EPR Study of KO2 as a Source of Superoxide and •BMPO-OH/OOH Radical That Cleaves Plasmid DNA and Detects Radical Interaction with H2S and Se-Derivatives

an ioxidan s A icle EPR S udy o KO2as a Sou ce o Supe oxide and •BMPO-OH/OOH Radical Tha Clea es Plasmid DNA and De ec s Radical In e ac ion wi h H2S and Se-De i a i es An on Misak 1, Vlas a B ezo a 2, Mi osla Cho anec 3, Ka ol Luspai 2, Muhammad Jawad Nasim 4, Ma ian G man 1, Lenka Tomaso a 1, Claus Jacob 4and Ka ol Ond ias 1,*   Ci a ion: Misak, A.; B ezo a, V.; Cho anec, M.; Luspai, K.; Nasim, M.J.; G man, M.; Tomaso a, L.; Jacob, C.; Ond ias, K. EPR S udy o KO2as a Sou ce o Supe oxide and •BMPO-OH/OOH Radical Tha Clea es Plasmid DNA and De ec s Radical In e ac ion wi h H2S and Se-De i a i es. An ioxidan s 2021,10, 1286. h ps://doi.o g/10.3390/ an iox10081286 Academic Edi o s: Kenne h R. Olson and Bulen Mu us Recei ed: 25 June 2021 Accep ed: 11 Augus 2021 Published: 13 Augus 2021 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2021 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). 1Biomedical Resea ch Cen e , Depa men o Molecula Physiology, Ins i u e o Clinical and T ansla ional Resea ch, Slo ak Academy o Sciences, Dúb a skáCes a 9, 84505 B a isla a, Slo akia; [email p o ec ed] (A.M.); [email p o ec ed] (M.G.); [email p o ec ed] (L.T.) 2Ins i u e o Physical Chemis y and Chemical Physics, Facul y o Chemical and Food Technology, Slo ak Uni e si y o Technology in B a isla a, Radlinského 9, 81237 B a isla a, Slo akia; [email p o ec ed] (V.B.); ka [email p o ec ed] (K.L.) 3 Biomedical Resea ch Cen e , Depa men o Gene ics, Cance Resea ch Ins i u e, Slo ak Academy o Sciences, Dúb a skáCes a 9, 84505 B a isla a, Slo akia; mi osla [email p o ec ed] 4Di ision o Bioo ganic Chemis y, School o Pha macy, Uni e si y o Saa land, D-66123 Saa b uecken, Ge many; [email p o ec ed] (M.J.N.); [email p o ec ed] (C.J.) *Co espondence: ka [email p o ec ed] Abs ac : Supe oxide adical anion (O 2•− ) and i s de i a i es egula e nume ous physiological and pa hological p ocesses, which a e ex ensi ely s udied. The aim o ou wo k was o u ilize KO 2 as a sou ce o O 2•− and he elec on pa amagne ic esonance (EPR) spin apping 5- e -bu oxyca bonyl-5- me hyl-1-py oline N-oxide (BMPO) echnique o he p epa a ion o • BMPO-OOH and/o • BMPO- OH adicals in wa e solu ion wi hou DMSO. The me hod dis inguishes he in e ac ions o a ious compounds wi h • BMPO-OOH and/o • BMPO-OH adicals o e ime. He e, we show ha he addi ion o a bu e ed BMPO-HCl mix u e o powde ed KO 2 o med ela i ely s able • BMPO-OOH and • BMPO-OH adicals and H 2 O 2 , whe e he • BMPO-OOH/OH a io depended on he pH. A a inal pH o ~6.5–8.0, he concen a ion o • BMPO-OOH adicals was ≥ 20 imes highe han ha o • BMPO-OH, whe eas a pH 9.0–10.0, he • BMPO-OH adicals p e ailed. The • BMPO-OOH/OH adicals e ec i ely clea ed he plasmid DNA. H 2 S dec eased he concen a ion o • BMPO-OOH/OH adicals, whe eas he selenium de i a i es 1-me hyl-4-(3-(phenylselanyl) p opyl) pipe azine and 1-me hyl-4-(4-(phenylselanyl) bu yl) pipe azine inc eased he p opo ion o • BMPO-OH o e he • BMPO-OOH adicals. In conclusion, he p esen ed app oach o using KO 2 as a sou ce o O 2•− /H 2 O 2 and EPR spin ap BMPO o he p epa a ion o • BMPO-OOH/OH adicals in a physiological solu ion could be use ul o s udy he biological e ec s o adicals and hei in e ac ions wi h compounds. Keywo ds: KO 2 ; an ioxidan s; EPR spec a simula ion; • BMPO-OOH spin adduc ; supe oxide; adical; hyd ogen sul ide; selenium-de i a i es; clea age DNA 1. In oduc ion Supe oxide anion adical (O 2•− ) is essen ial o he li e o ae obic o ganisms, egula ing nume ous physiological and pa hophysiological p ocesses. When an o e p oduc ion o O 2•− occu s and/o he an ioxidan de ense is de icien , oxida i e s ess may de elop, leading o oxida i e damage in many biological sys ems [ 1 , 2 ]. The e o e, he p oduc ion, biological in e ac ions, and con ol o he O 2•− concen a ion ha e all been in ensi ely s udied bo h in i o and in i o. Fo expe imen s in ol ing adical O 2•− eac ions, O 2•− can be gene a ed using a i- ous me hods [ 3 ], e.g., enzyma ically by xan hine/xan hine oxidase [ 4 , 5 ], which is a sys em oo complex o some ypes o O 2•− adical models. I can also be gene a ed by ionizing An ioxidan s 2021,10, 1286. h ps://doi.o g/10.3390/an iox10081286 h ps://www.mdpi.com/jou nal/an ioxidan s An ioxidan s 2021,10, 1286 2 o 22 adia ion o by dissol ing supe oxide sal s in ap o ic sol en s, e.g., KO 2 in anhyd ous DMSO; howe e , DMSO may no be desi able in some biological expe imen s [ 3 , 6 ]. On he o he hand, KO 2 dissol ed in an aqueous solu ion is a simple sou ce o O 2•− , bu i s li e ime is in he ange o milliseconds [ 7 ]. Fo hese easons, i would be p ac ical and app op ia e o ha e an easily accessible O 2•− sou ce ha could be used in physiological solu ions. The e o e, he aim o ou wo k was o examine KO 2 as a sui able sou ce o O 2•− , which can be used o eac wi h cyclic ni one 5- e -bu oxyca bonyl-5-me hyl-1-py oline- N-oxide (BMPO), gene a ing supe oxide ( • BMPO-OOH) o hyd oxyl ( • BMPO-OH) adical adduc s o u he s udies in aqueous sys ems. The elec on pa amagne ic esonance (EPR) spin apping echnique, using BMPO, is use ul o s udy sho -li ed O 2•− and hyd oxyl (HO • ) adicals [ 4 , 5 , 8 – 15 ]. By apping O 2•− , BMPO p oduces ela i ely s able • BMPO-OOH spin adduc s, which can be u he s udied as a po en ial model o o ganic hyd ope oxides. A sa u a ed KO 2 solu ion in DMSO has been used as a sou ce o O 2•− o o m • BMPO-OOH. The p esence o DMSO is con enien in s udies wi h wa e -insoluble compounds, bu i could be a limi a ion o biological s udies. Exogenously added and endogenously p oduced hyd ogen sul ide (H 2 S) in luences many physiological and pa hologic p ocesses, including oxida i e s ess, by eac ing wi h eac i e oxygen species [ 16 , 17 ]. O ganoselenium compounds ha e been known o se e as mul i unc ional agen s. Such compounds ha e been shown o p o ide an excellen an i-cance ac i i y by se ing as p o-oxidan s ha ul ima ely al e he edox homeos a- sis o cance cells, leading o apop osis. Many biological ac i i ies ha e been associa ed wi h o ganoselenium compounds, anging om simple an ioxidan s and an imic obials, o a he complica ed immuno-modula o y, an i-in lamma o y, and an i-nocicep i e e - ec s [ 18 ]. As o ganoselenium compounds ha e also been known o hei in e ac ion wi h adicals, he compounds RSe-1 and RSe-2 we e employed in his s udy. The e o e, in he p esen wo k, a new p ocedu e o he p epa a ion o ela i e s able • BMPO-OOH/OH adduc s om KO 2 as a sou ce o O 2•− wi hou he use o DMSO as a sol en is desc ibed. The • BMPO-OOH adical was mos ly obse ed a pH ~6.5–8.0. • BMPO-OOH/OH clea ed plasmid DNA and • BMPO-OOH/ • BMPO-OH o al adical concen a ions and hei p opo ion we e in luenced by H 2 S- and selenium-con aining o ganic compounds. 2. Ma e ials and Me hods 2.1. Chemicals, EPR Sample P epa a ion, and Measu emen Na 2 S was pu chased om DoJindo (SB01, Munich, Ge many), and he o he chemicals we e pu chased om Sigma-Ald ich (S einheim, Ge many). Na 2 S dissocia es in he solu ion and eac s wi h H + o yield H 2 S, HS − , and aces o S 2− . We used H 2 S o e e he o al mix u e o H 2 S, HS − , and S 2− . A s ock solu ion o 100 mmol L −1 Na 2 S was p epa ed in a gon-bubbled ul apu e deionized H 2 O, and was s o ed a − 80 ◦ C o a ew days and used immedia ely a e hawing. Two selenium de i a i es, 1-me hyl-4-(3-(phenylselanyl)p opyl)pipe azine (RSe-1) and 1-me hyl-4-(4-(phenylselanyl)bu yl)pipe azine (RSe-2), we e ob ained acco ding o he syn hesis ou es shown in Scheme 1; Scheme 2. Fo he compound RSe-1, me hyl pipe azine was alkyla ed, employing 1-b omo-3-chlo op opane in he p esence o anhyd ous K2CO3 and ace one as a sol en . The eac ion was pe o med a oom empe a u e (RT) and he p og ess o he eac ion was con olled by hin laye ch oma og aphy. The c ude p oduc was used o Se-alkyla ion in absolu e e hanol unde a ni ogen a mosphe e a oom empe a u e (Scheme 1). Fo he compound RSe-2, Se-alkyla ion o 1-b omo-4-chlo obu ane was pe o med unde a ni ogen a mosphe e and in absolu e e hanol a RT. The c ude p oduc was hen eac ed wi h me hyl pipe azine in he p esence o anhyd ous K 2 CO 3 and ace one a 50 ◦ C (Scheme 2) [ 19 ]. The s uc u e and pu i y o he inal compounds we e con i med ia spec oscopic and ch oma og aphic me hods. The inal p oduc s we e con e ed in o c ys alline hyd ochlo ic sal s o imp o e hei solubili y in wa e . An ioxidan s 2021,10, 1286 3 o 22 An ioxidan s 2021, 10, x FOR PEER REVIEW 3 o 23 K2CO3 and ace one a 50 °C (Scheme 2) [19]. The s uc u e and pu i y o he inal com- pounds we e con i med ia spec oscopic and ch oma og aphic me hods. The inal p od- uc s we e con e ed in o c ys alline hyd ochlo ic sal s o imp o e hei solubili y in wa e . Scheme 1. Syn hesis o compound RSe-1. (a)The i s s ep o syn hesis in ol es he alkyla ion o 1-me hylpipe azine wi h 1-b omo-3-chl op opane o p oduce 1-(3-chlo op opyl)-4-me hylpipe azine in he p esence o K2CO3 and ace one a oom empe a u e. (b) 1-(3-chlo op opyl)-4-me hylpipe azine is subsequen ly eac ed wi h diphenyl diselenide in an ine en- i onmen o N2, in he p esence o NaBH4 and absolu e e hanol a oom empe a u e o ob ain RSe-1 as inal p oduc . Scheme 2. Syn hesis o compound RSe-2. (a)The i s s ep o syn hesis in ol es he Se-alkyla ion o 1-b omo-4-chlo obu- ane wi h diphenyl diselenide in an ine en i onmen o N2, in he p esence o NaBH4 and absolu e e hanol a oom empe a u e o ob ain (4-chlo obu yl)(phenyl)selane. (b) In he second s ep (4-chlo obu yl)(phenyl)selane is eac ed wi h 1-me hylpipe azine in he p esence o K2CO3 and ace one a oom empe a u e o ob ain RSe-2 as inal p oduc . The spin- apping agen , 5- e -bu oxyca bonyl-5-me hyl-1-py oline N-oxide (BMPO, DoJindo B568, Munich, Ge many), was dissol ed in ul apu e deionized H2O (100 mmol L−1), s o ed a −80 °C, and used a e hawing. Fo he EPR samples, a buffe con- sis ing o 50 mmol L−1 sodium phospha e and 100 µmol L−1 die hylene iaminepen aace ic acid (DTPA; pH 7.4; 37 °C) was used. Powde ed KO2 (Sigma-Ald ich 278904, S einheim, Ge many) in amoun s o 0.5–1 mg was weighed in o ubes jus be o e being used. As he dissolu ion o KO2 in a phospha e bu e shi s om pH o alkaline alues, an adequa e olume o 1 mol L−1 HCl was added o he bu e con aining BMPO (20 mmol L−1) o ge he equi ed inal pH a e he addi ion o KO2. Twel e seconds a e adding he BMPO- con aining phospha e solu ion o he powde ed KO2, compounds Na2S in H2O and RSe-1 o RSe-2, p epa ed in he bu e , we e added. The pH in he samples was measu ed by pH indica o pape (Ahls om-Munksjö, Ge many) wi h an accu acy o ±0.2. The de ailed p o- cedu e o he sample p epa a ion o EPR measu emen is desc ibed in he Supplemen- a y Ma e ials. The sample was ans e ed o a s anda d ca i y aqueous EPR la cell (WG 808-Q, Wilmad-LabGlass, Vineland, NJ, USA), and he EPR spec a we e measu ed as de- sc ibed p e iously [6]. The i s EPR spec um was eco ded 100 ± 15 s a e he addi ion o he BMPO solu ion o powde ed KO2. The se s o indi idual EPR spec a o he BMPO Scheme 1. Syn hesis o compound RSe-1. ( a )The i s s ep o syn hesis in ol es he alkyla ion o 1-me hylpipe azine wi h 1-b omo-3-chl op opane o p oduce 1-(3-chlo op opyl)-4-me hylpipe azine in he p esence o K 2 CO 3 and ace one a oom empe a u e. ( b ) 1-(3-chlo op opyl)-4-me hylpipe azine is subsequen ly eac ed wi h diphenyl diselenide in an ine en i onmen o N2, in he p esence o NaBH4and absolu e e hanol a oom empe a u e o ob ain RSe-1 as inal p oduc . An ioxidan s 2021, 10, x FOR PEER REVIEW 3 o 23 K2CO3 and ace one a 50 °C (Scheme 2) [19]. The s uc u e and pu i y o he inal com- pounds we e con i med ia spec oscopic and ch oma og aphic me hods. The inal p od- uc s we e con e ed in o c ys alline hyd ochlo ic sal s o imp o e hei solubili y in wa e . Scheme 1. Syn hesis o compound RSe-1. (a)The i s s ep o syn hesis in ol es he alkyla ion o 1-me hylpipe azine wi h 1-b omo-3-chl op opane o p oduce 1-(3-chlo op opyl)-4-me hylpipe azine in he p esence o K2CO3 and ace one a oom empe a u e. (b) 1-(3-chlo op opyl)-4-me hylpipe azine is subsequen ly eac ed wi h diphenyl diselenide in an ine en- i onmen o N2, in he p esence o NaBH4 and absolu e e hanol a oom empe a u e o ob ain RSe-1 as inal p oduc . Scheme 2. Syn hesis o compound RSe-2. (a)The i s s ep o syn hesis in ol es he Se-alkyla ion o 1-b omo-4-chlo obu- ane wi h diphenyl diselenide in an ine en i onmen o N2, in he p esence o NaBH4 and absolu e e hanol a oom empe a u e o ob ain (4-chlo obu yl)(phenyl)selane. (b) In he second s ep (4-chlo obu yl)(phenyl)selane is eac ed wi h 1-me hylpipe azine in he p esence o K2CO3 and ace one a oom empe a u e o ob ain RSe-2 as inal p oduc . The spin- apping agen , 5- e -bu oxyca bonyl-5-me hyl-1-py oline N-oxide (BMPO, DoJindo B568, Munich, Ge many), was dissol ed in ul apu e deionized H2O (100 mmol L−1), s o ed a −80 °C, and used a e hawing. Fo he EPR samples, a buffe con- sis ing o 50 mmol L−1 sodium phospha e and 100 µmol L−1 die hylene iaminepen aace ic acid (DTPA; pH 7.4; 37 °C) was used. Powde ed KO2 (Sigma-Ald ich 278904, S einheim, Ge many) in amoun s o 0.5–1 mg was weighed in o ubes jus be o e being used. As he dissolu ion o KO2 in a phospha e bu e shi s om pH o alkaline alues, an adequa e olume o 1 mol L−1 HCl was added o he bu e con aining BMPO (20 mmol L−1) o ge he equi ed inal pH a e he addi ion o KO2. Twel e seconds a e adding he BMPO- con aining phospha e solu ion o he powde ed KO2, compounds Na2S in H2O and RSe-1 o RSe-2, p epa ed in he bu e , we e added. The pH in he samples was measu ed by pH indica o pape (Ahls om-Munksjö, Ge many) wi h an accu acy o ±0.2. The de ailed p o- cedu e o he sample p epa a ion o EPR measu emen is desc ibed in he Supplemen- a y Ma e ials. The sample was ans e ed o a s anda d ca i y aqueous EPR la cell (WG 808-Q, Wilmad-LabGlass, Vineland, NJ, USA), and he EPR spec a we e measu ed as de- sc ibed p e iously [6]. The i s EPR spec um was eco ded 100 ± 15 s a e he addi ion o he BMPO solu ion o powde ed KO2. The se s o indi idual EPR spec a o he BMPO Scheme 2. Syn hesis o compound RSe-2. ( a )The i s s ep o syn hesis in ol es he Se-alkyla ion o 1-b omo-4-chlo obu ane wi h diphenyl diselenide in an ine en i onmen o N 2 , in he p esence o NaBH 4 and absolu e e hanol a oom em- pe a u e o ob ain (4-chlo obu yl)(phenyl)selane. ( b ) In he second s ep (4-chlo obu yl)(phenyl)selane is eac ed wi h 1-me hylpipe azine in he p esence o K2CO3and ace one a oom empe a u e o ob ain RSe-2 as inal p oduc . The spin- apping agen , 5- e -bu oxyca bonyl-5-me hyl-1-py oline N-oxide (BMPO, DoJindo B568, Munich, Ge many), was dissol ed in ul apu e deionized H 2 O ( 100 mmol L−1 ), s o ed a − 80 ◦ C, and used a e hawing. Fo he EPR samples, a bu e consis ing o 50 mmol L −1 sodium phospha e and 100 µ mol L −1 die hylene iaminepen aace ic acid (DTPA; pH 7.4; 37 ◦ C) was used. Powde ed KO 2 (Sigma-Ald ich 278904, S einheim, Ge many) in amoun s o 0.5–1 mg was weighed in o ubes jus be o e being used. As he dissolu ion o KO 2 in a phospha e bu e shi s om pH o alkaline alues, an adequa e olume o 1 mol L −1 HCl was added o he bu e con aining BMPO (20 mmol L −1 ) o ge he equi ed inal pH a e he addi ion o KO 2 . Twel e seconds a e adding he BMPO-con aining phospha e solu ion o he powde ed KO 2 , compounds Na 2 S in H 2 O and RSe-1 o RSe-2, p epa ed in he bu e , we e added. The pH in he samples was measu ed by pH indica o pape (Ahls om-Munksjö, Ge many) wi h an accu acy o ± 0.2. The de ailed p ocedu e o he sample p epa a ion o EPR measu emen is desc ibed in he Supplemen a y Ma e ials. The sample was ans e ed o a s anda d ca i y aqueous EPR la cell (WG 808-Q, Wilmad-LabGlass, Vineland, NJ, USA), and he EPR spec a we e measu ed as desc ibed p e iously [ 6 ]. The i s EPR spec um was eco ded 100 ± 15 s a e he addi ion o he BMPO solu ion o powde ed KO 2 . The se s o indi idual EPR spec a o he BMPO spin adduc s we e eco ded as 15 o 30 sequen ial scans o 42 s each, wi h a o al acquisi ion ime o 11 o 22 min, espec i ely. The An ioxidan s 2021,10, 1286 4 o 22 EPR spec a o he BMPO spin adduc s we e measu ed on a B uke EMX spec ome e (Rheins e en, Ge many) essen ially as in ou p e ious s udy [ 6 ]. All o he EPR spec a we e eco ded a 37 ◦ C. The spec a we e simula ed using he EasySpin p og am wo king on he Ma Lab pla o m [ 20 ]. The concen a ion o • BMPO-OOH and • BMPO-OH adicals was e alua ed om a double in eg al o hei EPR spec a and compa ed wi h he spec a o 20 µ mol L −1 4-hyd oxy-2,2,6,6- e ame hylpipe idine-1-oxyl adical (TEMPOL, Fluka 42777, Munich, Ge many), which was used as a e e ence sample. To compa e he po ency o he in es iga ed compounds o dec easing he o e all apped adical concen a ion, a double in eg al o he o al EPR spec a in ensi y o he BMPO adduc s was e alua ed. The concen a ion o H 2 O 2 was measu ed by a Fluo ime ic Hyd ogen Pe oxide Assay Ki (MAK165, Me ck, B a isla a, Slo akia) acco ding o he manu ac u e ’s ins uc ions. The concen a ions o KO 2 and he BMPO/KO 2 mix u e we e dilu ed 1000 and 2000 imes, espec i ely, be o e he measu emen o H2O2. 2.2. Plasmid DNA (pDNA) Clea age Assay A pDNA clea age assay wi h he use o he pBR322 plasmid (N3033 L, New England BioLabs, Inc., Ipswich, MA, USA) was pe o med as epo ed p e iously, wi h some modi ica ions [ 6 ]. All o he inal samples con ained he s udied compounds and 0.2 µ g o pDNA in 20 µ L o he sodium phospha e bu e (25 mmol L −1 sodium phospha e, 50 µmol L−1 DTPA; pH 6.5, 7.4, 8.0, o 8.5). The p ocedu e o he sample p epa a ion o he pDNA clea age assay is desc ibed in de ail in he Supplemen a y Ma e ials. Likewise, in he EPR sample p epa a ion, he phospha e bu e was adjus ed by HCl o keep he inal pH a he equi ed alue a e being added o powde ed KO 2 (20 mmol L −1 ) (see P ocedu e 5 in Supplemen a y Ma e ials). In he p ocedu e ocused on he KO 2− BMPO in e ac ion, he powde ed KO 2 (20 mmol L −1 ) was dissol ed by 10 mmol L −1 BMPO in a 45 mmol L −1 phospha e bu e adjus ed by HCl, hen o exed o 10 s, and 10 µ L o he mix u e was added o a 10 µ L solu ion o pDNA in a 5 mmol L −1 phospha e bu e (see P ocedu e 6 in Supplemen a y Ma e ials). The pH condi ions we e also s anda dized in he clea age assay h ough he addi ion o 0.5 mol L −1 NaOH ( o pH 6.5) o 0.1/0.125 mol L −1 HCl ( o pH 8.0/8.5) o he pDNA solu ion o each pH 7.4 o all o he samples (see P ocedu e 7 in Supplemen a y Ma e ials). Fo he p epa a ion o he BMPO/KO 2 mix u e, in which, acco ding o EPR expe i- men s, • BMPO-OOH/OH adicals we e no o med, powde ed KO 2 (20 mmol L −1 ) was dissol ed in a 50 mmol L −1 phospha e bu e adjus ed by HCl, o exed o 10 s, and hen BMPO (10 mmol L −1 ) was added. As in he p e ious expe imen s, 10 µ L o he mix u e was added o 10 µ L o he pDNA solu ion (see P ocedu e 8 in Supplemen a y Ma e ials). All o he inal samples con ained 5 mmol L −1 BMPO and 10 mmol L −1 KO 2 in he sodium phospha e bu e (25 mmol L −1 sodium phospha e, 50 µ mol L −1 DTPA; pH 6.5, 7.4, 8.0 o 8.5). FeCl 2 (150 µ mol L −1 ), as a massi e pDNA clea age induc o , was used as a posi i e con ol in he assay. The esul ing mix u es we e incuba ed o 30 min a 37 ◦ C. A e incuba ion, he eac ion mix u es we e subjec ed o 0.6% aga ose gel elec opho esis. The in eg a ed densi ies o all pBR322 o ms in each lane we e quan i ied using Image S udio analysis so wa e (LI-COR Bio echnology, Bad Hombu g, Ge many) o es ima e he pDNA clea age po ency h ough he ela i e in ensi ies (I R ) o he nicked o m o pDNA (see Figu e S2 in Supplemen a y Ma e ials). 3. Resul s 3.1. pH-Dependen Composi ion o •BMPO-Adduc s o (BMPO+HCl)-KO2In e ac ion In he con ol expe imen , a phospha e bu e adjus ed wi h HCl was added o pow- de ed KO 2 ( inal 40 mmol L −1 KO 2 ) o ge a inal pH alue o 7.4. A e 10–13 s, when BMPO ( inal 20 mmol L −1 ) was added o he KO 2 –bu e solu ion, he EPR spec a o • BMPO adduc s we e no obse ed ( o de ails, see P ocedu e 1 in Supplemen a y Ma e i- als). Howe e , when BMPO (20 mmol L −1 ) in a phospha e bu e (pH 7.4) was added o powde ed KO 2 ( inal 40 mmol L −1 ), EPR spec a, depending on inal pH, we e obse ed An ioxidan s 2021,10, 1286 5 o 22 (Figu e 1). A e he BMPO-con aining phospha e solu ion (pH 7.4) was added o he powde ed KO 2 , he pH inc eased o ~10–11. The e o e, o s udy he pH dependence o he adical o ma ion om KO 2 , HCl was added o BMPO in he bu e , which ensu ed ha a e he BMPO solu ion was added o he powde ed KO 2 , he inal equi ed pH was ob ained ( o de ails, see P ocedu e 2 in Supplemen a y Ma e ials). The in ensi y and shapes o he • BMPO adduc s spec a depended on he pH. The spec al in ensi y o • BMPO adduc s ob ained a pH 9.0 dec eased in ime (Figu e 1a1–a3). In compa ison wi h pH 9.0, he EPR spec al in ensi y o he • BMPO adduc s a pH 7.7 was highe wi h a di e en ela i e abundance o indi idual species and slow decay o he adduc s. The • BMPO adduc s we e obse ed o mo e han 22 min a pH 7.7 (Figu e 1b1–c3). A simila e ec on he spec a o he • BMPO adduc s was also obse ed a pH 6.5 (Figu e 1d1–e3) and pH 2 (Figu e 1 1–g3). By compa ison, he in ensi y o he EPR spec a o he • BMPO adduc s o med in he mix u e o 5 mmol L −1 BMPO and 10 mmol L −1 KO 2 a pH 7.4 was quan i a i ely lowe (Figu e 1h1–h3), bu simila o ha o he highe BMPO–KO 2 concen a ion (Figu e 1b1–e3). The s able ni oxide adical TEMPOL (20 µ mol L −1 ) was used o de e mine he concen a ion o he adicals in he samples (Figu e 1i1–i3). Using he double in eg als o he TEMPOL calib a ion, he concen a ion o he o al • BMPO adduc adicals was high a pH 6.5–7.8 and low a pH 9.0–11.5, and dec eased in ime, wi h a hal ime (pH 6.5–7.8) o app oxima ely ~12 min (Figu e 2a). As desc ibed in de ail in Sec ion 3.2, he i s eco ded spec um was simula ed o ob ain he indi idual componen s o • BMPO-OOH (sum o con o me s 1 and 2) and • BMPO-OH (sum o con o me s 1 and 2) a 100 ± 15 s a e he sample p epa a ion (Figu e 2b). The a io o he componen popula ion signi ican ly depended on he pH; he po ions o • BMPO-OOH and • BMPO-OH we e ~95% and ~5%, espec i ely, o he whole concen a ion wi hin a pH o ~2–8, and ~10% and ~90% wi hin a pH o ~9–11. As H 2 O 2 is p oduced du ing he eac ion o KO 2 wi h H 2 O, i s concen a ion was measu ed in samples con aining KO 2 a pH 7.4. H 2 O 2 concen a ions o 8.9 ± 1.4 (n= 4) and 10.5 ± 0.7 (n= 4) mmol L −1 we e o med a e he addi ion o bu e + HCl o BMPO + HCl o he powde ed KO2( inal 20/40 BMPO/KO2in mmol L−1), espec i ely. 3.2. Simula ion o EPR Spec a o pH-Dependen BMPO Adduc s The shapes o he EPR spec a o he BMPO adduc s depended on he pH (Figu e 1) and changed o e ime, indica ing a dynamic supe posi ion o signals co esponding o he gene a ion o indi idual BMPO adduc s. The e o e, we analyzed he EPR spec a by simula ion, as in [ 6 ]. Two con o me s each o • BMPO-OOH and • BMPO-OH adduc s we e inse ed in o he spin Hamil onian calcula ions o he expe imen al spec a measu ed in he pH ange o 2–8. Howe e , in he EPR spec a measu ed in he solu ions a pH ≥ 9, wo addi ional low-in ensi y six-line signals we e de ec ed, which we e assigned o he BMPO adduc wi h a ca bon-cen e ed adical ( • BMPO-CR) [ 8 ] and • BMPO-CO 2− , mos likely o igina ing om he DTPA decomposi ion in he alkaline solu ions. The simula ed spec a shown in Figu e 3we e calcula ed using he hype ine coupling cons an s (h cc) elucida ed om he expe imen al spec a (Table 1). The compa ison o he expe imen al and simula ed spec a shows a good i when using he h cc. The de ailed simula ion analysis o he h cc o he indi idual BMPO adduc s e alua ed om he EPR spec a measu ed a a ious pH alues e ealed only a negligible pH e ec on he h cc. An ioxidan s 2021,10, 1286 6 o 22 An ioxidan s 2021, 10, x FOR PEER REVIEW 6 o 23 Figu e 1. Rep esen a i e pH-dependen EPR spec a o •BMPO adduc s a e he addi ion o 20 mmol L–1 BMPO + HCl o powde ed KO2 ( inal 40 mmol L–1 KO2). (a1–i1) Collec ion o 15 EPR spec- a o BMPO adduc s a anged back- o-back, each 42 s, wi h a s a ing acquisi ion o 100 ± 15 s a e sample p epa a ion. (a2–i2) The i s o i h accumula ed spec a and (a3–i3) las i e accumula ed spec a. HCl was added o BMPO (20 mmol L–1) in a bu e o he inal equi ed pH and he solu ion was added o powde ed KO2. (a1–a3) pH 9.0; (b1–b3) pH 7.7 and (c1–c3) con inua ion; (d1–d3) pH 6.5 and (e1–e3) con inua ion; ( 1– 3) pH 2.0 and (g1–g3) con inua ion. (h1–h3) •BMPO adduc s a e he addi ion o 5 mmol L–1 BMPO + HCl o powde ed KO2 ( inal 10 mM KO2) a pH 7.4. (i1–i3) The EPR spec a o s able adical TEMPOL (20 µmol L–1). In ensi ies o (a1–h1) ime-dependen EPR spec a and (a2–h3) he de ailed spec a a e app oxima ely compa able. Fo de ails o he sample p epa a ion, see P ocedu e 2 in Supplemen a y Ma e ials. Figu e 1. Rep esen a i e pH-dependen EPR spec a o • BMPO adduc s a e he addi ion o 20 mmol L −1 BMPO + HCl o powde ed KO 2 ( inal 40 mmol L −1 KO 2 ). ( a1 – i1 ) Collec ion o 15 EPR spec a o BMPO adduc s a anged back- o-back, each 42 s, wi h a s a ing acquisi ion o 100 ± 15 s a e sample p epa a ion. ( a2 – i2 ) The i s o i h accumula ed spec a and ( a3 – i3 ) las i e accumula ed spec a. HCl was added o BMPO (20 mmol L −1 ) in a bu e o he inal equi ed pH and he solu ion was added o powde ed KO 2 . ( a1 – a3 ) pH 9.0; ( b1 – b3 ) pH 7.7 and ( c1 – c3 ) con inua ion; ( d1 – d3 ) pH 6.5 and ( e1 – e3 ) con inua ion; ( 1 – 3 ) pH 2.0 and ( g1 – g3 ) con inua ion. ( h1 – h3 ) • BMPO adduc s a e he addi ion o 5 mmol L −1 BMPO + HCl o powde ed KO 2 ( inal 10 mM KO 2 ) a pH 7.4. ( i1 – i3 ) The EPR spec a o s able adical TEMPOL (20 µ mol L −1 ). In ensi ies o ( a1 – h1 ) ime-dependen EPR spec a and ( a2 – h3 ) he de ailed spec a a e app oxima ely compa able. Fo de ails o he sample p epa a ion, see P ocedu e 2 in Supplemen a y Ma e ials. An ioxidan s 2021,10, 1286 7 o 22 An ioxidan s 2021, 10, x FOR PEER REVIEW 7 o 23 Figu e 2. (a) Time and pH dependencies o he o al •BMPO adduc adical concen a ion o he BMPO + HCl/KO2 (20/40 in mmol L−1) mix u e. Each poin ep esen s he a e age o i e accumu- la ed subsequen EPR spec a. Time s a s a e he addi ion o BMPO + HCl o powde ed KO2. (b) pH-dependen no malized in eg al EPR in ensi y o indi idual •BMPO-OOH and •BMPO-OH com- ponen s elucida ed om he simula ion o he i s expe imen al EPR spec um eco ded 100 ± 15 s a e he addi ion o BMPO + HCl o powde ed KO2. Spec al simula ion is desc ibed in Sec ion 3.2. Spec al componen s: •BMPO-OOH (blue) and •BMPO-OH ( ed). The sum o indi idual BMPO ad- duc s o he i s spec a was no malized o 100%. Each pai (blue and ed) o wo complemen a y poin s ep esen s he indi idual sample p epa a ion. As desc ibed in de ail in Sec ion 3.2, he i s eco ded spec um was simula ed o ob ain he indi idual componen s o •BMPO-OOH (sum o con o me s 1 and 2) and •BMPO-OH (sum o con o me s 1 and 2) a 100 ± 15 s a e he sample p epa a ion (Figu e 2b). The a io o he componen popula ion signi ican ly depended on he pH; he po ions o •BMPO-OOH and •BMPO-OH we e ~95% and ~5%, espec i ely, o he whole concen- a ion wi hin a pH o ~2–8, and ~10% and ~90% wi hin a pH o ~9–11. As H2O2 is p oduced du ing he eac ion o KO2 wi h H2O, i s concen a ion was measu ed in samples con aining KO2 a pH 7.4. H2O2 concen a ions o 8.9 ± 1.4 (n = 4) and 10.5 ± 0.7 (n = 4) mmol L–1 we e o med a e he addi ion o bu e + HCl o BMPO + HCl o he powde ed KO2 ( inal 20/40 BMPO/KO2 in mmol L–1), espec i ely. 3.2. Simula ion o EPR Spec a o pH-Dependen BMPO Adduc s The shapes o he EPR spec a o he BMPO adduc s depended on he pH (Figu e 1) and changed o e ime, indica ing a dynamic supe posi ion o signals co esponding o Figu e 2. ( a ) Time and pH dependencies o he o al • BMPO adduc adical concen a ion o he BMPO + HCl/KO 2 (20/40 in mmol L −1 ) mix u e. Each poin ep esen s he a e age o i e accumu- la ed subsequen EPR spec a. Time s a s a e he addi ion o BMPO + HCl o powde ed KO 2 . ( b ) pH-dependen no malized in eg al EPR in ensi y o indi idual • BMPO-OOH and • BMPO-OH com- ponen s elucida ed om he simula ion o he i s expe imen al EPR spec um eco ded 100 ±15 s a e he addi ion o BMPO + HCl o powde ed KO 2 . Spec al simula ion is desc ibed in Sec ion 3.2. Spec al componen s: • BMPO-OOH (blue) and • BMPO-OH ( ed). The sum o indi idual BMPO adduc s o he i s spec a was no malized o 100%. Each pai (blue and ed) o wo complemen a y poin s ep esen s he indi idual sample p epa a ion. Table 1. The h cc o BMPO spin adduc s elucida ed om simula ions o expe imen al spec a measu ed in phospha e bu e solu ions con aining KO 2 . • BMPO-OOH and • BMPO-OH we e simula ed conside ing he p esence o wo con o me s. BMPO-Adduc aN, mT aHβ, mT aHγ, mT •BMPO-OH(1) 1.433 ±0.003 1.521 ±0.005 0.074 ±0.004 •BMPO-OH(2) 1.421 ±0.004 1.264 ±0.003 0.065 ±0.002 •BMPO-OOH(1) 1.341 ±0.003 1.198 ±0.004 – •BMPO-OOH(2) 1.340 ±0.001 0.967 ±0.006 – •BMPO-CR 1.515 ±0.008 2.077 ±0.024 – •BMPO-CO2−1.490 ±0.003 1.710 ±0.012 – An ioxidan s 2021,10, 1286 8 o 22 An ioxidan s 2021, 10, x FOR PEER REVIEW 8 o 23 he gene a ion o indi idual BMPO adduc s. The e o e, we analyzed he EPR spec a by simula ion, as in [6]. Two con o me s each o •BMPO-OOH and •BMPO-OH adduc s we e inse ed in o he spin Hamil onian calcula ions o he expe imen al spec a measu ed in he pH ange o 2–8. Howe e , in he EPR spec a measu ed in he solu ions a pH ≥ 9, wo addi ional low-in ensi y six-line signals we e de ec ed, which we e assigned o he BMPO adduc wi h a ca bon-cen e ed adical (•BMPO-CR) [8] and •BMPO-CO2–, mos likely o igina ing om he DTPA decomposi ion in he alkaline solu ions. The simula ed spec a shown in Figu e 3 we e calcula ed using he hype ine coupling cons an s (h cc) elucida ed om he expe imen al spec a (Table 1). The compa ison o he expe imen al and simula ed spec a shows a good i when using he h cc. The de ailed simula ion anal- ysis o he h cc o he indi idual BMPO adduc s e alua ed om he EPR spec a measu ed a a ious pH alues e ealed only a negligible pH e ec on he h cc. Figu e 3. Rep esen a i e no malized expe imen al EPR spec a o BMPO adduc s along wi h hei simula ions using he h cc summa ized in Table 1. Only he expe imen al spec a o he six h o en h accumula ed spec a a e shown (blue, measu ed 5.2–8.7 min a e sample p epa a ion); he simula ed spec a a e ed. (a) pH 9.0, (b) pH 7.7, (c) con inued measu emen du ing 15.7–19.2 min, and (d) pH 6.0. Table 1. The h cc o BMPO spin adduc s elucida ed om simula ions o expe imen al spec a meas- u ed in phospha e bu e solu ions con aining KO2. •BMPO-OOH and •BMPO-OH we e simula ed conside ing he p esence o wo con o me s. Figu e 3. Rep esen a i e no malized expe imen al EPR spec a o BMPO adduc s along wi h hei simula ions using he h cc summa ized in Table 1. Only he expe imen al spec a o he six h o en h accumula ed spec a a e shown (blue, measu ed 5.2–8.7 min a e sample p epa a ion); he simula ed spec a a e ed. ( a ) pH 9.0, ( b ) pH 7.7, ( c ) con inued measu emen du ing 15.7–19.2 min, and ( d ) pH 6.0. 3.3. Compa ison o pH-Dependen BMPO Adduc Spec a o BMPO−KO2In e ac ion Fo simplici y, he ela i e concen a ions o wo con o me s, • BMPO-OH(1) and • BMPO-OH(2), we e summed and desc ibed as • BMPO-OH. Analogously, • BMPO-OOH(1) and • BMPO-OOH(2) we e summed and desc ibed as • BMPO-OOH. As desc ibed in Sec- ion 3.1, he pH-dependen o ma ion o indi idual • BMPO-OOH and • BMPO-OH compo- nen s elucida ed om he simula ion o he i s expe imen al EPR spec um is shown in Figu e 2b. The ime dependence o he EPR in ensi y o he simula ed componen s o 1–5, 6–10, and 11–15 accumula ed expe imen al BMPO adduc spec a a di e en pH alues is shown in Figu e 4. The sum o indi idual • BMPO adduc s o he accumula ed spec a a a gi en ime was no malized o 100%. A pH 11.5, he • BMPO-OH componen dec eased in ime, and • BMPO-CR and • BMPO-CO 2− componen s we e p esen (Figu e 4a). Simila ly, mos ly • BMPO-OH, as well as a mino abundance o • BMPO-CR, • BMPO-CO 2− , and • BMPO-OOH componen s, we e obse ed a pH 9.0 (Figu e 4b). Howe e , a a lowe pH ( ≤ 8.0), he • BMPO-OOH componen p e ailed o e • BMPO-OH (Figu e 4c,d,e,g). The el- a i e concen a ion o • BMPO-OOH dec eased and • BMPO-OH inc eased g adually o e ime a pH 7.7–8.0 (Figu e 4c–e). The ela i e concen a ion o • BMPO-OOH in compa ison wi h •BMPO-OH was high a pH 6.0 and ela i ely s able o o e 11 min (Figu e 4g,h). An ioxidan s 2021,10, 1286 9 o 22 An ioxidan s 2021, 10, x FOR PEER REVIEW 10 o 23 Figu e 4. Compa ison o ime and pH-dependen no malized in eg al EPR in ensi y o indi idual BMPO adduc s elucida ed om he simula ion o expe imen al EPR spec a. Spec al componen s: •BMPO-OOH (blue), •BMPO-OH ( ed), •BMPO-CR (black), •BMPO-CO2- (g een) a (a) pH 11.5 (pH was adjus ed by NaOH), (b) pH 9.0, (c) pH 8.0, (d) pH 7.8, and (e) pH 7.7; ( ) con inued measu emen o (e) o he nex 11 min; (g) pH 6.0 and (h) con inued measu emen o (g) o he nex 11 min. Each poin ep esen s he a e age o i e accumula ed subsequen EPR spec a. Time s a s a e he ad- di ion o BMPO + HCl o powde ed KO2. The sum o indi idual BMPO adduc s o accumula ed spec a was no malized o 100%. Fo de ails o he sample p epa a ion, see P ocedu e 2 in Supple- men a y Ma e ials. To compa e he pH dependence o he o al adicals o he •BMPO adduc compo- nen s, double in eg al in ensi ies o he simula ed spec a we e applied (Figu e 5). The concen a ion o apped adicals o he •BMPO adduc s was e y low a pH ~9.0–11.5 (Figu e 5a,b). Howe e , a pH ~6.0–8.0, he numbe o apped adicals inc eased ~3–10 imes, pa icula ly because o he •BMPO-OOH componen (Figu e 5c–h). The amoun o •BMPO-OOH adical componen s was ~10 imes highe in compa ison wi h •BMPO-OH a pH ~6.0–8.0, 3.5 min a e he sample p epa a ion (Figu e 5c–h). The p esence o he •BMPO-OOH componen was s ill ~3 imes mo e abundan in compa ison wi h •BMPO- OH a e 22 min a pH ~6.0 (Figu e 5h). Figu e 4. Compa ison o ime and pH-dependen no malized in eg al EPR in ensi y o indi idual BMPO adduc s elucida ed om he simula ion o expe imen al EPR spec a. Spec al componen s: • BMPO-OOH (blue), • BMPO-OH ( ed), • BMPO-CR (black), • BMPO-CO 2- (g een) a ( a ) pH 11.5 (pH was adjus ed by NaOH), ( b ) pH 9.0, ( c ) pH 8.0, ( d ) pH 7.8, and ( e ) pH 7.7; ( ) con inued measu emen o ( e ) o he nex 11 min; ( g ) pH 6.0 and ( h ) con inued measu emen o ( g ) o he nex 11 min. Each poin ep esen s he a e age o i e accumula ed subsequen EPR spec a. Time s a s a e he addi ion o BMPO + HCl o powde ed KO 2 . The sum o indi idual BMPO adduc s o accumula ed spec a was no malized o 100%. Fo de ails o he sample p epa a ion, see P ocedu e 2 in Supplemen a y Ma e ials. To compa e he pH dependence o he o al adicals o he • BMPO adduc componen s, double in eg al in ensi ies o he simula ed spec a we e applied (Figu e 5). The concen a- ion o apped adicals o he • BMPO adduc s was e y low a pH ~9.0–11.5 (Figu e 5a,b). Howe e , a pH ~6.0–8.0, he numbe o apped adicals inc eased ~3–10 imes, pa icu- la ly because o he • BMPO-OOH componen (Figu e 5c–h). The amoun o • BMPO-OOH adical componen s was ~10 imes highe in compa ison wi h • BMPO-OH a pH ~6.0–8.0, 3.5 min a e he sample p epa a ion (Figu e 5c–h). The p esence o he • BMPO-OOH componen was s ill ~3 imes mo e abundan in compa ison wi h • BMPO-OH a e 22 min a pH ~6.0 (Figu e 5h). An ioxidan s 2021,10, 1286 16 o 22 An ioxidan s 2021, 10, x FOR PEER REVIEW 17 o 23 Figu e 12. Rep esen a i e no malized expe imen al EPR spec a o BMPO adduc s along wi h hei simula ions. A angemen o expe imen al and simula ed spec a is he same as in Figu e 3. EPR spec a a (a) pH 10.3, (b) pH 8.5, (c) pH 7.3, and (d) pH 6.5. Sample p epa a ion: BMPO in he bu e was added o powde ed KO2 and o exed, and 30 s la e HCl was added o adjus he inal pH o he solu ion. Figu e 12. Rep esen a i e no malized expe imen al EPR spec a o BMPO adduc s along wi h hei simula ions. A angemen o expe imen al and simula ed spec a is he same as in Figu e 3. EPR spec a a ( a ) pH 10.3, ( b ) pH 8.5, ( c ) pH 7.3, and ( d ) pH 6.5. Sample p epa a ion: BMPO in he bu e was added o powde ed KO 2 and o exed, and 30 s la e HCl was added o adjus he inal pH o he solu ion. An ioxidan s 2021,10, 1286 17 o 22 An ioxidan s 2021, 10, x FOR PEER REVIEW 18 o 23 Figu e 13. (Le column) Compa ison o ime- and pH-dependen no malized in eg al EPR in ensi y o indi idual BMPO adduc s elucida ed om he simula ion o he expe imen al EPR spec a. Spec- al componen s: •BMPO-OOH (blue), •BMPO-OH ( ed), •BMPO-CR (black), and •BMPO-CO2− (g een) a (a) pH 10.3, (b) pH 8.5, and (c) pH 7.3; (d) con inued measu emen o (c) o ano he 11 min; (e) pH 6.5. Each poin ep esen s he a e age o i e accumula ed subsequen EPR spec a. Time s a s a e he addi ion o BMPO o powde ed KO2. The sum o he indi idual BMPO adduc s o he accumula ed spec a was no malized o 100%. (Righ column) ( –j) Compa ison o ime- and pH-dependen absolu e in eg al EPR in ensi y ( o al quan i y o adicals in .u.) o indi idual BMPO adduc s o no malized in eg al EPR in ensi y shown in he le column. 3.6. pH-Dependen Clea age o pDNA by BMPO–KO2 In e ac ion To know whe he •BMPO adduc s ha e biological e ec s, he clea age o pDNA by •BMPO-OOH/OH was e alua ed (Figu es 14 and S2). pDNA clea age was negligible in he con ol bu e s a pH 6.5, 7.4, 8.0, and 8.5, bu signi ican ly inc eased in he p esence o FeCl2, which is known o clea e DNA [21,22]. Besides he single-s anded clea age o he supe coiled pDNA gene a ing he nicked ci cula o m, FeCl2 was no ably able o me- dia e double-s anded clea age, c ea ing linea o m (Figu e S2 in Supplemen a y Ma e- ials). The con ol samples con aining BMPO, H2O2, KO2, o he mix u e o H2O2 and KO2 did no induce pDNA clea age. Howe e , when BMPO + HCl was added o powde ed KO2, p oducing •BMPO-OOH/OH, and hen applied o pDNA, signi ican pDNA clea - age was obse ed a all o he pH alues s udied. The alues we e sca e ed, bu he pDNA clea age po ency had a endency o inc ease wi h he inc easing pH alues. To con i m he in ol emen o pH in he obse ed e ec , he solu ion o pDNA in he phospha e Figu e 13. (Le column) Compa ison o ime- and pH-dependen no malized in eg al EPR in ensi y o indi idual BMPO adduc s elucida ed om he simula ion o he expe imen al EPR spec a. Spec al componen s: • BMPO-OOH (blue), • BMPO-OH ( ed), • BMPO-CR (black), and • BMPO-CO 2− (g een) a ( a ) pH 10.3, ( b ) pH 8.5, and ( c ) pH 7.3; ( d ) con inued measu emen o ( c ) o ano he 11 min; ( e ) pH 6.5. Each poin ep esen s he a e age o i e accumula ed subsequen EPR spec a. Time s a s a e he addi ion o BMPO o powde ed KO 2 . The sum o he indi idual BMPO adduc s o he accumula ed spec a was no malized o 100%. (Righ column) ( – j ) Compa ison o ime- and pH-dependen absolu e in eg al EPR in ensi y ( o al quan i y o adicals in .u.) o indi idual BMPO adduc s o no malized in eg al EPR in ensi y shown in he le column. An ioxidan s 2021,10, 1286 18 o 22 An ioxidan s 2021, 10, x FOR PEER REVIEW 19 o 23 bu e was adjus ed by NaOH o HCl o s anda dize he pH o 7.4 a e he addi ion o (BMPO + HCl) + KO2 solu ions wi h pH 6.5, 8.0, and 8.5. A e he pH was adjus ed o 7.4, he e ec s o he mix u es we e s ill signi ican , bu he pH end was los . Based on he EPR measu emen o he adical concen a ion, pDNA was signi ican ly clea ed by •BMPO-OOH/OH a a concen a ion o ~18 µmol L−1, whe e he concen a ion a io o •BMPO-OOH o •BMPO-OH a he beginning o he expe imen s was ≥20. Taking in o conside a ion he sho li espan o O2•− in an aqueous solu ion, we p epa ed a con ol sam- ple ha , acco ding o he EPR s udy, did no o m adicals, as ollows: i s , powde ed KO2 was dissol ed in a phospha e bu e adjus ed wi h HCl o ge a 20 mmol L–1 KO2 so- lu ion wi h a co esponding pH, hen, 10 s la e BMPO ( inal 10 mmol L–1) was added, and his mix u e was applied a 1:1 olume a io o pDNA solu ion. In acco dance wi h he EPR da a, he mix u e p epa ed in his way had a negligible e ec on he pDNA clea age a all o he pH le els used (Figu e 14). Figu e 14. Po ency o compounds o induce pDNA clea age a pH 6.5 (g een), 7.4 ( ed), 8.0 (cyan), and 8.5 pH (pink) in a 25 mmol L−1 sodium phospha e bu e and 50 µmol L−1 DTPA a 37 °C. Con ol wi hou and wi h 150 µmol L–1 FeCl2; con ols wi h 5 mmol L–1 BMPO, 5 mmol L–1 H2O2, 10 mmol L–1 KO2, and 5 mmol L–1 H2O2 + 10 mmol L–1 KO2. Sample (BMPO + HCL) + KO2: BMPO ( inal 5 mmol L–1) in a phospha e bu e adjus ed by HCl ( o ge equi ed pH o BMPO/KO2 mix u e) was added o powde ed KO2 ( inal 10 mmol L–1), hen he mix u e was added o he pDNA solu ion (see P ocedu e 6 in Supplemen a y Ma e ials). Sample (BMPO + HCL) + KO2 adjus ed o pH 7.4: solu- ions we e p epa ed as o p e ious samples wi h a ious pH le els, bu HCl o NaOH we e added o he pDNA solu ion o s anda dize he pH o 7.4 (see P ocedu e 7 in Supplemen a y Ma e ials). Sample (Bu e + HCl) + KO2 + BMPO: phospha e bu e adjus ed by HCl was added o powde KO2 ( inal 10 mmol L–1), ollowed by he addi ion o BMPO ( inal 5 mmol L–1) 10 s la e , and he mix u e was applied o he pDNA solu ion (see P ocedu e 8 in Supplemen a y Ma e ials). The inal concen- a ion o pDNA was 0.2 µg in 20 µL. IR o nc DNA o m ep esen s he ela i e in ensi y o he nicked ci cula pDNA. Da a ep esen alues om indi idual samples. Ho izon al black ma ks in- dica e means ± SD. 4. Discussion O2•− and i s de i a i es a e in ol ed in adical signaling and, mo e signi ican ly, in he de elopmen o diseases due o oxida i e s ess-media ed damage o he cellula s uc- u es [1,2]. To s udy he molecula mechanism o i s biological ac ions, i is con enien o ha e a simple and ep oducible sou ce o O2•−. The e a e se e al me hods o p oduce O2•− Figu e 14. Po ency o compounds o induce pDNA clea age a pH 6.5 (g een), 7.4 ( ed), 8.0 (cyan), and 8.5 pH (pink) in a 25 mmol L −1 sodium phospha e bu e and 50 µ mol L −1 DTPA a 37 ◦ C. Con ol wi hou and wi h 150 µ mol L −1 FeCl 2 ; con ols wi h 5 mmol L −1 BMPO, 5 mmol L −1 H 2 O 2 , 10 mmol L −1 KO 2 , and 5 mmol L −1 H 2 O 2 + 10 mmol L −1 KO 2 . Sample (BMPO + HCL) + KO 2 : BMPO ( inal 5 mmol L −1 ) in a phospha e bu e adjus ed by HCl ( o ge equi ed pH o BMPO/KO 2 mix u e) was added o powde ed KO 2 ( inal 10 mmol L −1 ), hen he mix u e was added o he pDNA solu ion (see P ocedu e 6 in Supplemen a y Ma e ials). Sample (BMPO + HCL) + KO 2 adjus ed o pH 7.4: solu ions we e p epa ed as o p e ious samples wi h a ious pH le els, bu HCl o NaOH we e added o he pDNA solu ion o s anda dize he pH o 7.4 (see P ocedu e 7 in Supplemen a y Ma e ials). Sample (Bu e + HCl) + KO 2 + BMPO: phospha e bu e adjus ed by HCl was added o powde KO 2 ( inal 10 mmol L −1 ), ollowed by he addi ion o BMPO ( inal 5 mmol L −1 ) 10 s la e , and he mix u e was applied o he pDNA solu ion (see P ocedu e 8 in Supplemen a y Ma e ials). The inal concen a ion o pDNA was 0.2 µ g in 20 µ L. I R o nc DNA o m ep esen s he ela i e in ensi y o he nicked ci cula pDNA. Da a ep esen alues om indi idual samples. Ho izon al black ma ks indica e means ±SD. 4. Discussion O 2•− and i s de i a i es a e in ol ed in adical signaling and, mo e signi ican ly, in he de elopmen o diseases due o oxida i e s ess-media ed damage o he cellula s uc u es [ 1 , 2 ]. To s udy he molecula mechanism o i s biological ac ions, i is con enien o ha e a simple and ep oducible sou ce o O 2•− . The e a e se e al me hods o p oduce O 2•− [ 3 ]; howe e , some o hem a e no expe imen ally simple o a e no sui able o biological expe imen s. The e o e, KO 2 as a sou ce o O 2•− was s udied by he EPR spin apping me hod wi hou using non-biological componen s. As in ou aqueous expe imen al sys em he O 2•− is highly eac i e and spon aneously disp opo iona es wi hin 10 s, his s udy was limi ed mos ly o moni o ing he seconda y adicals (spin adduc s) • BMPO-OOH/ • BMPO-OH, and did no s udy he p oduc ion a es and a e o any p ima y adicals. Addi ionally, we a e conscious o he pH-dependen eac i i y o HO 2− /H 2 O 2 p oduced in he O 2•− disp opo iona ion in he p ocess o BMPO adduc gene a ion. The dissolu ion o KO 2 by a phospha e bu e p oduces O 2•− and H 2 O 2 . O 2•− is known o o m • BMPO-OOH o • BMPO-OH adicals, which a e de ec able by EPR and can be ob ained by spec al simula ion. F om he good i o he expe imen al spec a, i is ob ious ha he h cc a e sui able o spec al simula ion. The h cc alues a e in he An ioxidan s 2021,10, 1286 19 o 22 ange o da a ob ained unde simila expe imen al condi ions [ 4 , 6 , 8 , 9 , 11 ]. As ou me hod dis inguishes concen a ions o he ime-dependen changes o • BMPO-OOH and • BMPO- OH adicals in he same sample, i is app op ia e o ime-dependen s udies o compound in e ac ions wi h adicals. In • BMPO-OOH and • BMPO-OH ni oxide (aminoxyl) adicals, he unpai ed elec on is mos ly localized on he N-O moie y and he hype ine in e ac ions wi h ni ogen and β -hyd ogen nuclei domina e he EPR spec a (Table 1). The e o e, • BMPO-OOH and • BMPO-OH can be u he s udied as po en ial models o o ganic hyd ope oxide and alcohol, espec i ely. The dissolu ion o KO 2 in a wa e solu ion (e.g., pH 7.4) p oduces O 2•− wi h a sho li e ime (<ms) [ 3 , 7 , 23 ]. This was con i med unde ou expe imen al condi ions a pH 7.4, in which spin ap BMPO added 10–13 s a e he p epa a ion o he bu e /KO 2 solu ion did no show any • BMPO-adduc spec a. The same esul was ound when KO 2 in DMSO was added o a pH 7.4 bu e ed wa e solu ion ollowed by BMPO [6]. Using he spin ap BMPO, we obse ed ha he addi ion o a wa e solu ion o powde KO 2 p oduced adicals, whose composi ion and in ensi y signi ican ly depended on he pH le el du ing he dissolu ion o KO 2 . As KO 2 is a s ong base, he addi ion o a bu e ed wa e solu ion (50 mmol L −1 phospha e bu e , pH 7.4) o powde ed KO 2 ( inal 40 mmol L−1) inc eased he pH o 10–11. The e a e wo possible ways o dec ease he pH o he mix u e o he physiological alue o 7.4. When pH was dec eased o 6.5–8.5 h ough he addi ion o HCl o he p epa ed BMPO/KO 2 mix u e, • BMPO-OH adicals, compa ed wi h • BMPO-OOH, we e mos ly obse ed a pH ~7.3–10.3 (Figu e 13). On he o he hand, when he BMPO solu ion adjus ed wi h HCl was added o he powde ed KO 2 o ge a inal physiological pH o ~6–8, • BMPO-OOH p e ailed o e • BMPO-OH (Figu es 2b, 4and 10) . The • BMPO-OOH/ • BMPO-OH a io calcula ed om he i s eco ded EPR spec um was ≥ 20 (Figu e 2b). As he i s spec um was eco ded 100 ± 15 s a e he BMPO–HCl solu ion was mixed wi h powde ed KO 2 , i is e y p obable ha he • BMPO-OOH/OH a io was e en highe immedia ely a e he sample p epa a ion. Based on hese da a, i can be sugges ed ha KO 2 can be used as a sou ce o O 2•− and • BMPO-OOH, when i is ensu ed ha du ing he dissolu ion o KO 2 , he pH is wi hin he ange o 6.5–8.0. This can be done by applying acid du ing he dissolu ion o by using a s ong physiological bu e . Howe e , as a signi ican amoun o H 2 O 2 is p oduced du ing he dissolu ion o KO 2 by an aqueous bu e , i s in ol emen in adical–compound in e ac ions should be also aken in o accoun . I is supposed ha he obse ed hyd oxyl adduc , • BMPO-OH, was mos ly p o- duced by pH- and ime-dependen decomposi ion o • BMPO-OOH, which was o med by apping O 2•− du ing KO 2 dissolu ion. This sugges ion is suppo ed by he ime- and pH-dependen p og essi e inc ease in he ela i e concen a ion o • BMPO-OH, which is mo e s able han • BMPO-OOH, upon a p og essi e dec ease in he • BMPO-OOH ela i e concen a ion (Figu es 2b and 10). The signi ican p opo ion o he • BMPO-OH compo- nen in he i s eco ded spec um a pH ≥ 9.0 co ela ed well wi h published da a [ 8 ]. Howe e , he in ensi y o he • BMPO-OH spec a ansien ly inc eased o e ime in he mix u e o BMPO + H 2 O 2 (pH 9.0 and 12.2; Figu e S1), sugges ing ha he BMPO/H 2 O 2 in e ac ion con ibu ed o he • BMPO-OH componen . Addi ionally, he decomposi ion o H 2 O 2 in o eac i e oxygen species unde alkaline media may p omo e he p oduc ion o • BMPO-OH [ 24 – 27 ]. As he p esen s udy was ocused mainly on he p ac ical p epa a ion o • BMPO-OOH, he mechanism o hyd oxyl adduc o ma ion was no s udied in mo e de ail. Ou p e ious s udy desc ibed he p epa a ion o he • BMPO-OOH spin adduc as a model o he s udy o o ganic hyd ope oxide decomposi ion [ 6 ]. We used 10% DMSO ( / ) o p epa e he • BMPO adduc , which is use ul o e alua ing he an ioxidan po ency and e ec s o compounds insoluble in wa e on • BMPO-OOH. In he p esen s udy, • BMPO- OOH, as a po en ial model o hyd ope oxide, was p epa ed wi hou DMSO, and he an ioxidan po ency o compounds soluble in wa e , such as H 2 S, RSe-1, and RSe-2, was s udied. As in ou p e ious s udy wi h H 2 S in a 10% DMSO sol en [ 6 , 22 ], he wa e An ioxidan s 2021,10, 1286 20 o 22 solu ion o H 2 S changed he • BMPO-OOH/ • BMPO-OH p opo ion and dec eased he o e all adical concen a ions. RSe-1 and RSe-2 in e ac ed wi h • BMPO adduc s and inc eased he adical p opo ion o • BMPO-OH o e • BMPO-OOH in a ime-dependen manne , e en in he case when he o al ime-dependen • BMPO adduc s we e simila o he con ols. The de ailed mechanism o he chemical in e ac ions leading o changes in he concen a ion and/o a io o • BMPO-OOH/OH is no known a p esen and equi es u he examina ion. The oxida ion o DNA occu s h ough eac ions wi h eac i e oxygen species (ROS), e.g., HO • , O 2•− , single oxygen ( 1 O 2 ), pe oxyni i e (ONOO − ), o H 2 O 2 , which a e p o- duced du ing endogenous biological p ocesses [ 28 – 30 ]. The e o e, pDNA was used o es he biological e ec s o • BMPO adduc s. pDNA was signi ican ly clea ed by low • BMPO- OOH/ • BMPO-OH concen a ions, in which • BMPO-OOH p e ailed o e • BMPO-OH. BMPO, KO 2 , H 2 O 2 , and KO 2 + H 2 O 2 alone did no clea e pDNA, indica ing ha hey we e no in ol ed in he pDNA clea age e ec s o • BMPO-OOH/ • BMPO-OH. The mix u e p epa ed o 10 s, which delayed he addi ion o BMPO o he bu e + HCl + KO 2 solu ion, which did no o m a • BMPO adduc spec al signal, also did no clea e pDNA, con i ming ha nei he he componen s o he mix u e no hei non adical p oduc s we e able o clea e pDNA. Al oge he , his implies ha • BMPO-OOH/OH adicals a e esponsible o pDNA clea age. A de ailed cascade o he chemical in e ac ions leading o pDNA clea age by •BMPO-OOH/OH adicals is no known a he p esen and emains o be elucida ed. 5. Conclusions This s udy u ilized KO 2 as a sou ce o O 2•− adical anions and he EPR spin apping echnique wi h BMPO o moni o he gene a ion o he • BMPO-OOH spin adduc and i s con e sion o • BMPO-OH in a wa e solu ion wi hou DMSO as a po en ial model o o ganic hyd ope oxides and alcohols. The concen a ion and ela i e abundance o • BMPO-OOH and • BMPO-OH elucida ed om he EPR spec a s ongly depended on he p ocedu e o he sample p epa a ion and he pH alue. In he pH ange o 6.5–8.0, • BMPO-OOH p edomina ed o e • BMPO-OH. Mo eo e , he me hod also dis inguished he ime-dependen concen a ion changes o bo h • BMPO adduc s in he same sample, so i is app op ia e o s udies o ime-dependen in e ac ions o compounds wi h adicals. The • BMPO-OOH/OH adicals clea ed he plasmid DNA. H 2 S and selenium-con aining de i a i es inc eased he p opo ion o • BMPO-OH o e • BMPO-OOH adicals. This demons a es ha he p esen ed app oach can be used o s udy he biological e ec s o •BMPO-OOH/OH adicals and he in e ac ions o compounds wi h •BMPO-OOH/OH. Supplemen a y Ma e ials: The ollowing a e a ailable online a h ps://www.mdpi.com/a icle/ 10.3390/an iox10081286/s1, P ocedu es o p epa a ion o EPR samples. Figu e S1: Time- and pH- dependen EPR spec a o • BMPO adduc s o he 30 mmol L −1 BMPO in he p esence o 1 and 10 mmol L−1H2O2. Figu e S2: Rep esen a i e gels indica ing he e ec s o BMPO − KO 2 in e ac ion on pDNA clea age a a ious pH. Au ho Con ibu ions: Concep ualiza ion, K.O.; o mal analysis, A.M., V.B. and K.L.; in es iga ion, A.M., V.B., M.C., K.L., M.G. and K.O.; me hodology, A.M., V.B., M.C. and K.O.; p ojec adminis a ion, M.C. and K.O.; esou ces, K.L., M.J.N. and C.J.; supe ision, K.O.; alida ion, V.B., M.C., M.J.N., M.G., L.T. and K.O.; isualiza ion, A.M., V.B., M.C., M.G., L.T. and K.O.; w i ing—o iginal d a , K.O. All au ho s ha e ead and ag eed o he published e sion o he manusc ip . Funding: This esea ch was unded by he Slo ak Resea ch and De elopmen Agency, g an numbe APVV-19-0154 o K.O. and APVV-17-0384 o M.C., and he Scien i ic G an Agency o he Slo ak Republic, g an numbe VEGA 1/0064/21 o V.B., 2/0079/19 o M.G., 2/0091/21 o A.M., and 2/0053/19 o M.C. V.B. hanks Minis y o Educa ion, Science, Resea ch and Spo o he Slo ak Republic o unding wi hin he scheme “Excellen esea ch eams”. Ins i u ional Re iew Boa d S a emen : No applicable. In o med Consen S a emen : No applicable. An ioxidan s 2021,10, 1286 21 o 22 Da a A ailabili y S a emen : All indings and conclusions a e based on he p esen ed igu es in he main ex o in he Supplemen a y Ma e ials. O iginal sou ce iles can be sen om he co esponding au ho , D . Ka ol Ond ias, upon eques . Con lic s o In e es : The au ho s decla e no con lic o in e es . Re e ences 1. Ca oll, L.; Pa ison, D.I.; Da ies, J.B.; Ande son, R.F.; Lopez-Ala con, C.; Da ies, M.J. Supe oxide adicals eac wi h pep ide- de i ed yp ophan adicals wi h e y high a e cons an s o gi e hyd ope oxides as majo p oduc s. F ee Radic. Biol. Med. 2018 , 118, 126–136. [C ossRe ] 2. Chis e, R.C.; F ei as, M.; Me cadan e, A.Z.; Fe nandes, E. Supe oxide Anion Radical: Gene a ion and De ec ion in Cellula and Non-Cellula Sys ems. Cu . Med. Chem. 2015,22, 4234–4256. [C ossRe ] [PubMed] 3. Hayyan, M.; Hashim, M.A.; AlNashe , I. Supe oxide Ion: Gene a ion and Chemical Implica ions. Chem. Re . 2016 ,116, 3029–3085. [C ossRe ] [PubMed] 4. Zhao, H.; Joseph, J.; Zhang, H.; Ka oui, H.; Kalyana aman, B. Syn hesis and biochemical applica ions o a solid cyclic ni one spin ap: A ela i ely supe io ap o de ec ing supe oxide anions and glu a hiyl adicals. F ee Radic. Biol. Med. 2001 ,31, 599–606. [C ossRe ] 5. Béziè e, N.; Ha dy, M.; Poulhès, F.; Ka oui, H.; To do, P.; Oua i, O.; F apa , Y.-M.; Rockenbaue , A.; Bouche , J.-L.; Mansuy, D.; e al. Me abolic s abili y o supe oxide adduc s de i ed om newly de eloped cyclic ni one spin aps. F ee Radic. Biol. Med. 2014,67, 150–158. [C ossRe ] 6. Misak, A.; B ezo a, V.; G man, M.; Tomaso a, L.; Cho anec, M.; Ond ias, K. • BMPO-OOH Spin-Adduc as a Model o S udy o Decomposi ion o O ganic Hyd ope oxides and he E ec s o Sul ide/Seleni e De i a i es. An EPR Spin-T apping App oach. An ioxidan s 2020,9, 918. [C ossRe ] 7. Janik, I.; T ipa hi, G.N. The na u e o he supe oxide adical anion in wa e . J. Chem. Phys. 2013,139, 014302. [C ossRe ] 8. Villamena, F.A.; Zweie , J.L. Supe oxide adical apping and spin adduc decay o 5- e -bu oxyca bonyl-5-me hyl-1-py oline N-oxide (BocMPO): Kine ics and heo e ical analysis. J. Am. Chem. Soc. Pe kin T ans 1 2002, 1340–1344. [C ossRe ] 9. Tsai, P.; Ichikawa, K.; Maile , C.; Pou, S.; Halpe n, H.J.; Robinson, B.H.; Nielsen, R.; Rosen, G.M. Es e s o 5-Ca boxyl-5-me hyl-1- py olineN-Oxide: A Family o Spin T aps o Supe oxide. J. O g. Chem. 2003,68, 7811–7817. [C ossRe ] 10. S olze, K.; Udilo a, N.; Rosenau, T.; Ho inge , A.; Nohl, H. Syn hesis and Cha ac e iza ion o EMPO-De i ed 5,5-Disubs i u ed 1-Py oline N-Oxides as Spin T aps Fo ming Excep ionally S able Supe oxide Spin Adduc s. Biol. Chem. 2003 ,384, 493–500. [C ossRe ] 11. Tsai, P.; Ma a, J.M.; Pou, S.; Bowman, M.K.; Rosen, G.M. Is The e S e eoselec i i y in Spin T apping Supe oxide by 5- e - Bu oxyca bonyl-5-me hyl-1-py olineN-Oxide? J. O g. Chem. 2005,70, 7093–7097. [C ossRe ] 12. Spasoje i´c, I. F ee adicals and an ioxidan s a a glance using EPR spec oscopy. C i . Re . Clin. Lab. Sci. 2011 ,48, 114–142. [C ossRe ] 13. Abbas, K.; Ha dy, M.; Poulhès, F.; Ka oui, H.; To do, P.; Oua i, O.; Pey o , F. De ec ion o supe oxide p oduc ion in s imula ed and uns imula ed li ing cells using new cyclic ni one spin aps. F ee Radic. Biol. Med. 2014,71, 281–290. [C ossRe ] 14. Chang, J.; Taylo , R.D.; Da idson, R.A.; Sha mah, A.; Guo, T. Elec on Pa amagne ic Resonance Spec oscopy In es iga ion o Radical P oduc ion by Gold Nanopa icles in Aqueous Solu ions Unde X- ay I adia ion. J. Phys. Chem. A 2016 ,120, 2815–2823. [C ossRe ] 15. Suzen, S.; Gu e -O han, H.; Saso, L. De ec ion o Reac i e Oxygen and Ni ogen Species by Elec on Pa amagne ic Resonance (EPR) Technique. Molecules 2017,22, 181. [C ossRe ] [PubMed] 16. Olson, K.R. Hyd ogen sul ide, eac i e sul u species and coping wi h eac i e oxygen species. F ee Radic. Biol. Med. 2019 ,140, 74–83. [C ossRe ] 17. Co ino, A.; F ecen ese, F.; Magli, E.; Pe issu i, E.; San agada, V.; Scognamiglio, A.; Caliendo, G.; Fio ino, F.; Se e ino, B. T ends in H2S-Dono s Chemis y and Thei E ec s in Ca dio ascula Diseases. An ioxidan s 2021,10, 429. [C ossRe ] [PubMed] 18. Di Leo, I.; Sancine o, L.; Messina, F.; San i, C. O ganoselenium compounds, an o e iew on he biological ac i i ies beyond an ioxidan p ope ies. In P oceedings o he 20 h In e na ional Elec onic Con e ence on Syn he ic O ganic Chemis y (ECSOC), online, 1–30 No embe 2016. 19. Ali, W.; Spengle , G.; Kincses, A.; No é, M.; Ba is elli, C.; La acz, G.; S a ek, M.; D ˛ab owska, M.; Honkisz-O zechowska, E.; Romanelli, A.; e al. Disco e y o phenylselenoe he -hydan oin hyb ids as ABCB1 e lux pump modula ing agen s wi h cy o oxic and an ip oli e a i e ac ions in esis an T-lymphoma. Eu . J. Med. Chem. 2020,200, 112435. [C ossRe ] [PubMed] 20. S oll, S.; Schweige , A. EasySpin, a comp ehensi e so wa e package o spec al simula ion and analysis in EPR. J. Magn. Reson. 2006,178, 42–55. [C ossRe ] 21. Nagai, F.; Ushiyama, K.; Kano, I. DNA clea age by me aboli es o bu yla ed hyd oxy oluene. A ch. Toxicol. 1993 ,67, 552–557. [C ossRe ] [PubMed] 22. G man, M.; Misak, A.; Ku ako a, L.; B ezo a, V.; Cacanyio a, S.; Be enyio a, A.; Balis, P.; Tomaso a, L.; Kha ma, A.; Domínguez- Ál a ez, E.; e al. P oduc s o Sul ide/Seleni e In e ac ion Possess An ioxidan P ope ies, Sca enge Supe oxide-De i ed Radicals, Reac wi h DNA, and Modula e Blood P essu e and Tension o Isola ed Tho acic Ao a. Oxid. Med. Cell. Longe . 2019 ,2019, 9847650. [C ossRe ] An ioxidan s 2021,10, 1286 22 o 22 23. Bielski, B.H.J.; Cabelli, D.E.; A udi, R.L.; Ross, A.B. Reac i i y o HO 2 /O 2− Radicals in Aqueous Solu ion. J. Phys. Chem. Re . Da a 1985,14, 1041–1100. [C ossRe ] 24. Galbács, Z.M.; Csányi, L.J. Alkali-induced decomposi ion o hyd ogen pe oxide. J. Chem. Soc. Dal on T ans. 1983 , 2353–2357. [C ossRe ] 25. Csányi, L.J.; Nagy, L.; Galbács, Z.M.; Ho á h, I. Alkali-Induced Gene a ion o Supe oxide and Hyd oxyl Radicals om Aqueous Hyd ogen Pe oxide Solu ion. Z. Phys. Chem. 1983,138, 107–116. [C ossRe ] 26. Dong, D.; Vandeg i , G.F. Alkaline Pe oxide P ocessing o Low-En iched U anium Ta ge s o 99 Mo P oduc ion—Decomposi ion o Hyd ogen Pe oxide. Nucl. Sci. Eng. 1997,126, 213–223. [C ossRe ] 27. Mille , C.M.; Valen ine, R.L. Mechanis ic s udies o su ace ca alyzed H2O2 decomposi ion and con aminan deg ada ion in he p esence o sand. Wa e Res. 1999,33, 2805–2816. [C ossRe ] 28. Lo , S.; Poulsen, H.E. Cance isk and oxida i e DNA damage in man. J. Mol. Med. 1996,74, 297–312. [C ossRe ] 29. Miu a, T. The pe oxidase ac i i y o ADM-Fe 3+ coope a es wi h lipid pe oxida ion: The pa icipa ion o hyd ope oxide and hyd oxyl adicals in he damage o p o eins and DNA. Chem. Biol. In e ac . 2015,236, 67–73. [C ossRe ] [PubMed] 30. Jiang, D.; Rusling, J.F. Oxida ion Chemis y o DNA and p53 Tumo Supp esso Gene. Chemis yOpen 2019 ,8, 252–265. [C ossRe ] [PubMed]