Lag-phase in lipid peroxidation

Transition Metals in Oxidative Stress

19.33 THE EFFECT OF METALS ON THE AUTOXIDATION

OF BENZENETRIOL. Cu CHANGES THE FREE RADICAL PROPAGATED CHAIN REACTION BUT Fe DOES NOT. Luoping Zhang and Allan J. Davison Bioenergetics Research Laboratory, School of Kinesiology, Simon Fraser University, Burnaby, B.C. Canada, V5A 1$6

LAG-PHASE IN L I P I D P E R O X I D A T I O N H e l m w a r d Z o l l n e r , W e r n e r G. S i e m s , and Hermann Esterbauer I n s t i t u t e of B i o c h e m i s t r y , U n i v e r s i t y of G r a z , A - 8 0 1 0 A u s t r i a

1,2,4-Benzenetriol is a redox active benzene metaboiite. It increases frequencies of micronuclei in human lymphocytes and may cause loss of some specific human chromosomes. Its genotoxicity is presumed to result from its ability to activate 0 2 but surprisingly little is known of the mechanisms of its reactions with 0 2 We therefore compared candidate free radical propagators of its reaction with 0 2 when selected metals were (or were not) added to the reaction medium. When metals were omitted from the system, superoxide dismutase inhibited autoxidations and desferrioxamine stimulated. Catalytic amounts of Cu or Fe, accelerated the autoxidations of benzenetriol in a dose dependent manner. Fe (50 xJM) accelerated by about 60%. Cu at 10~uM concentration was 10 times as effective. In the Cucatalyzed reaction, superoxide dismutase neither inhibited nor accelerated, while desferrioxamine abolished Cu catalysis. When Fe was present, superoxide dismutase slowed the reaction, but desferrioxamine did not inhibit. Whether or not metals were added, the autoxidations are completely inhibited when both superoxide dismutase and desferrioxamine are present. Participation of a [benzenetrioI-Cu-O2] ternary complex may preclude superoxide release by allowing synchronous two electron transfer from benzenetriol to O;t via Cu. Apparently [benzenetrioI-Fe] complexes do not permit concerted electron transfer to 0 2 and thus most 0 2 reduction depends on propagation by superoxide, i.e. on one electron transfer. The actions of desferrioxamine are different in the case of Cu and Fe. Thus [Cu-desferrioxamine] has a reduction potential incompatible with redox cycling, whereas desferrioxamine decreases the reduction potential of Fe to a value more appropriate for redox cycling. Different conformation of Cu and Fe complexes of benzenetriol may explain their different sensitivities of their reactions with 0 2 to inhibition by superoxide dismutase.

A d e l a y of l i p i d p e r o x i d a t i o n (LPO) w a s found with different systems. We s t u d i e d L P O in m i c r o s o m e s stimulated with ferrous iron 5-100 uM by following oxygen uptake MDA formation and iron oxidation. Progress of LPO was threephasic. The three phases differed in t h e r a t e of i r o n o x i d a t i o n , o x y g e n u p t a k e a n d M D A f o r m a t i o n . In p h a s e 1 ( lag phase) oxygen uptake, MDA formation and iron oxidation was low or u n d e t e c t a b l e . In p h a s e 2 t h e r a t e of a l l parameters rapidly increased to an a l m o s t l i n e a r r a t e a n d w h e n all i r o n w a s oxidized oxygen uptake and MDA formation s l o w e d d o w n to a b o u t 5 to 10% of t h e m a x i m a l r a t e . A s e c o n d a d d i t i o n of i r o n stimulated LPO immediately. T h e lag w a s i n c r e a s e d b y i n c r e a s i n g t h e Fe +÷ c o n c e n t r a t i o n or the pH , by a d d i t i o n of Sn ÷÷ o r C u ++ a n d d e c r e a s e d b y increasing the protein concentration or c h e l a t o r s . As L P O d e t e r i o r a t e m e m b r a n e integrity and LPO proceeded without a lag a f t e r t h e s e c o n d i r o n a d d i t i o n it was concluded that membrane integrity my b e o n e r e a s o n for t h e lag. T h i s w a s p r o o v e n b y a d d i t i o n of T r i t o n X 100 w h a t c o m p l e t e l y a b o l i s h e d t h e lag.

19.35 POTENTIAL

MARKERS OF FREE RADICAL DAMAGE TO DNA Okezie I. Aruoma, Miral Dizdaroglu and Barry Halliwell Biochemistry Department, University of London King's College, Strand, London WC2R 2LS, UK and NIST, Gaithersburg, Maryland, USA

The fundamental mechanisms of mutagenesis centres around the genetic material, DNA. Identlflcation of products resulting from the attack of radiation-generated hydroxyl radical (.OH) on DNA led several scientists into using measurement of one or more of these products as an index of oxidative attack upon DNA in whole organisms or in isolated cells. Thus thymine and thymidine glycol were measured in human urine (Adelman et al 1988, PNAS, 85, 2706-2708), 8-hydroxyguanine was measured in DNA isolated from cells or from whole mice after irradiation (Kasai et al., 1986, Carcinogenesis, 7, 18491851), and an HPLC-based assay for 8-hydroxy-2'deoxyguanosine was develoPed (Floyd et al, 1986, FRRC, i, 163-172). However the use of gas chromatography-mass spectrometry with selected ion monitoring has enabled the identification and quantitative measurement of multiple products derived from pyrimidines and purines in DNA exposed to .OH generated by the hypoxanthine/ xanthine oxidase/iron EDTA system (Aruoma et al, 1989a, JBC, 264, 13024-13028), Fe(lll)-complexes in the presence of H20 a (Aruoma et al, 1989b, JBC, 264, 20509-20512) and by copper-ion/H20 a systems. The technique of GC/MS/SIM allows definitive studies of the underlying mechanisms of free radical damage to DNA in human diseases.

B A C T E R I O C I D A L A C T I V I T Y OF O R G A N I C P E R O X Y R A D I C A L S I D E N T I F I E D BY T H E SPIN T R A P M E T H O D W I T H ESR Takaaki Akaike, Keizo Sato, Sumiko ljiri, Yoichi Miyamoto, Masahiro Kohno#, and Hiroshi Maeda. Dept. Microbiology, Kumamoto Univ. Med. School, Kumamoto, Japan; #ESR Application Laboratory, Analytical Instruments Division, JEOL, Tokyo, Japan

Several organic peroxides can react readily with various iron chelating or heine conminig substances• During these reactions, generation of alkyl-, alkoxy- or alkyl peroxyradicals has been identified by the ESR techniques. The peroxide derived radicals have been reported to induce hemolysis and tumor promotion. Other biological potentials of the peroxide radicals are, however, still remains to be clarified. We, therefore, examined hacteriocidal activity of these radiacls. Various radical species were prepared in the mixture of t-butyl peroxide, cumene peroxide or methy ethyl ketone(MEK) peroxide with cytochrome c, methemoglobin, metmyoglobin, heroin or DTPA-Fe++ complex in sodium phosphate buffer, pH 7.5. Each radical was identified by the spin trap method with ESR using highly purified 5,5-methyl-l-pyrroline-l-oxide(DMPO). Luminol dependent chemiluminescence with iron-catalyzing redox reaction of peroxides was also measured. Bacteriocidal actions of peroxide radicals were tested using various gram - or + bacteria by colony formation. The signals of redox reactions of organic peroxides with heine and heine proteins were found to be spin adducts of alkoxyl- and alkyl peroxy-radicals, while that of DTPAFe'~ + was alkoxyl-radical. In case of MEK peroxide only alkyl peroxy-radical was generated with heine proteins. Luminol dependent chemiluminescence by redox reaction of peroxides with heine or heine protein was about 100-fold higher than those with DTPA-Fe++ at the same iron concentration. In addition, a strong bacreriocidal action was observed only in the reaction of peroxides with heine proteins, in which alkyl peroxy-radical was identified with ESR explicitly and intense chemiluminescence was observed. Bacteriocidal effect of the DTPA-Fe++ catalyzing reactions was only negligible. Furthemore, these bacteriocidal actions of peroxide radicals were completely inhibited by luminol and DMPO which consume the radicals• Taken ali together, we concluded that alky peroxy-radical has the most potent cytocidal activity in the iron-catalyzing redox reactions of organic peroxides.

185

19.34

19.36