Cytogenetic effects of singlet oxygen

J. Photochem. Photobiol. B: Biol., I6 (1992) 381-384

Preliminary

381

Note

Cytogenetic effects of singlet oxygen Walter

C. Eisenberg

and Kevin Taylor+

Department of Chemistry IlIinois Institute of Technology, Chicago, IL 60616 (USA) Robert

R. Guerrero+

Life Science Division, IIT Research Institute, Chicago, IL 60616 (USA) (Received January 8, 1992; accepted July 8, 1992)

Abstract Singlet oxygen was generated in the gas phase at atmospheric pressure by the method of heterogeneous photosensitization. In vitro exposure of human lung WI-38 fibroblasts to gas-phase singlet oxygen resulted in sister chromatid exchange.

Keywords: Hydrogen peroxide, hydroxyl radical, singlet oxygen, superoxide anion. 1. Introduction Reactive oxygen species: superoxide anion; hydrogen peroxide; hydroxyl radical; and singlet oxygen, have all been implicated as agents causing injury to cells and

tissues, ultimately giving rise to adverse health effects. Interest in the biological effects of singlet oxygen was stimulated by the development of laboratory sources of “chemically pure” singlet oxygen [l, 21 and, more recently, by the knowledge that singlet oxygen is the cytocidal agent in the promising new cancer treatment, photoradiation therapy involving porphyrins [3]. To our knowledge, the cytotoxicity of “pure” gas-phase singlet oxygen was first demonstrated in this laboratory [4, 51. Tracheal explants exposed to gas-phase singlet oxygen showed a dose-dependent decrease in cilia beating frequency, and focal ciliostasis. A statistically significant decrease in ciliary activity occurred at singlet oxygen concentrations as low as 154 ppb after a two h exposure. We have also observed evidence for the mutagenicity of singlet oxygen [6]. A positive audiographic response for unscheduled DNA synthesis was obtained when tracheal explants were treated for two hours with gas phase singlet oxygen and H3 thymidine. Hartman et al. [7] reported the killing of various strains of Salmonella typhitnurium and Escherichia coli exposed to “pure” gas-phase singlet oxygen generated using the separated surface sensitizer (SSS) method, but did not observe any evidence of the mutagenicity of exogenous singlet oxygen. Sister chromatid exchange was detected after treatment ofAllium cepa meristematic cells with red light and hematoporphyrin or meso-tetra (Cpyridyl)porphine [8]. Moan +Present addresses: K. Taylor, IIT Research Institute, Chicago, IL., R. Guerrero, AMDL Inc., Pasadena, CA.

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382

[9] observed sister chromatid exchange and DNA single strand breaks in human NHIK 3025 cells following treatment with hematoporphyrin and light. Piette et al. [lo] reported preliminary results that lymphocytes in aqueous suspension exposed to gas-phase singlet oxygen, generated using a modified SSS system, showed an increase in the frequency of sister chromatid exchange. We now report the observation of sister chromatid exchange when human lung WI-38 fibroblasts were exposed to “pure” singlet oxygen. The cells were grown on an inert material and exposed to gas-phase singlet oxygen that was externally generated using the flow generation system.

2. Experimental

procedures

2.1. Ceils Human lung WI-38 cells in the 27th passage (2 ml) were seeded in triplicate of 1X105 cells culture into 100 mm sterilized PyrexTM petri dishes at a concentration ml-‘. The cells were grown in Eagle’s MEM culture medium containing 10% heat inactivated fetal bovine serum (FBS), 100 U ml-’ penicillin, 100 ug ml-’ streptomycin, 25 mM Hepes buffer and 24 mM sodium bicarbonate (complete medium). The cells were incubated at 37 “C in a humidified 5% CO2 incubator for 24 h. Prior to the experiment, the medium was drawn off and replaced with 10 ml of Eagle’s MEM medium containing 100 U ml-’ penicillin, 100 ug ml-’ streptomycin, 10 ug ml-’ fungizone, 25 mM Hepes buffer and 24 mM sodium bicarbonate. 2.2. Singlet oxygen generation and exposure

Singlet oxygen was generated in the gas phase at atmospheric pressure by the method of heterogeneous photosensitization. A dry film of rose bengal was exposed to strong visible radiation while in contact with a 98% N,-2% O2 gas mixture flowing of the gas-phase ‘02 was measured by monitoring at 8.2 L min- ‘. The concentration the 1.27 pm emission using a liquid nitrogen-cooled germanium photodiode. The detector was calibrated in chemical trapping experiments with tetramethylethylene [ll]. The generator gas containing 0.528 ppm ‘02 was passed directly into the top of a cube-shaped exposure chamber (1 X 1 X 1 ft3) for one hour. Three uncovered culture dishes, each containing WI-38 cells with 10 mL of medium were supported on a rack in the middle of the chamber. The chamber was placed on a rocker platform (Bellco Glass, Inc.) that rocked at 10 cycles min-‘, allowing the explants to contact both ‘02 and the medium. Medium was added to the dishes after each hour of exposure to compensate for evaporation loss. Additional details of the generation system and the parameters affecting ‘02 gas phase generation are provided in a recent review 1121. 2.3. Sister-chromatid

exchange assay Following the experiment, the WI-38 cells were incubated in 20.0 ml of complete medium supplemented with 10 ug ml-’ fungizone and 10 ug ml-’ bromodeoxyuridine for 48 h (two cell cycles) at 37 “C in a humidified 5% CO1 incubator. During the last hours of incubation, the cells were arrested at metaphase with 0.05 ug ml-’ colcemid. The cell suspensions were then centrifuged at 250 g for 7 min, and the cell pellet was resuspended in hypotonic KC1 solution. The cells were pelleted out of the hypotonic solution and fixed with Carnoy’s solution which comprised of absolute methanol/glacial acetic acid (3/l, v/v). Slides were then prepared and stained using the fluorescence plus Geisma technique [13]. Thirty well-spread second cycle metaphases

383

were scored for SCE per culture. Means and standard deviations were calculated and the data were analyzed for significant difference from the incubator negative control with a two tailed t test. A p- ~0.05 was considered significant.

3. Results

and discussion

An increase in SCE was observed in human lung WI-38 cells following exposure to gas-phase ‘OZ. These results are presented in Table 1. The incubator control received no ‘02 treatment. The positive control was exposed to 1.2 ug ml-’ Mitomycin C. In the ‘02 experiment, three culture plates were simultaneously exposed to 0.582 ppm ‘02 for 1 h. The generator control was an identical experiment without ‘OZ. The SCE response for the incubator control was 7.53 mean SCE/dose which is normal for the laboratory and significantly below the positive control (38.10 SCE/dose). The SCE response for the generator control was virtually the same as for the incubator control, e.g., 7.58kO.16 SCE/dose. Thus, the generator gas did not contain any mutagenic contaminants. The SCE response for the singlet oxygen-exposed cells was 10.97 SCE/ dose which was a significant increase (Pi;O.OOl). It is worth noting that while the cells tolerated the one h exposure, both the generator control and singlet oxygen exposed cells showed some evidence of biological stress, e.g. decreased mitotic activity and some cell detachment was common to both. However, the singlet oxygen exposed cells showed more uneven cell margins, some blebbing and an increase in cytoplasmic granulation which presumably reflects an increase in vacuolization. All of these observations are suggestive of cell membrane damage. At the chromosome level, in addition to the increase in SCE, there was a subjective increase in chromatid type aberrations and a definite increase in endoreduplication which suggests spindle fiber effects. This last observation is not surprising since the spindle fiber apparatus is known to reside in the cell membrane.

TABLE

1

SCE results on WI-38 Test article Incubator

cells exposed Total Met. scored

control

Mitomycin c” Generator control

Singlet oxygenb

to singlet oxygen in vitro Total SCE

x SCEkulture

x SCEldose

*SD

*SD

30

228

30 30 30

1143 231 229

7.53 f 1.96 38.1Ok11.37 7.70f 2.23 7.63 f 2.46

7.53 f 1.96 38.10* 11.37

30 30 30 30

222 336 350 301

7.40 f 2.53 11.2OIt3.34’ 11.67+2.73’ 10.03 f 2Hd

7.58 f 0.16

“Positive control 1.2 ug ml-’ bl h exposure to 0.528 ppm gas phase ‘Oa ‘Significantly different from incubator control dSignificantly different from incubator control

at p g 0.0001 at p 6 0.0009

significance level significance level

10.97 f 0.84

384 4. Conclusions The one h exposure to 0.582 ppm singlet oxygen in the gas phase caused detectable cell membrane toxic effects and an increase in cytogenetic damage. Singlet oxygen is classified as a weak SCE inducer under the conditions tested. However, when coupled with the other cytogenetic damage observed, the conclusion is that exposure to 0.582

ppm for one h poses a significant genotoxic risk. We are currently examining the nature and extent of the chromosomal abberations resulting from the singlet oxygen exposure. It is not clear whether singlet oxygen or a secondary toxic species is the genotoxic agent in these experiments. Although there is evidence that singlet oxygen can pass though the cell membrane [14], it may not survive the aqueous environment of the cell. Clearly, lipid peroxides form as ‘02 passes through the cell membrane. These compounds are known to decompose to yield free radical species which could lead to the observed genotoxic effects. Experiments are underway to clarify this point.

References 1 W. C. Eisenberg, A. Snelson, R. Butler, J. Veltman and R. W. Murray, Gas phase generation of singlet oxygen at atmospheric pressure, Tetrahedron. Let& 22 (1981) 377-380. 2 W. R. Midden and S. Y. Wang, Singlet oxygen generation for solution kinetics: clean and simple, .I. Am. Chem. Sot., 91 (1983) 4129-4135. 3 T. J. Dougherty, Photosensitizers: therapy and detection of malignant tumors, Photochem. PhotobioL, 45 (1987) 879-889. 4 W. C. Eisenberg, K. Taylor and L. J. Schiff, Biological effects of singlet delta oxygen on respiratory tract epithelium, @erimerrria, 40 (1986) 514-515. 5 L. J. Schiff, W. C. Eisenberg, J. Dziuba, J. K. Taylor and S. J. Moore, Cytotoxic effects of singlet oxygen, Envirort. Health Perspect., 76 (1987) 199-203. 6 L. J. Schiff, W. C. Eisenberg and K. Taylor, Evidence for DNA repair in organ cultures of hamster tracheal epithelium following exposure to gas phase singlet oxygen, Mutation Res., 142 (1985) 41-44. 7 P. E. Hartman, T. A. Dahl and W. R. Midden, Pure singlet oxygen toxicity for bacteria, Photochem. Photobiol., 46 (1987) 345-352. 8 M. J. Hazen, A. Villanueva and J. C. Stockert, Induction of sister chromatid exchange in Allium cepu meristematic cells exposed to meso-tetra (4-pyridyl)porphine and hematoporphyrin photoradiation, Photochem. Photobiol., 46 (1987) 463-467. 9 J. Moan, H. Waksvik and T. Christensen, DNA single-strand breaks and sister chromatid exchanges induced by treatment with hematoporphyrin and light or X-rays in human NHIK 3025 cells, Cancer Res., 40 (1987) 2915-2918. 10 J. Piette, D. Decuyper-Debergh, C. Laurent and A. van der Vorst, Cytotoxic and genotoxic effects of extracellular generated singlet oxygen in human lymphocytes in vitro, Mutation Res., 225 (1989) 11-14. 11 W. C. Eisenberg, K Taylor and R. W. Murray, Gas-phase kinetics of the reaction of singlet oxygen with ole8ns at atmospheric pressure, J. Phys. Chem., 90 (1986) 1945-1948. 12 W. C. Eisenberg, Atmospheric gas phase generation of singlet delta oxygen, in A. Baumstark (ed.), Advances in oxygenated processes, Vol. 3, JAI Press, London, 1991, pp. 71-113. 13 P. Perry and S. Wolff, New Giesma method for the differential staining of sister chromatids, Nature, 2.51 (1974) 156-158. 14 J. Moan, On the diffusion length of singlet oxygen in cells and tissues, J. Photo&m. Photobiol. B: Biol., 6 (1990) 343-344. 15 A. C. Nye, G. M. Rosen, E. W. Gabrielson, J. F. Keana and V. S. Prabhu, Diffusion of singlet oxygen into human bronchial epithelial cells, B&him. Biophys. Actu, 928 (1987) 1-7.