VISIBLE LIGHT ANAEROBIC PHOTOCONVERSION OF TYROSINE SENSITIZED BY RIBOFLAVIN. CYTOTOXICITY ON MOUSE TUMORAL CELLS

Photochemistry and Photobiology, Vol. 62, No. 6, pp. 104-1045, 1995 Printed in the United States. All rights reserved

0031-8655/95 $05.00+0.00 0 1995 American Society for Photobiology

RESEARCH NOTE

VISIBLE LIGHT ANAEROBIC PHOTOCONVERSION OF TYROSINE SENSITIZED BY RIBOFLAVIN. CYTOTOXICITY ON MOUSE TUMORAL CELLS

EDUARDO SILVA*’, SONIA FURSTI, ANA M. ED WARDS^, M. INES BECKER? and ALFREW E. DE IOANNES~

‘Facultad de Quimica (536) and ZFacultad de Ciencias BioMgicas, P. Universidad Cat6lica de Chile, Casilla 306 Santiago, Chile

(Received 1.5 May 1995; accepted 2 August 1995)

Abstract-The anaerobic phototransformation of tyrosine under visible light sensitized by riboflavin is reported. The cytotoxicity of the anaerobic photoproducts on in vitro-cultured myeloid mouse tumoral cells was demonstrated. A radical mechanism is proposed. Dityrosine was identified as one of the main anaerobic photoproducts by using absorption, emission and ‘H-NMR spectra.

INTRODUCTION

Near UV light’ and daylight fluorescent light2 produce lethal effects on mammalian cells in culture. This cytotoxicity has been attributed to products of the riboflavin (RF)t-sensitized photooxidation of Trp and/or Tyr.’.” Because the vitamin and both amino acids are usual components of the tissue culture medium, it is important to study the chemical and biochemical mechanisms that produce cytoxicity. From the photochemical viewpoint, two mechanisms, type I and type 11, have been proposed to explain the dye-sensitized photooxidation process. In type I mechanisms, the substrate reacts initially with an excited triplet sensitizer and thereafter with oxygen, via a radical intermediate. In type I1 mechanisms, energy is transferred to molecular oxygen to form singlet oxygen, which, in turn, reacts with the substrate. A mixed type I-type I1 mechanism has been observed when lumiflav- in4 and RF5 have been used as sensitizers in the photooxidation of indolic compounds. Type I process in these systems was far more efficient than the competing type I1 process, irrespective of the indole concentration. One reason for this behavior is that the redox potential of the triplet flavin is estimated to be shifted to 1.7 V4 (basal state: 0.3 V), which is higher than the redox potential of indole’hdole (1.50 V).6 Considering that the Tyr redox potential is 0.93 V7 it is highly probable that triplet R F can be effectively reduced by Tyr, with the concomitant generation of the flavin radical anion and the tyrosyl radical cation. From a biochemical viewpoint, the subcellular targets and molecular mechanism for the induction of cytotoxicity by the photoproducts have not been precisely identified. However, when myeloid and teratocarcinoma mouse cell lines were grown in an irradiated culture medium enriched with Trp and RF, cytoplasmic and nuclear damage was revealed by transmission electron mi-

*To whom correspondence should be addressed. tAbbrevinrions: RF, riboflavin; Tyr/RF/N,, irradiated solution of Tyr

plus RF in anaerobic atmosphere: TyrRF/Oz, irradiated solution of Tyr plus RF in oxygen-saturated condition.

croscopy,” similar to those described for apoptotic lymphoma cells as a consequence of photodynamic the rap^.^

In this work we have investigated the RF-sensitized photoconversion of Tyr under an anaerobic atmosphere and the effect of the anaerobic and aerobic products of the irradiation of Tyr solutions in the presence of R F on an in vifro myeloid cell line named NS0I2.Io

MATERIALS AND METHODS

Amino acids, antibiotics, Dulbecco’s culture medium, Sephadex (3-15, RF and salts were purchased from Sigma Chem. Co. (St. Louis, MO). Epon and glutaraldehyde were supplied by Polysci- ences (Warrington, PA) and fetal calf serum was obtained from GIB- CO BRL (Maryland, CA). All reagents were of the highest available purity and were employed as received.

Solutions of RF (3.5 X 10-5M) and Tyr (1.75 X 10-4M) in 0.05 M phosphate buffer, pH 7.0, were irradiated in a 1 cm light path cell thermostated at 37°C. The anaerobic condition was reached by nitrogen bubbling. Light from a 150 W slide projector lamp, equipped with an interference filter, was employed for irradiation at 450 nm. The energy flow rate was 4.0 J m-z s-’.

Gel filtration was carried out on a 1.8 X 75 cm Sephadex G-15 column equilibrated with distilled water. High-performance liquid chromatography was performed by using a FBondapak C-18 column (Waters Associates, Inc., Milford, MA, 2 mm [ID] X 30 cm). A Waters Associates, Inc. instrument equipped with a model 6000 A pump, a 746 data module and a 441 absorbance detector (254 nm) was utilized. The solvent system consisted of two eluents: A: 0.05 M sodium acetate, 0.05 M citric acid (pH 3.6) : methanol (95:5); and B: 0.05 M sodium acetate, 0.05 M citric acid (pH 3.6) :methanol (75:25). During the first 5 min solvent A was employed and then B was used as eluent.’H-NMR spectrum (D,O, 200 MHz) of dityrosine was obtained using a Bruker AC-200 P RMN spectrophotometer (Karlsriihe, Germany).

Cells of the NS012 myeloid line, deficient in the enzyme hypo- xanthine-guanine-phosphoribosyl transferase (donated by Dr. C. Milstein, Molecular Biology Laboratory, Cambridge, England) were kept under exponential growth in high glucose Dulbecco’s modified Eagle minimal essential medium supplemented with 10% fetal calf serum, nonessential amino acids, 100 (*g/mL streptomycin and 100 I U / d penicillin. For the assays, 100 p,L suspensions of lo4 cells/ mL were seeded on 96 well tissue culture plates at 37°C. Assays were performed at the time of cell seeding, adding in duplicate and at different concentrations (between 10 and 50 kL/well) irradiated solutions of Tyr (2.1 X 10-3M) plus RF (2.1 X 10-4M) in anaerobic

lMl

1042 ED~JARDO SILVA et a1

atmosphere (Tyr/RF/N,) and in oxygen-saturated conditions (Tyr/ RF/02). The controls were irradiated solutions of Tyr/N,, Tyr/Oz, RF/O, and RF/N, as well as nonirradiated FW, Tyr/RF and Tyr solutions. All the solutions were prepared in 0.005 M phosphate buffer. The cultures were incubated at 3 7 T , 10% CO, and 1 0 0 % humidity for 48 h. To quantify the cells, the ones of each well were resus- pended. The count was done in duplicate using a Nikon (Tokyo, Japan) inverted phase-contrast microscope, placing 3 pL of each suspension in a hemocytometer.

For transmission electron microscopy purposes the controls and the treated NS0/2 cells were grown in culture flasks, collected by centrifugation at lo00 rpm during 10 min at room temperature and then fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4 for 24 h, postfixed with 1 % OsO,, dehydrated in graded acetone and embedded in Epon according to Luft." Thin sections ( 4 W 5 0 0 p\) were stained with 4% uranyl in methanol and lead citrate according to Reynolds.'z The preparations were observed under a Philips EM- 300 electron microscope (Eindhoven, Holland).

RESULTS

The irradiation with polychromatic visible light of a Tyr solution (1.75 X M) in the presence of RF (3.5 X 1 O-s n/r, in an anaerobic atmosphere produces alterations in the absorption spectrum; a decrease in intensity is observed in the band at 447 nm and an increase in the zone between 300 and 400 nm, with the formation of an isosbestic point. Con- comitantly, there is a decrease in the fluorescent emission of Tyr, at 310 nm. When the irradiated samples are excited at 320 nm, a new fluorescent emission band is observed, with a maximum at 410 nm; its intensity increases with the time of irradiation. These results indicate that, in the absence of molecular oxygen, there is a phototransformation of Tyr sensitized by RF. When the same Tyr solution is irradiated in the absence of RF, maintaining the other conditions, none of the phenomena described above occur.

We studied the effect of adding Tyr solutions previously irradiated in the presence of RF, in aerobic (Tyr/RF/O,) and anaerobic (Tyr/RF/N2) atmospheres, to mouse NS0/2 tumoral cells cultures, and a dose-response study was carried out. Dif- ferent concentrations of a previously irradiated Tyr plus RF solutions were added to the cultured cells. A cell count was performed in each case in order to quantify the effect on cel- lular proliferation in the same experiment described above. The dose-response curve shown in Fig. 1 was obtained using these data. A decrease in the cell number is clearly seen, which is more drastic in the solutions irradiated under an anaerobic atmosphere (TyrlRFIN,). This effect is also observed when adding solutions irradiated under aerobic atmosphere (Tyr/RF/Oz). These results clearly show that the photoproducts from the anaerobic irradiation of Tyr in the presence of RF have cytotoxic effects on mouse NS0/2 cell cultures.

In order to investigate at the ultrastructural level the cytotoxic effects described above, NS0/2 cells incubated during 24 h and 70 h in Tyr/RF/N, and TyrW/O, were observed by transmission electron microscopy and the results are shown in Fig. 2 . A number of cells with a similar aspect to those of the control cells (Fig. 2a) was observed under both experimental conditions being fewer in the cultures treated with Tyr/RF/N,. In the cell population showing a cytotoxic effect, the more remarkable alterations were found in mitochondria, showing a swelling aspect with diminished cristae (Fig. 2b). Furthermore, the appearance of cytoplasmic vacuoles was observed, the number of which increased with incubation time (Fig. 2c,d).

'f 100,

80

60

40

To characterize and identify the photoproducts of the anaerobic irradiation of Tyr solutions in the presence of RF, the irradiated samples was applied to a G-15 Sephadex column. The elution profile from this column is shown in Fig. 3A; that of nonirradiated Tyr and RF solutions in Fig. 3B, nonirradiated RF in Fig. 3C and that of RF irradiated at the same conditions indicated above, in Fig. 3D.

Figure 3A discloses the presence of two fractions (I and 11) corresponding to photoproducts. Peaks I11 and IV correspond to unreacted Tyr and RF, respectively, which is consistent with the profile shown in Fig. 3B. This was confirmed by absorption and emission spectroscopy and by the elution times on an HPLC-C,, column. Fraction I in Fig. 3D corresponds to an RF photoreaction product with an elution volume that coincides with that of peak I shown in Fig. 3A. In order to establish the purity of the photoproducts found, an HPLC-C,, column was employed. Peak I (Fig. 3A) pre- sents four main signals, two of which coincide with those observed when applying the peak I shown in Fig. 3D, which corresponds to RF photoreaction products. Peak I1 exhibits only one signal on the HPLC chromatogram. Its absorption spectrum showed two absorption bands at 284 nm and 320 nm. This compound showed a fluorescent emission with a maximum at 410 nm (characteristic of dityrosine), when excited at 320 nm.13

The IH-NMR spectrum (200 MHz at 25°C) of peak I1 (Fig. 3A) taken in D,O showed the following signals: 6 = 3.15 ppm (1H X 2, dd, J = 14.5 and 7.5 Hz, Hb), 3.33 ppm (1 H X 2, dd, J = 14.5 and 5.4 Hz, Hc), 4.04 ppm (1 H X 2, dd, J = 7.8 and 4.8 Hz, Ha), 6.99 pprn (1 H X 2, d, J = 8.1 Hz, Hf), 7.27 ppm (1 H X 2, dd, J = 8.1 and 2.7 Hz, He) and 7.43 pprn (1 H X 2, d, J = 2.0 Hz, Hd), indicating that this compound corresponds to dityr0~ine.I~

Research Note 1043

Figure 2. Transmission electron micrograph of NS0/2 cells treated with TyrRF photoproducts: (a) control cells; (b) cells treated with Tyd RF/O, during 24 h; (c,d) cells treated with Tyr/RF/N, during 24 h and 70 h, respectively (bars, 1.0 pm).

The effect of the Tyr and Rf irradiation photoproducts on the NS0/2 cells was also studied. Peaks I and I1 (Fig. 3A) were concentrated by lyophilization and added to the cells at the time of seeding. At the conditions used, no cytotoxic effect was observed.

DISCUSSION

When a Tyr plus RF solution is irradiated with polychromatic visible light under an anaerobic atmosphere, there are alterations in the absorption spectrum; the Tyr fluorescent emission decreases, and a product appears that emits at 410 nm, indicating that the amino acid is being transformed and the process does not require the presence of molecular oxygen. Riboflavin photoconversion also occurs.

Riboflavin plays a sensitizing role, as these phototrans- formations do not occur in its absence. The photochemical

reaction must be initiated by the interaction between the excited RF and Tyr. The sensitizer must be in the triplet state because the fluorescence of RF is not affected by the presence of the amino acid. This is consistent with the very efficient intersystem crossing of RF (+Isc = 0.7)15 and its long phosphorescence lifetime (0.14.2 s).I6 The same type of photoprocesses, but in a 40% less amount, occur when using monochromatic light (h = 450 nm). These results are the first evidence of Tyr transformation mediated by visible light and sensitized by RF in an anaerobic atmosphere. These anaerobic photoprocesses may be explained on account of the redox potential of the triplet flavin (1.7 V)? which is higher than the redox potential of Tyr (0.93 V).7 Thus, an electron would be transferred from Tyr to the triplet RF, with the consequent formation of a tyrosyl radical.

It is known that visible light may induce lethal effects on

1044 EDLIARDO SILVA et a f .

/

4- ”

mL Figure 3. Elution patterns in a Sephadex G-15 column (78 X 1.8 cm) of solutions of (A) Tyr plus RF irradiated under a nitrogen atmosphere in phosphate buffer, (B) an unirradiated mixture of Tyr and RF, (C) unirradiated RF and (D) RF irradiated under the same conditions as in (A).

biological systems. Toxic effects on cells in culture have been attributed to products of the RF-sensitized photooxidation of Tyr.’r3 In this sense, our results, shown in Figs. 1 and 2, demonstrate that Tyr photoproducts sensitized by RF in anaerobic atmosphere induce cytotoxic effects on mouse NS0/2 tumor cells. The dose-response study demonstrated that cytotoxicity was higher when the added photoproducts had been obtained anaerobically; although Tyr phototransformation was greater under aerobic conditions.” Our main interest to study the ultrastructure of cells treated with the photoproducts of Tyr and RF was to approach the identification of the subcellular targets of the photoproducts, in the future allowing the determination of its mechanism of action at the molecular level. Previous work carried out in our laboratory has demonstrated in vitro high toxicity of the photoproducts from Trp sensitized by RF.8J8 However, at the ultrastructural level, the toxic effect of the Tyr/RF photoproducts is less evident than the ultrastructural damage caused by the Trp/RF photoproducts on the same NS0/2 plasmacytoma cell line.8 The ultrastructural analysis of the cells in the Trp/RF case show evidence of an apoptotic process, similar to the one reported on lymphoma cells that underwent photodynamic therapy with chloroaluminum phthal~cyanine.~ In contrast, at the subcellular level the toxic effect of Tyr/RF is characterized by a swelling of mitochondria; a similar effect has been reported earlier by Gomer er u L . , ’ ~ on carcinoma and melanoma cells treated with photodynamic therapy. Therefore, it is possible that Trp and Tyr photoproducts hit different subcellular targets. Actually, the photoproducts responsible for the cytotoxicity described above have not been precisely identified. Efforts in this sense are made in our laboratory.

Irradiation with visible light of Tyr solutions sensitized by RF in an anaerobic atmosphere results in a mixture of photoproducts. Figure 3A shows two fractions corresponding to products o higher molecular weight than those of the amino acid and the vitamin. The one of highest molecular weight is formed by at least four components with different reten- tion times on an HPLC-C,, column. Two of these photoproducts would correspond to flavinic type aggregateszo,”

with similar characteristics to the photoproducts of the RF photoconversion shown in Fig. 3C. The precise chemical nature of the products of tyrosinic origin found in fraction I of Fig. 3A has not yet been established, but they must be originated from the tyrosyl radical mentioned above.

Fraction I1 of Fig. 3A corresponds to dityrosine, whose structure is demonstrated by its absorption, emission and ‘H- NMR spectra. The formation of dityrosine from the tyrosyl radical has been already reported in studies where the radical form of the amino acid was generated by means of UV pho- t o l y s i ~ ~ ~ , ~ ~ or through the peroxidase/H,02 s y ~ t e m . ’ ~ , ~ ~ - ~ ~

It is noteworthy that the presence of dityrosine has also been observed in proteins of human aged lenses and also in the insoluble protein fraction from human cataractous lenses.26 This formation of dityrosine in vivo may be produced by RF-sensitized modification of Tyr, because the vitamin is normally present in ocular lenses that have a low oxygen c~ncentration.~’

Acknowledgements-The authors thank Dr. Eduardo Lissi (Univer- sidad de Santiago, Chile) for critical reading of the manuscript. This work was supported by Fondo Nacional de Investigaci6n Cientifica y Tecnoldgica (FONDECYT grant 193057 1).

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Research Note 1045

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VISIBLE LIGHT ANAEROBIC PHOTOCONVERSION OF TYROSINE SENSITIZED BY RIBOFLAVIN. CYTOTOXICITY ON MOUSE TUMORAL CELLS