photosensitized hemolysis of human erythrocytes, the photosensitized inhibition of tritiated thymidine ([SH]-Td) incorporation in lymphocytes and the photocytotoxicity towards a human bladder carcinoma cell line (MGH-U1). Erythrocytes lack nuclei and are a well-established model for membrane damage of various kinds including 0 2 - toxicity [21, 22]. Lymphocytes have little cytoplasm, a low rate of protein synthesis and a limited number of cell organelles. Mitogenstimulated [3H]-Td incorporation and its inhibition is a standard procedure for assessing proliferative response and its inhibition. MGH-U1 cells are transformed cells from the transitory epithelium of the urinary bladder [23] with abundant protein synthesis. Their response to photo-

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toxicdamage has been well characterized in our laboratories [24, 25l. Rf (vitamin Be; Fig. 1) is a nutritional factor found in milk, eggs, vegetables, yeast and other food. Its active forms, flavin mononucleotide (FMN) and flavin-adenine dinucleotide (FAD), are cofactors for a multitude of enzymes. It is bright yellow and has absorption maxima at 266, 372 and 446 u m and fluoresces with an emission maximum at 572 um (Fig. l(b)). The green fluorescence of Rf disappears on irradiation owing to concomitant cleavage of the side-chain (photodealkylation) [26]. The photoproduct is dependent on the pH of the environment, with lumiflavin (Fig. 1) being the photoproduct at basic pH and lumichrome in neutral or acidic conditions. Rf excited states can react with molecular oxygen. Superoxide anion produced by this interaction [27] has been demonstrated by SD-inhibitable reduction of cytochrome c (cyc) and nitroblue tetrazolium (NBT) [28]. Photosensitization resulting in photohemolysis or cell killing has traditionally been attributed to Oe- production [29, 30]. Rf has also been shown to bleach N~V-dimethylp-nitrosoa~iline (RNO) in the presence of histidine, which is consistent with singlet oxygen (102) production [31]. There is also evidence that flavin excited triplet states are reactive without involvement of oxygen intermediates [32]. 2. M a t e r i a l s

and methods

2.1. C h e m i c a l s

Rf, type I xanthine oxidase, type HI cytochrome c (cyc), bovine superoxide dismutase, xanthine, RNO, sodium azide (NaNs), mannitol, sodium benzoate, catalase, potassium iodide (KI), nitroblue tetrazolium (NBT), phytohemagglutinine (PHA), ethidium bromide, fluorescein diacetate, sodium hydrosulfite, potassium phosphate and sodium bicarbonate were purchased from Sigma Chemical Company (St. Louis, MO) and were used as received. All reagents were dissolved in double-distilled deionized water. Hanks buffered salt solution without calcium and magnesium (HBSS), Dulbecco's phosphate-buffered saline (DPBS) and McCoy's modified 5a medium containing 25 mM N-(2-hydroxethyl) piperazine-N'-(2-ethanesulfonic acid) (HEPES) buffer were purchased from Gibco Laboratories, Grand Island, NY. Tritiated thymidine ([SH]-Td; specific activity, 20.0 Ci m m o l - 1 ) was obtained from NEN Research Products, Boston, MA. Mndf was prepared as described by Darr et al. [19] by stirring a suspension of ground manganese dioxide in an aqueous solution of desferioxamine mesylate (desferal, CIBA Pharmaceutical Company, Summit, NJ). For i n v i t r o cellular experiments Mndf was sterilized by filtration through a 0.22 ~ m pore size sterile filter (Nalge Company, Rochester, NY). 2.2. R a d i a t i o n s o u r c e s

The radiation source for solution experiments consisted of a bank of UVA fluorescent tubes with a broad peak at 365 nm (Elder Pharmaceuticals,

264

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265 Bryan, OH). The typical irradiance at the level of the specimens was 0.9-1.2 mW cm -2 at a distance of 60 cm. For the experiments with MGH-U1 cells and lymphocytes and for photohemolysis, a solar simulator equipped with a 2500 W xenon arc lamp was used. The radiation was passed through a liquid filter (6 cm path; mixture of 7% cupric sulfate and 8% cobalt sulfate) and two I nun glass filters (WG-345 and UG-5 from Schott Glass Technologies, Inc., Duryea, PA). The radiation was reflected by a dichroic mirror in order to reduce the longer wavelengths further. This resulted in a bell-shaped spectrum with a peak at 372 nm. The irradiance was typically around 25 mW cm -2 at a distance of 60 cm. Dosimetry was performed using an IL 1700 photometer equipped with a photodiode with peak sensitivity at 365 n m (International Light Inc., Newburyport, MA).

2.3. Spectrophotometer A Hewlett Packard 8451A diode array spectrophotometer was used for all spectrophotometric determinations. 2.4. Cells Human erythrocytes were obtained from healthy volunteers by centrifugation of freshly drawn, heparinized blood. The plasma was saved and the erythrocytes washed twice in DPBS. Human lymphocytes were isolated from freshly drawn blood of healthy volunteers by centrifuging over a FicoU/Hypaque density gradient. After washing twice with HBSS at 4 °C, cells were resuspended in HBSS. MGH-U1 cells, a human urinary bladder carcinoma cell line, were donated by Dr. Chl-Wei Lin, Department of Urology, Massachussets General Hospital, Boston, MA~ They were maintained in McCoy's modified 5a medium containing 5% fetal calf serum, in a 59/0 C02 humidified atmosphere at 37 °C. 2.5. Photosensitization of cells 2.5.1. Erythrocyte suspensions Erythrocyte suspensions used in the hemolysis experiments were prepared by adding 2% plasma and 0.1% packed erythrocytes to the Rf solution (100 ~M Rf in 1.29% sodium bicarbonate buffer (pH 8.9)). Aliquots of the suspensions were transferred to 35 m m Petri dishes and kept in the dark for at least 15 min before irradiation. Each sample was then placed on a slightly inclined rotational device in order to keep the erythrocytes suspended throughout the exposure. The plates were returned to the dark for another 150 min before the samples were transferred to test-tubes and the remaining erythrocytes were spun down; 1 ml of the supernatant was mixed with 100 ~1 of a 5% sodium hydrosulfite solution to reduce the hemoglobin, and the absorbance was recorded at 556 run. Controls included buffer plus irradiation and buffer, Rf and htmitlavin in the dark. A dark control was measured with each combination of sensitizer and quenchers. Quenchers were added to the buffer at the same time as the Rf. The concentrations of the quenchers used were 5 mM NAN3, 100 mM sodium benzoate, 20 mM mannitol, 100 and 500 /xM Mndf, 500 U SD and 500 U SD plus 1000 U catalase.

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2.5.2. Lymphocyte suspensions Lymphocyte suspensions with an approximate density of 106 ceils m i - 1 were used in the phototoxicity experiment; suspensions (120 /zl; 1.2 × 105 cells) were plated into a 96 well microtiter plate. After addition of the reactants the cells were kept in the dark for 15 min. Rf was used in concentrations from 5 to 30/~M and Mndf was added in concentrations from 5 to 1000 /~M. After irradiation, the supernatant was r e m o v e d and RPMI 1640 medium (Gibco) containing penicillin, streptomycin, 10% fetal calf serum (FCS) and 2.5 /~g ml -~ PHA was added. After 65 h incubation 1 /xCi of a solution o f [aH]-Td was added for 6 h. The cells were then harvested and the radioactive label was counted in a Beckmann LS 3801 scintillation counter (Beckmann Instruments, Inc., FuUerton, CA). Controls included lymphocytes treated with Rf or Mndf alone but not irradiated and cells irradiated without any reagents added.

2.5.3. MGH-U1 cells MGH-U1 ceils were plated 24 h before the experiment at a density of 5 × 105 cells p er 35 mm Petri dish and left overnight for attachment. After removal of the medium and rinsing with DPBS, 1 ml of the reaction mixture (in DPBS) was added at certain times before irradiation. Light exposures were p e r f o r med at r o o m temperature. The plates were then rinsed twice with DPBS and 2 ml of medium were added for further incubation. Controls included cells that were treated in the same way but not irradiated and cells that were not treated at all. The surviving fraction was determined approximately 24 h after irradiation. Cells were trypsinized with 0.05% trypsin and 0.53 mM ethylenediaminetetraacetic acid (EDTA); the cell n u m b e r was determined by counting an aliquot of the sample with a Coulter count er (Coulter Electronics, Inc., Hialeah, FA). The cell suspension was then mixed with 10% (v/v) of 50 /xg m1-1 fluorescein diacetate in DPBS, and after 5 min the same quantity of a 0.004% solution of ethidium bromide in DPBS was added. The fraction of surviving cells was determined with a fluorescence m i croscope by counting the percentage of green-fluorescing (live) and orange-fluorescing (dead) cells. Incorporation of [aH]-Td was determined typically 24 h after irradiation. After removal of the medium, 1 ml of medium containing 0.25% of a solution of [3H]-Td (specific activity, 20.0 Ci -1 raM; concentration, 0.05 /zM -1 ml; NEN) was added. After 4 h the [3H]-Td-contalning medium was removed, the plates were rinsed twice with DPBS and the cells were trypsinized as described above. The cell num be r and the fraction of surviving cells were determined as described above. The radioactive label was counted in a Beckmann LS 3801 scintillation count er after adding a 500 /~1 aliquot to 3 ml of a scintillation liquid (Ready Gel, Beckmann Instruments, Inc.).

2.5.4. Determination of SD activity This was carried out according to the original m et hod described by McCord and Fridovich [1]. Briefly, final concentrations of 10 /xM cyc and

267 50 p2Vl xanthine in 3 ml of a 50 mM potassium phosphate buffer (pH 7.8) were reacted at 25 °C with a sufficient quantity of xanthine oxidase to give an increase in the absorbance at 550 nm of 0.02 min -1. Typically 12 /~1 of a 1/30 dilution of the stock xanthine oxidase were used. One unit of SD present in this reaction mixture reduces the increase in absorbance to 0.01 min -1. The only modification was the omission of EDTA because of its reported effect on Mndf [19].

2.5. Solution photochemistry The photodegradation of Rf was followed spectrophotometrically in 50 mM potassium phosphate buffer at pH 9.0. The influence of oxygen was determined by irradiating air-saturated samples and solutions which had been bubbled with either oxygen or nitrogen gas for at least 15 rain. The Rf-photosensitized reduction of cyc was performed typically at concentrations of 10 ~M cyc and 10 ~M Rf at pH 9 in 50 mM potassium phosphate buffer. NBT was used under similar conditions. RNO (50 /~M) at pH 9 in potassium phosphate was reacted with 25 /~M Rf in the presence and absence of 10 mM L-histidine. Bleaching of RNO at 440 nm was followed spectrophotometrically.

3. R e s u l t s

3.1. In vitro The dose dependence of Rf photohemolysis is described by a sigmoidal curve (Fig. 2). At threshold fluences the completion of the hemolytic process may require several hours. Hemoglobin was routinely assayed in the supernatant at 2.5 h after irradiation. Protection is recorded as a delay (right shift) and flattening of the sigmoidal hemolysis curve. Variation in the absolute dosimetry was observed for individual experiments, but the percentage changes

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Investigations of a manganese-containing mimic of superoxide dismutase in riboflavin phototoxicity in human cells in vitro.

The activity and specificity of a manganese-containing low molecular weight mimic of superoxide dismutase (manganese desferioxamine (Mndf)) were inves...
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