A Familial Amyotrophic Lateral Sclerosis-associated A4V Cu,Zn-Superoxide Dismutase Mutant Has a Lower K for Hydrogen Peroxide

The effect of doxorubicin on oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) by lactoperoxidase and hydrogen peroxide has been investigated. It was found that: (1) oxidation of ABTS to its radical cation (ABTS •+ ) is inhibited by doxorubicin as evidenced by its induction of a lag period, duration of which depends on doxorubicin concentration; (2) the inhibition is due to doxorubicin hydroquinone reducing the ABTS •+ radical (stoichiometry 1: 1.8); (3) concomitant with the ABTS •+ reduction is oxidation of doxorubicin; only when the doxorubicin concentration decreases to a near zero level, net oxidation of ABTS could be detected; (4) oxidation of doxorubicin leads to its degradation to 3-methoxysalicylic acid and 3-methoxyphthalic acid; (5) the efficacy of doxorubicin to quench ABTS •+ is similar to the efficacy of p-hydroquinone, glutathione and Trolox C. These observations support the assertion that under certain conditions doxorubicin can function as an antioxidant. They also suggest that interaction of doxorubicin with oxidants may lead to its oxidative degradation. KeywordsABTS; anthracyclines; doxorubicin; EPR; glutathione; hydroquinone; inactivation; lactoperoxidase; oxidation; Trolox C The biological action of the anticancer anthracyclines doxorubicin (DOX, Adriamycin) and daunorubicin (DNR) is frequently linked to their ability to induce oxidative stress through generation of free radicals and ROS. This property results from the presence in the drugs' structures of a quinone moiety (Fig. 1, ring C), which may undergo metabolic reduction and, via aerobic redox cycling, generate superoxide and, subsequently, other more reactive forms of oxygen [1][2][3]. It is believed that ROS may play a role in the anthracycline-induced cardiotoxicity [4].Several recent studies demonstrated that anthracyclines also possess reducing capabilities since they react with oxidants. This is not surprising, as the drugs contain in their chromophores an electron-donating hydroquinone moiety (Fig. 1, ring B Fax: (513) 558-0852, E-mail: reszkakj@ucmail.uc.edu. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The above reports prompted us to explore further the putative antioxidant properties of anthracyclines. In the present study we investigated the effect of DOX on oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) by LPO and hydrogen peroxide (H 2 O 2 ). ABTS is an excellent substrate for peroxidases [14][15][16] and is frequently used to study antioxidant properties of natural compounds [17][18][19] and electron transfer reactions involving free radicals [2...

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“…ABTS •+ is a persistent radical which can be readily detected and identified by its characteristic EPR spectrum [29,30]. Fig.…”

Section: Epr Studymentioning

confidence: 99%

Doxorubicin inhibits oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) by a lactoperoxidase/H2O2 system by reacting with ABTS-derived radical

Reszka

Britigan²

2007

Archives of Biochemistry and Biophysics

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confidence: 63%

“…In the presence of H202 and radical scavengers, especially with anionic character, both free 9 radicals and scavenger-derived radicals are produced (15, 16). We also found that the free radical-generating function of several FALS-associated Cu,Zn-SOD mutants is enhanced relative to that of the wild-type enzyme, while the dismutation activities remain unchanged (17,18).To monitor the production of free radicals, two spin traps, 5,5-dimethyl-l-pyrroline N-oxide (DMPO) and Ntert-butyl-c~-phenylnitrone (PBN), were used in our studies (15) to convert transient free radicals to stable free radicals adducts according to the following reactions:The nature of the trapped free radical was identified by EPR spectroscopy. When O2-" (dissolved KO 2 in dried dimethylsulfoxide) was added to the NaHCOJCO 2 buffer at pH 7.6 containing 100 mM DMPO, EPR OH adducts.…”

mentioning

confidence: 66%

“…Therefore, the enhanced free radical-generating activity induced by a burst of H202 formation, elevated level of Cu,Zn-SOD, or mutation of the enzyme may exert the deleterious effects in vivo. We demonstrated such an effect in FALS (17,18). Most of the FALS mutants have point-mutation sites in conserved interaction regions critical to the subunit fold and dimer contact, rather than residues in the active-site or in the electrostatic active channel (19)(20).…”

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confidence: 80%

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Pro-oxidant activity of Cu,Zn-superoxide dismutase

Yim

Chock

et al. 1998

AGE

Self Cite

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Whereas Cu,Zn-superoxide dismutase (Cu,Zn-SOD) is an essential antioxidant enzyme for superoxide dismutation in vivo(1 ), many reports have shown that an elevated level of Cu,Zn-SOD induces cell killing (2, 3), increases lipid peroxidation (4,5), and interferes with the transport of neurotransmitters (4, 6). All of these observations are archetypical results inducible by oxygen radicals. Cu,Zn-SOD (7) and FALS mutants (8) are known to develop neurodegeneration. These findings suggest that under certain conditions Cu,Zn-SOD may behave directly or indirectly as a pro-oxidant.In addition to the usual superoxide dismutation activity, Cu,Zn-SOD is known to exhibit anion binding capacity (9, 10), inactivation by its own reaction product H202 (11-13), and the purported peroxidase activity (14). Here we summarize our findings that Cu,Zn-SOD has free radical ('OH and scavenger-derived radicals)-generating activity (15,16). TheactiveCu,Zn-SOD can catalyze the generation of free 9 radicals from H202. In the presence of H202 and radical scavengers, especially with anionic character, both free 9 radicals and scavenger-derived radicals are produced (15, 16). We also found that the free radical-generating function of several FALS-associated Cu,Zn-SOD mutants is enhanced relative to that of the wild-type enzyme, while the dismutation activities remain unchanged (17,18).To monitor the production of free radicals, two spin traps, 5,5-dimethyl-l-pyrroline N-oxide (DMPO) and Ntert-butyl-c~-phenylnitrone (PBN), were used in our studies (15) to convert transient free radicals to stable free radicals adducts according to the following reactions:The nature of the trapped free radical was identified by EPR spectroscopy. When O2-" (dissolved KO 2 in dried dimethylsulfoxide) was added to the NaHCOJCO 2 buffer at pH 7.6 containing 100 mM DMPO, EPR OH adducts. DMPO-OH formation also required active Cu,Zn-SOD, because heat-inactivated Cu,Zn-SOD or Mn-SOD failed to produce these radical adducts. The direct addition of H202 in place of O2-" produced similar results. In the presence of .OH radical scavengers with anionic character such as HCO 2 or N3-, DMPO adducts of the scavenger-derived radicals, DMPO-CO2-" or DMPO-N3", were observed in addition to DMPO-OH. The concentration ratio between DMPO-OH and DMPO-scavenger-derived radical adducts observed in these experiments reflects competition reactions between DMPO and scavengers for free "OH radicals. Surprisingly, with neutral'OH radical scavengers, such as ethanol, the formation of DMPO-hydroxyethyl radical adducts was insignificant and the concentration of DMPO-OH formed was unaffected. In contrast, with a different spin trap, PBN, in place of DMPO, ethanol yields PBNhydroxyethyl radical adducts as expected in the competition reaction for free "OH radicals. These seemingly contradicting results may be caused by the different affinities of these spin traps for the positively charged active channel leading to the active site of Cu,Zn-SOD, where the'OH radicals are generated. To prove this hypo...

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“…It has been reported that the FALS mutants, G93A and A4V, exhibits an enhanced free radicalgenerating activity, while its dismutation activity is identical to that of the wild-type enzyme [12][13][14]. Previous results showed that the hydroxyl radical was generated in the reaction of human Cu,Zn-SOD with H202 [ 15].…”

Section: Resultsmentioning

confidence: 99%

The free radical‐generating function of a familial amyotrophic lateral sclerosis‐associated D90A Cu,Zn‐superoxide dismutase mutant

Kim¹,

Eum²,

Kwon³

et al. 1998

IUBMB Life

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SUMMARYThe free radical-generating functions of the D90A Cu,Zn-superoxide dismutase (SOD) associated with Swedish familial amyotrophic lateral sclerosis (FALS) patients are investigated. The results show that both the wild-type and mutant enzymes have identical dismutase activity, while the free radical-generating activity of the D90A mutant is enhanced relative to that of the wild-type enzyme. The studies suggest that the active channel of the D90A mutant is larger than that of the wild-type enzyme. A higher free radical-generating activity of the mutant enzyme led to the release of copper ions from the damaged protein. The generation of strand breaks in plasmid DNA was enhanced more effectively by the D90A mutant Cu,Zn-SOD than by the wildtype enzyme. The results suggest that the pathology of FALS may be attributed to oxidative damage caused by the gain-of-function of FALS Cu,Zn-SOD mutant.Key words: copper,zin-superoxide dismutase, mutant, hydroxyl radical, copper. 1191All rights of reproduction in any form reserved, Vol. 46, No. 6, 1998 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONALAndersen et al. [7] reported a Cu,Zn-SOD exon 4 mutation (D90A) with novel features in Swedish FALS patients and the disease has only been observed in patients homozygous for this mutation, whereas heterozygotes remained unaffected. Furthermore, there was essentially no reduction in erythrocyte Cu,Zn-SOD dismutation activity. Thus, [7] suggested that this Cu,Zn-SOD mutation caused ALS by a gain-of-function rather than by the loss of dismutation activity.In view of these considerations, the present work represents an investigation of the free radical-generating functions of the D90A mutant and the oxidative damage to DNA, as a first attempt to establish the extent of any correlation between the gain-of-function and the pathology of FALS. MATERIALS AND METHODS Expression and purification of wild-type and mutated human Cu, Zn-SOD

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A Familial Amyotrophic Lateral Sclerosis-associated A4V Cu,Zn-Superoxide Dismutase Mutant Has a Lower K for Hydrogen Peroxide

Cited by 157 publications

References 22 publications

Doxorubicin inhibits oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) by a lactoperoxidase/H2O2 system by reacting with ABTS-derived radical

Doxorubicin inhibits oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) by a lactoperoxidase/H2O2 system by reacting with ABTS-derived radical

Pro-oxidant activity of Cu,Zn-superoxide dismutase

The free radical‐generating function of a familial amyotrophic lateral sclerosis‐associated D90A Cu,Zn‐superoxide dismutase mutant

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