Prevention of singlet oxygen-induced DNA damage by lipoate

Chem.-Biol. Interactions, 86 (1993)79-92 Elsevier Scientific Publishers Ireland Ltd.

79

PREVENTION OF SINGLET OXYGEN-INDUCED DNA DAMAGE BY LIPOATE

T H O M A S P.A. D E V A S A G A Y A M a'b,M A Y I L V A H A N A N D I N K A R S. P R A D H A N b and H E L M U T SIES a

SUBRAMANIAN

b,

aInstitut J~r Physiologische Chemic L Universitdt Diisseldarf, Moorenstrafle 5, W-4000 Diisseldarf (Germany) and bBiochem~stry Division, Bhabha Atomic Research Center, Bombay 400 085 (India) (Received January 15th, 1992) (Revision receivedNovember 20th, 1992) (Accepted November 26th, 1992)

SUMMARY

Among the several biologically and pharmacologically active sulfur compounds examined, only lipoic acid and dihydrolipoic acid provided protection to plasmid DNA against singlet molecular oxygen (102). 102 was generated in phosphate buffer by the thermal dissociation of the endoperoxide of 3,3'-(1,4-naphthylidene) dipropionate (NDP02). The protecting effect of lipoic acid was time- and pH-dependent and significant protection was seen even at 50 ~M. The antioxidant effect was adversely affected by temperatures above 45°C. Superoxide dismutase and catalase marginally enhanced this effect. Metal chelation with EDTA decreased the protection by lipoate, indicating that metal ions my be involved. The protective effect was diminished when the disulfide was added after single-strand breaks were induced by 102. The formation of 8-oxoguanosine from guanosine upon exposure to NDP02 was not altered by lipoate. Key words: Singlet molecular oxygen; Lipoate; 3,3'-(1,4-naphthylidene) dipropionate; Single-strand breaks; Plasmid pBR322 INTRODUCTION

Singlet molecular oxygen (102) is an electronically excited species of oxygen produced under various pathophysiological conditions in mammalian tissues. It Correspondence to: Prof. Dr. Helmut Sies, Institut ffir Physiologische Chemie I, Universit~t Dfisseldorf, Moorenstrat{e 5, W-4000 Dfisseldorf, Germany. Abbreviations: EDTA, ethylene diamine tetra-acetic acid; EGTA, ethylene glycol bis (betaaminoethylether) N,N,N' ~'-tetraacetic acid; NDP, 3,3'-(1,4-naphthylidene) dipropionate; NDPO2, endoperoxide of 3,3'-(1,4-naphthylidene) dipropionate; pD, refers to pH of deuterated buffer; 102, singlet molecular oxygen.

0009-2797/93/$06.00

80

has attracted considerable attention in the recent years due to its possible involvement in tissue damage in different disease conditions and as a major cytotoxic agent in the photodynamic therapy of malignant tumors [1-4]. In biological systems it may be generated by dark reactions due to chemi-excitation which include enzymatic reactions and free radical reactions and by light reactions involving photoexcitation [3,5,6]. Due to its relatively long half-life, in the range of 10-50 #s, 102 is capable of travelling an appreciable distance in the cellular environment and damaging crucial biological molecules like lipids, proteins and DNA [2,3,7]. In DNA 102 causes single-strand breaks resulting in the loss of biological activity [8 - 13]. Our earlier studies [14] as well as those of others [15 - 17] have shown that 102 is capable of damaging DNA bases resulting in the formation of 8-hydroxydeoxyguanosine (8-oxodG). This finding assumes significance since DNA residues containing 8-oxodG causes misreading of the DNA template [18,19] resulting in possible mutagenicity and carcinogenicity. Moreover, 8oxodG is an indicator of in vivo DNA damage in mammals [20-22]. 102 was shown to cause mutagenicity in bacteria and mammalian shuttle vectors [23,24]. The possible mutagenic and genotoxic properties of singlet oxygen have been recently reviewed [12,13,25]. There is an interest in biomolecules which can prevent 102-induced DNA damage. Water-soluble antioxidants such as glutathione and dithiothreitol protect DNA against other reactive oxygen species such as the hydroxyl radical ('OH) or hydrogen peroxide (H202) generated during radiation [26]. These same antioxidants, however, enhance the DNA strand-breaks induced by 102 [11]. In contrast, lipoate protects DNA. The present study examines factors influencing the protection of DNA by lipoate against singlet oxygen and the possible mechanisms involved. MATERIALS AND METHODS

Chemicals Deuterium oxide (99.8 atom%), N-acetylcysteine, cysteine, cystine, dimethylcysteine, mercaptopropionylglycine, dithiothreitol, dithioerythritol, glutathione, superoxide dismutase, catalase, agarose and mannitol were from Sigma Chemical Co. (Deisenhofen, Germany). Mesna (sodium 2-mercaptoethane sulfonate), dimesna, racemic lipoate, R-lipoate, S-lipoate and dihydrolipoate were gifts from Asta Medica (Frankfurt, Germany). The compound S-2-(3-aminopropylamino) ethylphosphoorthioic acid was a gift from Dr. A. Nagele of the Institut ffir Klinische Hamatologie, GSF (Munich, Germany). All other chemicals used were of analytical grade from Merck (Darmstadt, Germany). The endoperoxide of the disodium salt of NDP was prepared as described [27]. The product NDP02, was identified by 1H nuclear magnetic resonance (NMR) and IR spectroscopy. Absorption measurements were performed with an LKB spectrophotometer (model Ultrospec 4050). Stock solutions were kept at -70°C until use. Singlet oxygen was generated by the thermal dissociation of NDPO2 as

81 described [27]. NDPO2 on thermal dissociation yields 3,3'-(1,4-naphthylidene) dipropionate (NDP) and molecular oxygen. One-half of the oxygen liberated is in the triplet state, the other half being in the excited singlet state. Plasmid pBR322 DNA was prepared utilising Escherichia coli cells and the Qi a gen Plasmid kit-Pack 500 (Diagen, Dfisseldorf, Germany). The yield was 200- 240 ~g per batch and the preparation contained 93-100% Form I (supercoiled form) and 0 - 7 % Form II (open circular form). To study the effect of lipoate or other antioxidants, 2 - 3 ~g pBR322 DNA was incubated with 40 mM NDP02 and 10 mM freshly prepared antioxidant in deaerated 50 mM sodium phosphate buffer (pD 7.4), in D20 at 37°C with shaking. pD was taken as the pH measured with a glass electrode plus 0.4 pH units [28]. To study the effect of inhibitors, sodium azide (10 mM) or superoxide dismutase (60 U/assay) or catalase (20 U/assay) or mannitol (100 mM) were present. The effect of metal chelation was studied by including ethylene diamine tetraacetic acid (EDTA) (10 mM) or ethylene glycol-bis(beta-aminoethyl ether) N 2 / ~ ' ~'-tetraacetic acid (EGTA) (10 mM). Separation of the different forms of DNA, Form I (native supercoiled form) and Form II (open circular form resulting from single-strand breaks) was performed using 0.7% agarose gels in 89 mM Tris-borate/2 mM EDTA buffer (pH 8.3) [10]. DNA bands were stained with ethidium bromide and evaluated by scanning reverse photonegatives with Shimadzu model CD-9000 densitometer. To examine the effect of lipoate on the formation of 8-oxoguanosine, the reaction mixture contained 1 mM guanosine in 50 mM sodium phosphate buffer in D20 (pD 7.4), at 37°C. The reaction was started by adding NDP02 at a final concentration of 40 mM and stopped by adding 10 mM sodium azide. The 8-oxoG formed was identified and quantitated by use of high pressure liquid chromatography as described previously [14]. RESULTS

Singlet molecular oxygen, generated by the thermal dissociation NDP02, induced a significant amount of single-strand breaks, as evidenced by the increase in the percentage of open circular form of pBR322 (Table I). Among the various thiols and disulfides of biological and pharmacological interest examined in this study, only lipoate and dihydrolipoate gave significant protection against 102induced single-strand breaks. The protection by lipoate was more pronounced than that by dihydrolipoate. No significant differences were observed between the two lipoate stereoisomers. Most of the other sulfur compounds examined, including glutathione, mesna, captopril, WR-2721 and dithiothreitol, enhanced the strand-breaking activity of 10 2. Dimesna and glutathione disulfide showed much less enhancing effect than their respective thiols. The effects of 102 or 102 plus lipoate on single-strand breaks in plasmid DNA as a function of time are shown in Fig. la and b. Protection by lipoate was observed as early as 10 min and continued up to 90 min. Lipoate seems to affect both the rate of formation of single-strand breaks as well as the number of breaks observed at the end of incubation time. Fig. 2 shows the concentration

82 TABLE I ENHANCING OR PROTECTING EFFECT OF SOME BIOLOGICALLY AND PHARMACOLOGICALLY ACTIVE THIOL-DISULFIDES (10 mM) ON THE SINGLET OXYGENINDUCED SINGLE-STRAND BREAKS IN pBR322 DNA Compound

Mesna Dimesna Captopril Glutathione Glutathione disulfide WR-2721 L-Dithiothreitol Ergothioneine Dihydrolipoate rac-Lipoate R-Lipoate S-Lipoate

Strand breaks (% increase in Form II)

(A) Without NDP02

(B) With NDPOz

(C) Enhancing (+) or Protecting ( - ) effect over NDPO2

0.2 0.0 1.4 9.2 1.1 2.7 52.8 3.9 3.9 0.0 0.0 0.0

90.0 40.1 66.5 67.9 39.1 54.8 c.d. 44.7 18.9 13.4 14.9 14.7

+ + + + + + + + -

± 0.1 ± ± ± ± ± ± ±

0.2 1.8 0.2 0.4 3.8 0.4 0.6

± ± ± ± ± ±

4.9 1.8 4.3 4.2 2.8 5.3

± ± ± ± ±

6.2 1.3 1.0 1.6 2.1

51.9 2.2 27.2 20.8 0.1 14.2 9.3 2.9 15.1 24.5 23.0 23.2

Values (mean ± S.E., n = 4) are given as percent increase in open circular form (Form II) over pBR322 control. The control values of Form II were pBR322, 5.2 ± 0.8, NDP control, 5.4 ± 0.3. Value with NDPOz over pBR control was 37.9 ± 3.6. The enhancing or protecting effect (Column C) is percent increase in strand breaks above that observed with NDP02 (Column B minus (Column A + 37.9). All other thiols or disulfides of biological or commercial importance studied (25 in number) enhance the singlet oxygen induced DNA damage by 1 to 100% (complete breakdown of DNA). c.d. -- complete damage to DNA.

dependence of protection by lipoate. There was a steep increase in the protection afforded between the concentrations of I and 25 mM. At the latter concentration there was almost 90% protection. Lipoate addition gives protection to DNA only if added within the first 10 min of incubation (Fig. 3). This corresponds to the period of maximum increase in single-strand break formation due to 102, as is seen in Fig. 1. Delay in the addition significantly diminished the ability to protect DNA against 10 z. Temperature is another factor influencing the protection by lipoate (Fig. 4). Increasing the temperature above 40°C diminished the ant±oxidant ability, pD of the incubation medium used also influenced the protective effect of lipoate (Fig. 5). The protection, as measured by decrease in open circular form upon exposure to NDPO2, varied from 19.6% at pD 6.0 to 32.5% at pD 6.8. The maximum protection by lipoate, whose pKa is 5.4, is around neutral pH. Table II shows the effect of quenchers of reactive oxygen species on 10z- or lO z plus lipoate-induced DNA damage. Superoxide dismutase (for O~-), cata-

83

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Fig. 1. (a) Agarose gel electrophoretic separation of pBR322 DNA exposed to NDPO 2 or NDPO 2 plus lipoate as a function of time. Conditions same as Table I. (b) Effect of lipoate on singlet oxygen induced strand breaks in plasmid pBR322 DNA as a function of time.

lase (H202) and mannitol (for " OH) did not significantly alter singlet oxygeninduced DNA damage, excluding the possibility that the strand-breaks are due to these reactive species. Sodium azide, a compound reacting with 102, gave significant protection. Protection by lipoate was only marginally enhanced by superoxide dismutase and catalase and not affected by other inhibitors studied. Data presented in Fig. 6 show that EDTA had little effect on the damage caused by 102 but diminished the protection afforded by lipoate, pointing to the possibility of the involvement of metal ions in the antioxidant activity. EGTA,

84

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Fig. 2. Concentration dependent protection of singlet oxygen induced single-strand breaks in pBR322 DNA by lipoate. Conditions same as Table I.

a preferential chelator of calcium ions, on the other hand, had little effect on the ability of lipoate to protect DNA against 102. The effect of lipoate on 102 plus glutathione-induced single-strand breaks is shown in Fig. 7. At equimolar concentrations (10 mM each), lipoate completely prevented the enhancement in DNA damage due to glutathione, the major biological thiol in mammalian tissues. Data presented in Table III show that glutathione enhances the 102-induced formation of 8-oxoG from guanosine, whereas lipoate had no significant effect. These results indicate that the protection by lipoate may not be effected through the inhibition of the formation of this DNA damage product. DISCUSSION

Our earlier results [10,11,24] as well as those of others [9] have established that 102 is capable of inducing single-strand breaks. One of these studies [10] has used 102 generated by different methods to test the ability of this reactive species to induce strand-breaks. In a recent review on the biological consequences of DNA oxidation mediated by 102, Piette [12] has stated that the ques-

85

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Fig. 3. Ability of lipoate added after NDP02 to protect plasmid DNA against singlet oxygen induced single-strand breaks. Values are means ± S.E. from three independent experiments. The total time of incubation for NDP02 was 90 rain. Protection obtained when lipoate was added at 0 min was taken as 100%.

tion of the ability of 102 to induce strand-breaks in DNA is not fully resolved. One of the arguments put forward is that Nieuwint et al. [29] who used the same method of generation of 102 as Di Mascio et al. [10] and Devasagayam et al. [11], namely thermal dissociation of NDPO2, did not observe significant increase in strand-breaks. This discrepancy could be explained by the fact that the DNA samples of Nieuwint et al. [29] contained a high amount of open-circular form even before incubation with NDPO2. The other item raised by Piette [12] was that in these studies the participation of superoxide in the formation of NDPO2-induced strand-breaks was not ruled out. Present results (see Table II) as well as that in an earlier report [11] show that superoxide dismutase did not alter the strand-breaking activity of NDPO2-generated 102. Hence, it is reasonable to conclude that the single-strand breaks observed were due to 102 . Sulhtr-containing compounds such as thiols, disulfides and related substances are known to fulfill fundamental biological functions. Aminothiols and their derivatives exhibit a pronounced ability to protect cells against oxidative dam-

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Fig. 4. Temperature dependence of the ability of lipoate to protect plasmid pBR322 DNA against singlet oxygen induced single-strand breaks. Incubation conditions were as described in the legend to Table I. Values are means ± S.E. from 3 or 4 experiments.

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87 TABLE II EFFECT OF SCAVENGERS OF REACTIVE OXYGEN SPECIES ON THE ABILITY OF LIPOATE TO PROTECT AGAINST SINGLET OXYGEN INDUCED DNA DAMAGE, AS STUDIED BY INCREASE IN STRAND BREAKS IN PLASMID pBR322 DNA Treatment over NDPO 2

Percent open circular form

Increase (+) or decrease ( - ) over NDP02

None Lipoate Superoxide dismutase (SOD) SOD + lipoate Catalase Catalase + lipoate Azide Azide + lipoate Mannitol Mannitol + lipoate

34.7 ± 4.7 13.2 ± 1.2 32.8 ± 2.9

--21.5 -1,9

11.4 33.3 10.5 10.1 14.9 32.6 17.0

-23.3 -1.4 -24.2 -24.6 -19.8 -2.1 -17.7

± ± ± ± ± ± ±

2.2 2.2 0.4 2.0 1.0 1.1 1.8

Increase (+) or decrease ( - ) over NDP02 + lipoate

-2.2 -2.7 +1.7 +3.8

Data are means ± S.E. of four independent observations. Incubations were carried out as described in the legends to Table I. The concentrations of the scavengers used were: superoxide dismutase, 60 U/assay; catalase, 20 U/assay; azide, 10 raM; and mannitol, 100 mM.

age, ionizing radiation and xenobiotics [26,30,31]. Thiols can act as free radical scavengers and, in addition, can quench singlet molecular oxygen [32- 34]. The compounds used in the present study are biologically or pharmacologically active. Mesna is used as a mucolytic agent and captopril as an antihypertensive drug. WR-2721 is a potential radioprotector in cancer therapy and glutathione is a biologically abundant antioxidant. Most of these sulfur-compounds enhanced the 102-induced DNA damage. This finding is in contrast to the normally found and well-documented protective role of thiols. It is worth mentioning here that the thiols dithiothreitol, cysteine, cysteamine and glutathione have enhancing effects on the 102-induced formation of 8-oxoG from guanosine [14]. Such 8-oxo derivatives in DNA are believed to be mutagenic. One reason why only lipoate gives protection is that compared to other sulfur compounds it has a relatively high 102 quenching constant (kq + k r = 140 × 10 6 M-is -1) [35,36]. An explanation for the protection by lipoate may be the unique chemical nature of lipoate among the biological sulfur-compounds. The disulfides with a cyclic five-membered ring, exemplified by lipoate, show significantly different features compared to their open-chain analogues. The biological action of lipoate as a coenzyme is based on redox reactions of the disulfide [37]. In five-membered-ring structures the S--S bond has been found to be subject to a much faster reductive and/or nucleophilic attack than in the open-chain derivatives [38]. CO2 =, for example, readily reduces lipoic acid to the lip(S" --S)- three-electron bonded radical anion [39], while such a reaction has not been observed for cystamine, cystine

88

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Fig. 6. Effect of metal ion chelation on the ability of lipoate to protect against 102-induced singlestrand breaks in plasmid pBR322 DNA. Data are means + S.E. of 4 observations. Incubations were carried out as described in the legend to Table I, EDTA and EGTA were included at the concentration of 10 mM.

or glutathione disulfide [40,411. Though the above-mentioned disulfides did not protect plasmid DNA against 102, lipoate protected it [10]. The main reason for this general difference in behavior may be a smaller torsional angle in the cyclic structures which results in a comparatively higher electron density of the disulfide bridge. Experimentally this has been corroborated by photoionization and chemical experiments which show that lipoic acid and similar five-membered ring systems are much easier to oxidize than the non-cyclic disulfides [37]. Our present results show that protection by lipoate requires optimum temperature, pD and certain metal ions. It is also known that metal ions such as Cu 2+ by themselves induce strand-breaks [42]. Our study indicates that certain other metal ions, which can be chelated by EDTA, are essential for protection by lipoate. The protection by lipoate is effected only if the antioxidant was added before the occurrence of strand-breaks, indicating that the observed effect is

89

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TABLE III EFFECT OF LIPOATE AND GLUTATHIONE ON SINGLET OXYGEN-INDUCED FORMATION OF 8-OXOGUANOSINE FROM GUANOSINE Additions

8-oxoguanosine (~M)

Change as compared to NDPO 2

None NDP NDPO 2 Glutathione NDP02+ glutathione Lipoate NDPO 2 + Lipoate

0.0 0.0 15.2 + 1.3 0.0 60.7 ± 4.5 0.0 14.9 ± 1.3

---+45.5 (300%) -- 0 . 3 (3%)

The incubation medium contained 1 mM guanosine, 40 mM NDP02 and the respective thiols (10 mM) in 50 mM phosphate buffer in D20 (pD 7.4). Incubation was for 1 h at 37°C. Values are means ± S.E. from four observations.

90

preventive rather than through repair of damaged DNA. The protective effect of this disulfide is also not mediated through its influence on the formation of DNA damage products like 8-oxodG. It is likely that lipoate protects DNA by interfering in an early event in the sequence of 102-induced oxidation of DNA which leads to strand-breaks. Our study also points out that lipoate can protect DNA against 102 plus glutathione-induced strand breaks. This may be due to the fact that lipoate can act much faster against reductive and/or nucleophilic attack than other open chain disulfides such as glutathione, so that it may be able to scavenge/quench the reactive species, which, if allowed to react with glutathione, can lead to enhanced DNA damage. The biological significance of lipoate or dihydrolipoate mediated protection against 102 may be more prominent in the mitochondria where lipoate containing ketoacid dehydrogenases are located. Mitochondrial DNA is particularly exposed to damaging agents and has a higher levels of damage products than nuclear DNA [43]. Lipoic acid (-- thioctic acid) is one among the central coenzymes of the three dehydrogenase complexes in mitochondria. It functions as a cofactor for the pyruvate dehydrogenase complex, covalently bound to its dihydrolipoamide acetyltransferase component. Lipoate is pharmacologically employed in diverse disease conditions such as diabetes mellitus, myocardial infarction, ischemiareperfusion injury, heavy-metal poisoning, amanita poisoning, radiation damage, epilepsy, AIDS, photodamage, inflammation and ageing [44,45]. Evidence implicating reactive oxygen species in some disease states are accumulating and anti-radical interventions using biological antioxidants are being evaluated. There are protective effects of dihydrolipoate on microsomal lipid peroxidation [46-48]. Lipoate has been shown to quench singlet oxygen as well as inhibit photoperoxidation [49]. ACKNOWLEDGEMENTS

Part of the work was supported by the National Foundation for Cancer Research, Bethesda, USA. Help in densitometric tracings by Dr. W. Altekar and Miss V. Rangaswamy is appreciated. REFERENCES 1 2 3 4

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