Biochimtca et Bwphyswa Acta, 1088 (1991) 409-412 <'~ 1991 Elsevier ~iencc I'ubli:,hcrs BV. 0167-4781/91/$03.50 ADONIS ()167478191001081~ 409 BBAEXP 92229 Singlet oxygen induced single-strand breaks in plasmid pBR322 DNA: the enhancing effect of thiols Thomas P.A. Devasagayam, Paolo Di Mascio, Stephan Kaiser and Helmut Sies Institut fiir Physiologische Chemie I, Universitlit Diisseldorf, DiisseMorf (F. R. G.) (Received 8 October 1990) Key words: Singlet oxygen; Single-strand break: DNA damage: Thiol The biologically occurring thiolso glutathione, cysteaminc and cysteinc, significantly enhance the single-strand breaks in plasmid pBR322 DNA induced by singlet molecular oxygen (IO 2) generated by the thermodissociation of the endoperoxide of 3,3'-(I,4-naphthylidene)dipropionate. The enhancing effect was also observed with chemically related sulfh)dryl compounds but not by disulfides. In contrast, dihydrolipoate and its disulfide lipoate protected the plasmid DNA. Metal chelators as well as superoxide dismutase or catalase had no effect, whereas mannitol or sodium azide, decreased the thiol-~Oz-induced strand breaks. It is concluded that the observed effects are mediated by reactive oxidation products arising from the IO2-oxidation of thiols. Introduction Singlet molecular oxygen (~O2) is produced in mam- malian cells unde~ normal and pathophysiologicai con- ditions [1], The photodynamic action of some drugs is t mediated through 0 2 [2]. Singlet oxygen is capable of damaging biomolecules including DNA, mainly its guanine moiety [3], and the generation of 8-hydroxy- guanine with methylene blue and light has been attri- buted to IO 2 [4]. DNA damage, measured as single- strand breaks, leads to the loss of biological activity and to mutagenicity [5-9]. Under in vivo conditions the DNA damaging effect of tO 2 is likely to be modulated by other biomolecules. Thiols and related solfur-containing compounds are known to fulfill fundamental biological functions [10]. Though exhibiting significant antioxidant properties, they can give rise to mutagenic activity [11,121. Our recent studies as well as those of others have shown that biological thiois are highly reactive towards 10 2 result- ing in several oxidation products [13,141. Though thiols can reduce 1,2-dioxetanes [15] and protect DNA from radicals produced during radiation [16], they generate thiyl radicals in the presence of photosensitizer and Abbreviation: NDPO 2, endoperoxide of 3,3'-(l,4-naphthylidene)di- propionate. Correspondence: H. Sies, Institut far Physiologische Chemic l, Om- versit~it Diisseldod, Moorenstrasse 5, D-4000 Diisseldorf, F.R.G. light, involving singlet oxygen production [171. Besides, their reaction with molecular oxygen leads to several highly reactive radical species [181. It is likely that even more reactive species, capable of damaging DNA, are produced by the reaction of to 2 with thiols. Due to the high concentration of thiols in the biological tissues their oxidation by singlet oxygen in the vicinity of DNA can have serious consequences. The present study ex- amines such an eventuality, using plasmid DNA as a model. Materials and Methods Singlet oxygen was generated by the thermodissocia- tion of the endoperoxide of 3,3'-(1,4-naphthylidene)di- propionate (NDPO2), as described previously [19]. Plasmid pBR322 DNA was prepared utilising the Escherichia coil culture and purified using a Qiagen Plasmid kit-Pack 500 (Diagen, Diisseldorf, F.R.G.). The preparation contained 92 to 100% Form 1 (supercoiled form) and 0 to 8% Form 11 (open circular form). 2-3 pg pBR322 DNA (20 pl) was exposed to 40 mM NDPO 2 and/or 10 mM freshly dissolved thioi in a deaerated 50 mM sodium phosphate buffer, pD 7.4, in DzO. To study the effect of inhibitors, sodium azide (10 mM) or superoxide dismutase (60 units/assay) or cata- lase (20 units/assay) or mannitoi (100 mM) were pre- sent. Heat-denatured catalase was obtained by treat- ment at 90 °C for 30 rain. Ethylene diamine tetraacetic acid (EDTA) was added at a concentration of 10 mM. ,-rl. l,e incubation wa:. at 37°C for !t"d) ham wilh ~haki~3.