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Sensitivity of mitochondrial transcription to different free radical species
Free Radical Biology & Medicine, Vol. 16, No. 3, pp. 323-329, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0891-5849/94 $6.00 + .00
Pergamon
0891-5849(94)E0004-3
Original Contribution SENSITIVITY
OF MITOCHONDRIAL TRANSCRIPTION FREE RADICAL SPECIES
TO
DIFFERENT
BRUCE S. KRISTAL, JUANJUAN CHEN, and BYUNG PAL Y u Department of Physiology,The University of Texas Health ScienceCenter, San Antonio, TX, USA (Received 12 April 1993; Revised 30 June 1993;Accepted 21 July 1993)
Abstract--Proper mitochondrial function requires the continual maintenance of the integrity of the mitochondrial gene expression system. The sensitivity of both mitochondrial lipids and mitochondriai respiration to free radicals has been well recognized, but the effect of free radicals and lipid peroxidation on mitochondrial transcription has not yet been examined. Using an in vitro mitochondrial transcription assay, we tested the ability of fiveprooxidants to affectmitochondrial transcription. Results show that mitoehondrial transcription is extremelysensitiveto inhibition by peroxylradicalsgeneratedby either 2,2'-azobis-(2-amidinopropane) (AAPH) or 2,2'-azobis-(2,4-dimethylvaleronitrile)(AMVN), and that this inhibition occurs prior to detectableevidenceof lipid peroxidationas measuredby thiobarbituric acid (TBA)-reactivesubstances, 4-hydroxynonenai accumulation, and oxygenconsumption. Furthermore, although mitochondrial transcription was sensitiveto 4-hydroxynonenal, it was resistant to malondiaidehydeas wellas high levelsof lipid peroxidationinduced by ADP/Fe/NADPH. Together, these results suggestthat the repressionof mitochondrialtranscription is differentiallysensitiveto specificmodes of free radical reaction. Keywords--Mitochondria, Transcription, Free radicals, AAPH, AMVN, Lipid peroxidation, 4-Hydroxynonenal, Malondialdehyde
(MDA) and the more reactive 4-hydroxynonenal (HNE). 7 It has been shown that peroxidizing lipids and the byproducts o f lipid peroxidation can act directly to biochemically alter and functionally damage DNA. s-~6 Furthermore, in vitro induction o f lipid peroxidation on intact mitochondria can alter the electrophoretic mobility o f m t D N A (refs. 17,18), and increase levels o f the oxidized base 8-hydroxyguanosineJ 9 These results suggest that lipid peroxidation products might mediate oxidative damage to mtDNA, but provide no evidence directly addressing whether free radicals/lipid peroxidation can inhibit mitochondrial transcription, or whether lipid peroxidation products can act as mediators o f oxidant-induced repression o f mitochondrial function. Our study addressed these questions using four independent and chemically distinct challenges that were assessed using a modified version o f the in vitro mitochondrial transcription system developed by Gaines and Attardi. 2° Our results demonstrated that free radicals can inhibit mitochondrial transcription and that mitochondrial transcription and mitochondrial lipid peroxidation may display differential sensitivity to specific free radical species.
INTRODUCTION Mitochondrial impairment resulting from oxidantinduced damage has been proposed to play a critical, possibly causative role in both the aging process and m a n y age-related diseases. 1-6 Proper mitochondrial function requires continual maintenance o f the integrity o f m a n y systems, including those involved in mitochondrial gene expression. However, the proximity o f the inner mitochondrial m e m b r a n e to the relatively unprotected mitochondrial genome, and the accessory proteins required for the transcription process, suggest that mitochondrial transcription could be sensitive to inhibition induced by either free radicals, by the products o f lipid peroxidation, or by both. Lipid peroxidation occurs as a chain reaction in which polyunsaturated fatty acids undergo a series o f oxidation and cleavage reactions that yield several kinds o f breakdown products, including lipid radicals and aldehydic metabolites such as malondialdehyde Address correspondenceto: ByungPal Yu, Department of Physiology, The Universityof Texas Health ScienceCenter, 7703 Floyd Curl Drive, San Antonio, TX 78284-7756, USA. 323
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Fig. 1. (A) This figure shows the gel used to obtain the values graphed in Figures IB-ID. Lane 1-4: AMVN 75, 150, 300, 600 #M; Lane 5-9: AAPH 2, 4, 8, 16, 32 raM; Lane 10-14: HNE 20, 40, 80, 160, 320 uM; Lane 15-16: ADP/Fe/NADPH (l.7/0.1/0.1 mM); Lane 17:Control (untreated). By comparison with the data in Gaines and Attardi,2°and Fernandez-Silvaet al.,26Band A contains the mitochondrial RNA encoding ND5, the bands at B contain the species encoding ND4, CoI, and the 16S RNA. The diffuse series of bands at C include cyt b, ND2, NDI, Colll, A6/8, Coll, and the 12S RNA. Band D appears to be the 7s RNA. The rRNAs are observable due to the use of 1 mM ATP (ref. 20). B-D: Dose response curves for AAPH, AMVN, and HNE. Following transcription reaction and RNA purification as described, samples were electrophoresed through sequencing thickness, gradient 3.5-4.5% polyacrylamidegels (l x TBE, 60W, 1400V, 6-8 h). Radioactivity present in bands C-D (that contain most major processed mitochondrial transcripts) were quantitated on a Molecular Dynamics Phospho-Imager and expressed as a percentage of an untreated sample. As mitochondrial transcription utilizes only a single, bidirectional promotor and yields two primary transcripts that are processed to yield individual messages, no individual analysis of transcripts was done. This rationale is supported by longer gel runs that suggest there is no consistent message to message variability following oxidant challenge for the longer messages. Data shown in panels B-D is from the single, representative experiment shown in Figure IA, and is expressed as a percentage of total activity present in untreated controls. (B) Treatment with AAPH: samples were pretreated with 2, 4, 8, 16, and 32 mM AAPH, respectively. (C) Treatment with AMVN: samples were pretreated with 80, 160, 320, and 640 uM AMVN, respectively. (D) Treatment with HNE: samples were pretreated with 20, 40, 80, 160, and 320 uM HNE, respectively.
MATERIALS AND METHODS
Animals M a l e F i s c h e r 344 rats were o b t a i n e d f r o m C h a r l e s R i v e r ( W i l m i n g t o n , M A ) at 90 d a y s o f age. A n i m a l s were m a i n t a i n e d for 2 weeks in o u r u n i v e r s i t y l a b o r a t o r y care facility p r i o r to sacrifice b y d e c a p i t a t i o n . A f t e r sacrifice, livers were i m m e d i a t e l y r e m o v e d , frozen in l i q u i d n i t r o g e n (in 20 m M H e p e s (7.4) w i t h 140 m M KC1), a n d s t o r e d at - 7 0 ° C .
Mitochondrial transcription M i t o c h o n d r i a were isolated b y s t a n d a r d differential c e n t r i f u g a t i o n t e c h n i q u e s , q u i c k frozen in l i q u i d
A
nitrogen, a n d s t o r e d at - 7 0 ° C u n t i l n e e d e d . M i t o c h o n d r i a l t r a n s c r i p t i o n was p e r f o r m e d essentially as b y G a i n e s a n d A t t a r d i , 2° e x c e p t for t w o m o d i f i c a tions: t h e t r a n s c r i p t i o n buffer was u s e d at 1.3x c o n c e n t r a t i o n s a n d s u p p l e m e n t e d with 100 m M KC1 a n d 1 m M a d e n o s i n e t r i p h o s p h a t e (ATP), (which a l l o w e d m i t o c h o n d r i a l r R N A t r a n s c r i p t i o n , as s h o w n b y G a i n e s a n d A t t a r d i ) , a n d t h e assay u s e d frozen m i t o c h o n d r i a . T h e use o f frozen m i t o c h o n d r i a h a d n o significant effect e i t h e r o n t o t a l t r a n s c r i p t i o n a l a c t i v i t y or on oxidant induced repression of transcription (tested o n A A P H only, d a t a n o t shown). T o e x a m i n e t h e effects o f p r o o x i d a n t s o n m i t o c h o n d r i a l t r a n s c r i p tion, 5 m g m i t o c h o n d r i a were p r e i n c u b a t e d in t h e p r e s e n c e o f the o x i d a n t for 15 r a i n at 3 7 ° C in a t o t a l
325
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volume of 1 ml, unreacted oxidant removed by centrifugation, and the mitochondria incubated for 30 min in the transcription buffer. Labeled RNA was isolated, separated by PAGE, and gels were analyzed using a Molecular Dynamics Phospho-Imager. The existence of a micrococcal nuclease digestion step insures that that the RNA being purified was transcribed in intact mitochondria.
Quantitation of liNE by HPLC The method of isolation and quantitation of HNE from mitochondria was essentially the same as described by Esterbauer and Cheeseman. 2~ Oxidation was accomplished identically to that done in the transcription assays, and the sample was then diluted to 20 ml. This 20 ml mitochondrial suspension was combined with 20 ul of 1% ethanolic butylated hydroxytoluene (BHT) and 200 ul of 1% aqueous desferal solution to prevent formation of additional HNE during the following solid-phase extraction procedure. The mitochondrial suspension was poured onto an Extrelut column and twice extracted with 20 ml of dichloromethane. The eluate was collected in a flask containing 2 ml of 0.1M acetate buffer, pH 3. After
the dichloromethane was removed under nitrogen gassing, the aqueous phase was applied quantitatively to an octadecylsilyl (ODS) extraction column preconditioned with 3 ml of methanol and equilibrated with 3 ml of water. The nonpolar material, which interferes with HNE analysis, was removed by eluting the column with 2 ml of hexane. HNE was eluted with 2 ml of methanol/water (80:20) into a 2 ml volumetric tube. Isocratic high performance liquid chromatography (HPLC) analysis was performed with a Waters HPLC system using a Spherisorb $ 5 0 D S 2 column with acetonitrile/water (50/50) as eluant, detector wavelength set at 220 nm, and a flow rate of 1 ml/ rain. Peak identification and quantitation was done relative to reference chromotograms of standard HNE solutions.
Determination of MDA MDA was determined in an 1 ml aliquot taken from the same mitochondrial suspension used for HNE quantitation. The method used was a modification of the TBA assay reported by Buege et al.22 Briefly, 1 ml of the mitochondrial suspension was combined with 10 ul of 2% BHT and 2 ml TBA solu-
B.S. KRISTALel aL
326
tion (15% trichoroacetic acid; 0.375% thiobarbituric acid; 0.25 N hydrochloric acid). The mixture was heated for 15 rain in a boiling water bath. After cooling, the mixture was centrifuged at 1000 g for l0 min. The absorbance of the supernatant was read at 535nm, and the MDA concentration was determined relative to the reference absorbance of an MDA standard.
Data analysis Data was analyzed using the Macintosh statistical package SuperAnova. P values of 0.05 and 0.01 were assessed by Duncan's New Multiple Range Test. If a value was found significant at p < 0.01 by this test, it was then examined by Fisher's Protected LSD to examine p < 0.001 and p < 0.0001.
Determination of lipid peroxidation by oxygen consumption Lipid peroxidation was determined by measuring the oxygen consumption by mitochondria upon addition of prooxidants, according to the method of Estabrook et al.23 and Yu et al. 24 A Gilson 5/6 oxygraph connected to a circulating water bath was used. Oxygen uptake was measured polarographically with a Clark-type oxygen electrode at 37°C. Mitochondrial suspensions (in 140mM KC1, 20mM Hepes, pH 7.4) were added into the reaction chamber. Oxygen uptake was initiated with the addition of the prooxidants, which induce lipid peroxidation in the mitochondrial membrane. RESULTS AND DISCUSSION Rat liver mitochondria were preincubated with varying concentrations of two well-defined free radical generators, AAPH (2,2'-azobis-(2-amidinopropane) hydrochloride) and AMVN (2,2'-azobis-(2,4-dimethylvaleronitrile)), which decompose at a constant rate to generate hydrophilic and lipophilic peroxyl radicals, respectively. 25 Use of these compounds allows regulation of both the nature and the intensity of the oxidative stress applied to each sample. Dose response curves of inhibition of mitochondrial transcription by these hydrophilic and lipophilic free radicals are shown in Figures 1A and lB. We confirmed that these agents were inhibiting mitochondrial transcription via a free radical mediated mechanism by determining that several antioxidants could partially protect mitochondria from their effects. Specifically, we found that the presence of glutathione (60-240 ~M), a-tocopherol (0.25-1 mM), and/or ascorbate (2.5- l 0
mM) could increase the amount of activity remaining after challenge by approximately 60%.* We also tested whether the well characterized lipid peroxidation byproducts MDA and HNE could repress transcription. MDA had no effect on transcription in concentrations as high as 10 mM (data not shown), but HNE was inhibitory (Fig. 1C). Because dose response analysis showed that mitochondrial transcription was more sensitive to the lipophilic AMVN (approximate LDso = 220 uM) than the hydrophilic AAPH (approximate LDso = 8mM), and because exogenously added HNE could inhibit transcription, we next examined the effect of lipid peroxidation itself on mitochondrial transcription using the ADP/Fe/NADPH system. This system induces high levels of lipid peroxidation by treatment of the target with 1.7 mM ADP, 0.1 mM NADPH, and 0.1 mM FeC13 for 15 min at 37°C. Surprisingly, this lipid peroxidation system did not inhibit mitochondrial transcription, but actually appears to enhance it (550 _+ 200% of controls). Preliminary evidence suggests this enhancement may be related to ADP or NADPH, but we have not examined this in detail. The inability of ADP/Fe/NADPH to inhibit mitochondrial transcription suggests that lipid peroxidation itself is insufficient to inhibit mitochondrial transcription. To determine whether lipid peroxidation played any role in the observed inhibition of mitochondrial transcription, we determined whether lipid peroxidation was induced by ADP/Fe/NADPH, AAPH, AMVN, or HNE. Chemical challenges were performed under identical conditions to those utilized for the transcription assay, and the levels of MDA and HNE produced were determined (Figs. 2A and 2B). Our results showed that AAPH, AMVN, and HNE induced little lipid peroxidation, even at concentrations that inhibited 50% of mitochondrial transcription, whereas ADP/Fe/NADPH, which does not repress transcription, induced high quantities of lipid peroxidation. Specifically, the ADP/Fe/NADPH system generated over 30 times more MDA and four times more HNE than concentrations of AMVN and AAPH that inhibit 50% of mitochondrial transcription. Treatment with HNE did not appear to induce further lipid peroxidation. Together, these results show that the highly reactive HNE can repress transcription, but not at the concentrations generated by AAPH or AMVN treatment. Finally, AAPH does generate some MDA and HNE at concentrations that repress 100% of mitochondrial transcription, and AMVN may also be generating HNE at this point,
*B.S.K./B.P.Y., manuscript in preparation.
Mitochondrial transcription
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Fig. 2. Quantification of lipid peroxidation byproducts relative to the inhibition they induce. (A) The amount of MDA generated by three concentrations of AAPH, AMVN, or HNE (noted above corresponding column), or by the ADP/Fe/NADPH system, was quantitated by the TBA test and plotted vs. the percentage of transcriptional activity remaining following challenge with that concentration of oxidant. The data in this graph represents four independent experiments. The horizontal line represents MDA levels of untreated mitochondria. ADP/Fe/NADPH values are significant at p < 0.0001; no other values are significant, although 32 mM AAPH approaches significance (p ~ 0.06). (B) The amount of HNE generated by three concentrations of AAPH, and AMVN, or by the ADP/Fe/NADPH system, was quantitated by HPLC and plotted vs. the percentage of transcriptional activity remaining following challenge with that concentration of oxidant. Fifty-fold higher levels of HNE were isolated (from samples treated with concentrations of HNE that repressed 50% of transcription) than were observed in the ADP/Fe/ NADPH-treated samples. To clarify the figure, these data are not graphed. The horizontal line represents HNE levels in untreated mitochondria. The data shown is from a single experiment, but is consistent with the data in Figure 2A and Figure 3, and thus, due to the generally observed approximate correlations between these measurements, it was felt that HNE quantitation need not be repeated.
although, based on the ADP/Fe/NADPH data, the levels induced are not sufficient to explain the inhibition observed. The extent of lipid peroxidation induced was further quantitated by measuring the oxygen uptake by mitochondrial membranes following challenge. Under the conditions used, this measurement is an established index of lipid peroxidation, 24and serves to further confirm the independence of the inhibition of mitochondrial transcription from the process of lipid peroxidation in samples treated with AAPH, AMVN, or ADP/Fe/NADPH. As shown in Figure 3, the ADP/ Fe/NADPH system that generates lipid peroxidation also consumes more oxygen than samples treated with concentrations of AAPH or AMVN that com-
pletely repress transcription. This data reaffirms the MDA and HNE studies and supports the hypothesis that lipid peroxidation does not play a causative role in the inhibition of mitochondrial transcription induced by free radicals generated by AAPH and AMVN. Questions concerning the mechanism of the observed inhibition fall into two classes: "What is the critical target?" and "Why is there a differential sensitivity of different readouts (the induction of lipid peroxidation and the repression of mitochondrial transcription) to different free radical species?" Our mitochondria had been freeze-thawed, and thus do not display coupled respiration, which eliminates the possibility that inhibition of ATP production mediates
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Fig. 3. Quantification of lipid peroxidation by oxygen consumption. Lipid peroxidation was assessed by measuring oxygen consumption by mitochondrial samples treated with AAPH, AMVN, or ADP/Fe/NADPH. Data was obtained using the protocol described elsewhere, in which oxygen consumption is due to lipid peroxidation and not respiration. Lipid peroxidation is expressed as nMoles oxygen/mg protein/rain, with higher levels of oxygen consumption being indicative of higher levels of lipid peroxidation. The horizontal line represents oxygen consumption by untreated mitochondria. Data shown is from two experiments and is consistent with other data that could not be directly compared. Oxygen consumption by ADP/Fe/NADPHtreated mitochondria is significantly different from all others at p < 0.0001, 32 mM AAPH-induced oxygen consumption is significant at p < 0.001 against all other treatments except 10 mM AAPH (p = 0.014). No other comparisons are significant.
this effect and suggests that a component of the transcription complex--either mtDNA or the proteins involved in transcription--is directly attacked by the free radical challenge. Our findings, when compared to those of others, suggest that lipid peroxidation-mediated mtDNA damage is not causally involved in the observed repression ofmitochondrial transcription by AAPH, AMVN, or HNE. This is supported by the observation that even concentrations of AAPH, AMVN, and HNE that are sufficient to completely inhibit transcriptional activity did not consistently alter either the electrophoretic mobility of mtDNA or its quantitative yield, as assayed by Southern blot.* These observations do not, however, eliminate the
*B.S.K./B.P.Y., unpublished data.
possibility that the specific species of free radicals used do oxidatively damage mtDNA, but it must be considered that while many examples of oxidative damage to DNA (such as 8-hydroxy-deoxyguanosine) are known to cause miscoding problems, they are not generally associated with transcription inhibition. One could speculate that the reason for the differential sensitivity observed is that the accessibility of these free radicals to their targets modulates their effects. It is clear, for example, that either the initial chemical, or some signal or damage it generates, must penetrate both the outer and inner mitochondrial membranes to inhibit transcription. The lipophilic nature of both AMVN and HNE may explain why they are over a log-and-a-half more efficient at inhibiting mitochondrial transcription than the hydrophilic AAPH. Similarly, AAPH may be able to inhibit tran-
Mitochondrial transcription scription because at these doses s o m e percentage o f the free radicals generated by A A P H d e c o m p o s i t i o n or the p r o d u c t s generated b y their interaction with the m e m b r a n e can cross the lipid bilayer, whereas those p r o d u c t s generated b y A D P / F e / N A D P H are preferentially m a i n t a i n e d in the m e m b r a n e , thus explaining w h y A D P / F e / N A D P H induces so m u c h lipid peroxidation, yet fails to inhibit m i t o c h o n d r i a l transcription. It also r e m a i n s to be tested h o w other m e a n s o f i n d u c i n g lipid peroxidation w o u l d b e h a v e in these assays. SUMMARY AND CONCLUSIONS T h e results o f this study demonstrate, for the first time, that free radicals can inhibit in vitro m i t o c h o n drial transcription, a n d that this inhibition m a y be free radical species-specific. This is based o n observations that c o n c e n t r a t i o n s o f specific free radical species that inhibit m i t o c h o n d r i a l transcription are insufficient to i n d u c e lipid peroxidation, a n d that treatm e n t with A D P / F e / N A D P H , despite generating high levels o f lipid peroxidation, does n o t inhibit transcription. Together, these results suggest that detectable inhibition o f m i t o c h o n d r i a l transcription a n d the detectable i n d u c t i o n o f m i t o c h o n d r i a l lipid peroxidation can o c c u r independently, a n d that these processes are differentially sensitive to specific free radical species. This suggests the possibility that, d e p e n d i n g on the source o f oxidative challenge, s o m e factor or factors involved in m i t o c h o n d r i a l transcription m a y be a m o n g the first c o m p o n e n t s o f the mitoc h o n d r i a to be affected b y free radicals during periods o f increased oxidative stress. F u r t h e r w o r k c o n c e r n ing the m e c h a n i s m o f the transcriptional inhibition a n d its prevention with antioxidants is in progress. Acknowledgements - - The authors wish to thank Dr. H. Esterbauer
for providing 4-HNE, and Drs. H. Bertrand, J. Herlihy, A. Heydari, P. Hornsby, R. McCarter, J. Nelson, A. Richardson, M. Salih, W. Ward, and the UT Health Science Center (San Antonio) aging group for helpful discussions and comments on the manuscript. This work was supported in part by an American Federation for Aging Grant (AFAR to B.S.K.), training grant (NIA T32 AG00205), the Glenn Foundation, and research grant (NIA AG01188). REFERENCES
1. Harman, D. The biologic clock: The mitochondria? J. Amer Ger Soc. 20:145-147; 1972. 2. Fleming, J. E.; Miquel, J.; Cottell, S. F.; Yengoyan, L. S.; Economos, A. C. Is cell aging caused by respiration dependent injury to the mitochondrial genome? Gerontology 28:44-53; 1982.
329
3. Miquel, J. An update on the mitochondrial DNA mutation hypothesis of cell aging. Mut. Res. 275:209-216; 1992. 4. Miquel, J. An integrated theory of aging as the result of mitochondrial DNA mutations in differentiated cells. Arch. Gerontol. Geriatrics 12:99-117; 1991. 5. Wallace, D. C. Mitochondrial genetics: A paradigm for aging and degenerative diseases. Science 256:628-632; 1992. 6. Linnane, A. W.; Marzuki, S.; Ozawa, T.; Tanaka, M. Mitochondrial DNA mutations as an important contributor to aging and degenerative diseases. Lancet i:642; 1989. 7. Esterbauer, H.; Schaur, R. J.; Zollner, H. Chemistry and biochemistry of 4-hydroxynonenal, malondialdehyde and related aldehydes. Free Radic. Biol. Med. 11:81-128; 1991. 8. Weitberg, A. B., Corvese, D. Hydroxy- and hydroperoxy6,8,11,14-eicosatetraenoic acids induce DNA strand breaks in human lymphocytes. Carcinogenesis 10:1029-1031; 1989. 9. Cerutti, P. A.; Emerit, A. P. Membrane mediated chromosomal damage. In: Weinstein, I. B.; Vogel, H. J., eds. Genes and proteins in oncogenesis. New York: Academic Press; 1983:5567. 10. Fraga, C. G.; Tappel, A. L. Damage to DNA concurrent with lipid peroxidation in rat liver slices. Biochem. J. 252:893-896; 1988. 11. Inouye, S. Site specific cleavage of double stranded DNA by hydroperoxide oflinoleic acid. FEBSLett. 172:231-232; 1984. 12. Ueda, K.; Kobayahi, S.; Morita, K. Site-specific DNA damage caused by lipid peroxidation products. BBA 824:341-348; 1985. 13. Fujimoto, K.; Neff, W. E.; Frankel, E. NI The reaction of DNA with lipid oxidation products, metals, and reducing agents. BBA 795:100-107; 1984. 14. Reiss, U.; Tappel, A. Fluorescent product formation and changes in structure of DNA reacted with aeroxidizing arachidonic acid. Lipids 8:199-202; 1972. 15. Akasaka, S. Inactivation of transforming activity of plasmid DNA by lipid peroxidation. BBA 867:201-208; 1986. 16. Park, J.-W.; Floyd, R. A. Lipid peroxidation products mediate the formation of 8-hydroxydeoxyguanosine in DNA. Free Radic. Biol. Med. 12:245-250; 1992. 17. Hruszkewycz, A. M. Lipid peroxidation and mtDNA degeneration: A hypothesis. Mut. Res. 275:243-248; 1992. 18. Hruszkewycz, A. M. Evidence for mitochondrial DNA damage by lipid peroxidation. BBRC 153:19 l- 197; 1988. 19. Hruszkewycz, A. M.; Bergtold, D. S. The 8-hydroxyguanine content of isolated mitochondria increases with lipid peroxidation. Mut. Res. 244:123-128; 1990. 20. Gaines, G.; Attardi, G. Highly efficient RNA-synthesizing system that uses isolated human mitochondria: New initiation events and in vivo-like processing patterns. M C B 4:16051617; 1984. 21. Esterbauer, H.; Cheeseman, K. Determination of aldehydic lipid peroxidation products: Malondialdehyde and 4-hydroxynenal. Methods Enzymol. 186:407-421; 1990. 22, Buege, J. A.; Aust, S. D. Microsomal lipid peroxidation. Methods Enzymol. 52:302-310; 1978. 23. Estabrook, R. W. Mitochondrial respiratory control and the polarographic measurement of ADP:O ratios. Methods Enzytool. 10:41-47; 1967. 24. Yu, B. P.; Suescun, E. A.; Yang, S. Y. Effect of age-related lipid peroxidation on membrane fluidity and phospholipase A2: Modulation by dietary restriction. Mech. Age. Dev. 65:17-33; 1992. 25. Niki, E.; Yamamoto, Y.; Komuro, E.; Sato, K. Membrane damage due to lipid oxidation. Am. ,L Clin Nutr. 53:201S205S; 1991. 26. Fernandez-Silva, P.; Petruzzella, V.; Fracasso, F.; Gadaleta, M. N.; Cantatore, P. Reduced synthesis of mtRNA in isolated mitochondria or senescent rat brain. B B R C 176:645-653, 1991.
Pergamon
0891-5849(94)E0004-3
Original Contribution SENSITIVITY
OF MITOCHONDRIAL TRANSCRIPTION FREE RADICAL SPECIES
TO
DIFFERENT
BRUCE S. KRISTAL, JUANJUAN CHEN, and BYUNG PAL Y u Department of Physiology,The University of Texas Health ScienceCenter, San Antonio, TX, USA (Received 12 April 1993; Revised 30 June 1993;Accepted 21 July 1993)
Abstract--Proper mitochondrial function requires the continual maintenance of the integrity of the mitochondrial gene expression system. The sensitivity of both mitochondrial lipids and mitochondriai respiration to free radicals has been well recognized, but the effect of free radicals and lipid peroxidation on mitochondrial transcription has not yet been examined. Using an in vitro mitochondrial transcription assay, we tested the ability of fiveprooxidants to affectmitochondrial transcription. Results show that mitoehondrial transcription is extremelysensitiveto inhibition by peroxylradicalsgeneratedby either 2,2'-azobis-(2-amidinopropane) (AAPH) or 2,2'-azobis-(2,4-dimethylvaleronitrile)(AMVN), and that this inhibition occurs prior to detectableevidenceof lipid peroxidationas measuredby thiobarbituric acid (TBA)-reactivesubstances, 4-hydroxynonenai accumulation, and oxygenconsumption. Furthermore, although mitochondrial transcription was sensitiveto 4-hydroxynonenal, it was resistant to malondiaidehydeas wellas high levelsof lipid peroxidationinduced by ADP/Fe/NADPH. Together, these results suggestthat the repressionof mitochondrialtranscription is differentiallysensitiveto specificmodes of free radical reaction. Keywords--Mitochondria, Transcription, Free radicals, AAPH, AMVN, Lipid peroxidation, 4-Hydroxynonenal, Malondialdehyde
(MDA) and the more reactive 4-hydroxynonenal (HNE). 7 It has been shown that peroxidizing lipids and the byproducts o f lipid peroxidation can act directly to biochemically alter and functionally damage DNA. s-~6 Furthermore, in vitro induction o f lipid peroxidation on intact mitochondria can alter the electrophoretic mobility o f m t D N A (refs. 17,18), and increase levels o f the oxidized base 8-hydroxyguanosineJ 9 These results suggest that lipid peroxidation products might mediate oxidative damage to mtDNA, but provide no evidence directly addressing whether free radicals/lipid peroxidation can inhibit mitochondrial transcription, or whether lipid peroxidation products can act as mediators o f oxidant-induced repression o f mitochondrial function. Our study addressed these questions using four independent and chemically distinct challenges that were assessed using a modified version o f the in vitro mitochondrial transcription system developed by Gaines and Attardi. 2° Our results demonstrated that free radicals can inhibit mitochondrial transcription and that mitochondrial transcription and mitochondrial lipid peroxidation may display differential sensitivity to specific free radical species.
INTRODUCTION Mitochondrial impairment resulting from oxidantinduced damage has been proposed to play a critical, possibly causative role in both the aging process and m a n y age-related diseases. 1-6 Proper mitochondrial function requires continual maintenance o f the integrity o f m a n y systems, including those involved in mitochondrial gene expression. However, the proximity o f the inner mitochondrial m e m b r a n e to the relatively unprotected mitochondrial genome, and the accessory proteins required for the transcription process, suggest that mitochondrial transcription could be sensitive to inhibition induced by either free radicals, by the products o f lipid peroxidation, or by both. Lipid peroxidation occurs as a chain reaction in which polyunsaturated fatty acids undergo a series o f oxidation and cleavage reactions that yield several kinds o f breakdown products, including lipid radicals and aldehydic metabolites such as malondialdehyde Address correspondenceto: ByungPal Yu, Department of Physiology, The Universityof Texas Health ScienceCenter, 7703 Floyd Curl Drive, San Antonio, TX 78284-7756, USA. 323
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Fig. 1. (A) This figure shows the gel used to obtain the values graphed in Figures IB-ID. Lane 1-4: AMVN 75, 150, 300, 600 #M; Lane 5-9: AAPH 2, 4, 8, 16, 32 raM; Lane 10-14: HNE 20, 40, 80, 160, 320 uM; Lane 15-16: ADP/Fe/NADPH (l.7/0.1/0.1 mM); Lane 17:Control (untreated). By comparison with the data in Gaines and Attardi,2°and Fernandez-Silvaet al.,26Band A contains the mitochondrial RNA encoding ND5, the bands at B contain the species encoding ND4, CoI, and the 16S RNA. The diffuse series of bands at C include cyt b, ND2, NDI, Colll, A6/8, Coll, and the 12S RNA. Band D appears to be the 7s RNA. The rRNAs are observable due to the use of 1 mM ATP (ref. 20). B-D: Dose response curves for AAPH, AMVN, and HNE. Following transcription reaction and RNA purification as described, samples were electrophoresed through sequencing thickness, gradient 3.5-4.5% polyacrylamidegels (l x TBE, 60W, 1400V, 6-8 h). Radioactivity present in bands C-D (that contain most major processed mitochondrial transcripts) were quantitated on a Molecular Dynamics Phospho-Imager and expressed as a percentage of an untreated sample. As mitochondrial transcription utilizes only a single, bidirectional promotor and yields two primary transcripts that are processed to yield individual messages, no individual analysis of transcripts was done. This rationale is supported by longer gel runs that suggest there is no consistent message to message variability following oxidant challenge for the longer messages. Data shown in panels B-D is from the single, representative experiment shown in Figure IA, and is expressed as a percentage of total activity present in untreated controls. (B) Treatment with AAPH: samples were pretreated with 2, 4, 8, 16, and 32 mM AAPH, respectively. (C) Treatment with AMVN: samples were pretreated with 80, 160, 320, and 640 uM AMVN, respectively. (D) Treatment with HNE: samples were pretreated with 20, 40, 80, 160, and 320 uM HNE, respectively.
MATERIALS AND METHODS
Animals M a l e F i s c h e r 344 rats were o b t a i n e d f r o m C h a r l e s R i v e r ( W i l m i n g t o n , M A ) at 90 d a y s o f age. A n i m a l s were m a i n t a i n e d for 2 weeks in o u r u n i v e r s i t y l a b o r a t o r y care facility p r i o r to sacrifice b y d e c a p i t a t i o n . A f t e r sacrifice, livers were i m m e d i a t e l y r e m o v e d , frozen in l i q u i d n i t r o g e n (in 20 m M H e p e s (7.4) w i t h 140 m M KC1), a n d s t o r e d at - 7 0 ° C .
Mitochondrial transcription M i t o c h o n d r i a were isolated b y s t a n d a r d differential c e n t r i f u g a t i o n t e c h n i q u e s , q u i c k frozen in l i q u i d
A
nitrogen, a n d s t o r e d at - 7 0 ° C u n t i l n e e d e d . M i t o c h o n d r i a l t r a n s c r i p t i o n was p e r f o r m e d essentially as b y G a i n e s a n d A t t a r d i , 2° e x c e p t for t w o m o d i f i c a tions: t h e t r a n s c r i p t i o n buffer was u s e d at 1.3x c o n c e n t r a t i o n s a n d s u p p l e m e n t e d with 100 m M KC1 a n d 1 m M a d e n o s i n e t r i p h o s p h a t e (ATP), (which a l l o w e d m i t o c h o n d r i a l r R N A t r a n s c r i p t i o n , as s h o w n b y G a i n e s a n d A t t a r d i ) , a n d t h e assay u s e d frozen m i t o c h o n d r i a . T h e use o f frozen m i t o c h o n d r i a h a d n o significant effect e i t h e r o n t o t a l t r a n s c r i p t i o n a l a c t i v i t y or on oxidant induced repression of transcription (tested o n A A P H only, d a t a n o t shown). T o e x a m i n e t h e effects o f p r o o x i d a n t s o n m i t o c h o n d r i a l t r a n s c r i p tion, 5 m g m i t o c h o n d r i a were p r e i n c u b a t e d in t h e p r e s e n c e o f the o x i d a n t for 15 r a i n at 3 7 ° C in a t o t a l
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volume of 1 ml, unreacted oxidant removed by centrifugation, and the mitochondria incubated for 30 min in the transcription buffer. Labeled RNA was isolated, separated by PAGE, and gels were analyzed using a Molecular Dynamics Phospho-Imager. The existence of a micrococcal nuclease digestion step insures that that the RNA being purified was transcribed in intact mitochondria.
Quantitation of liNE by HPLC The method of isolation and quantitation of HNE from mitochondria was essentially the same as described by Esterbauer and Cheeseman. 2~ Oxidation was accomplished identically to that done in the transcription assays, and the sample was then diluted to 20 ml. This 20 ml mitochondrial suspension was combined with 20 ul of 1% ethanolic butylated hydroxytoluene (BHT) and 200 ul of 1% aqueous desferal solution to prevent formation of additional HNE during the following solid-phase extraction procedure. The mitochondrial suspension was poured onto an Extrelut column and twice extracted with 20 ml of dichloromethane. The eluate was collected in a flask containing 2 ml of 0.1M acetate buffer, pH 3. After
the dichloromethane was removed under nitrogen gassing, the aqueous phase was applied quantitatively to an octadecylsilyl (ODS) extraction column preconditioned with 3 ml of methanol and equilibrated with 3 ml of water. The nonpolar material, which interferes with HNE analysis, was removed by eluting the column with 2 ml of hexane. HNE was eluted with 2 ml of methanol/water (80:20) into a 2 ml volumetric tube. Isocratic high performance liquid chromatography (HPLC) analysis was performed with a Waters HPLC system using a Spherisorb $ 5 0 D S 2 column with acetonitrile/water (50/50) as eluant, detector wavelength set at 220 nm, and a flow rate of 1 ml/ rain. Peak identification and quantitation was done relative to reference chromotograms of standard HNE solutions.
Determination of MDA MDA was determined in an 1 ml aliquot taken from the same mitochondrial suspension used for HNE quantitation. The method used was a modification of the TBA assay reported by Buege et al.22 Briefly, 1 ml of the mitochondrial suspension was combined with 10 ul of 2% BHT and 2 ml TBA solu-
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tion (15% trichoroacetic acid; 0.375% thiobarbituric acid; 0.25 N hydrochloric acid). The mixture was heated for 15 rain in a boiling water bath. After cooling, the mixture was centrifuged at 1000 g for l0 min. The absorbance of the supernatant was read at 535nm, and the MDA concentration was determined relative to the reference absorbance of an MDA standard.
Data analysis Data was analyzed using the Macintosh statistical package SuperAnova. P values of 0.05 and 0.01 were assessed by Duncan's New Multiple Range Test. If a value was found significant at p < 0.01 by this test, it was then examined by Fisher's Protected LSD to examine p < 0.001 and p < 0.0001.
Determination of lipid peroxidation by oxygen consumption Lipid peroxidation was determined by measuring the oxygen consumption by mitochondria upon addition of prooxidants, according to the method of Estabrook et al.23 and Yu et al. 24 A Gilson 5/6 oxygraph connected to a circulating water bath was used. Oxygen uptake was measured polarographically with a Clark-type oxygen electrode at 37°C. Mitochondrial suspensions (in 140mM KC1, 20mM Hepes, pH 7.4) were added into the reaction chamber. Oxygen uptake was initiated with the addition of the prooxidants, which induce lipid peroxidation in the mitochondrial membrane. RESULTS AND DISCUSSION Rat liver mitochondria were preincubated with varying concentrations of two well-defined free radical generators, AAPH (2,2'-azobis-(2-amidinopropane) hydrochloride) and AMVN (2,2'-azobis-(2,4-dimethylvaleronitrile)), which decompose at a constant rate to generate hydrophilic and lipophilic peroxyl radicals, respectively. 25 Use of these compounds allows regulation of both the nature and the intensity of the oxidative stress applied to each sample. Dose response curves of inhibition of mitochondrial transcription by these hydrophilic and lipophilic free radicals are shown in Figures 1A and lB. We confirmed that these agents were inhibiting mitochondrial transcription via a free radical mediated mechanism by determining that several antioxidants could partially protect mitochondria from their effects. Specifically, we found that the presence of glutathione (60-240 ~M), a-tocopherol (0.25-1 mM), and/or ascorbate (2.5- l 0
mM) could increase the amount of activity remaining after challenge by approximately 60%.* We also tested whether the well characterized lipid peroxidation byproducts MDA and HNE could repress transcription. MDA had no effect on transcription in concentrations as high as 10 mM (data not shown), but HNE was inhibitory (Fig. 1C). Because dose response analysis showed that mitochondrial transcription was more sensitive to the lipophilic AMVN (approximate LDso = 220 uM) than the hydrophilic AAPH (approximate LDso = 8mM), and because exogenously added HNE could inhibit transcription, we next examined the effect of lipid peroxidation itself on mitochondrial transcription using the ADP/Fe/NADPH system. This system induces high levels of lipid peroxidation by treatment of the target with 1.7 mM ADP, 0.1 mM NADPH, and 0.1 mM FeC13 for 15 min at 37°C. Surprisingly, this lipid peroxidation system did not inhibit mitochondrial transcription, but actually appears to enhance it (550 _+ 200% of controls). Preliminary evidence suggests this enhancement may be related to ADP or NADPH, but we have not examined this in detail. The inability of ADP/Fe/NADPH to inhibit mitochondrial transcription suggests that lipid peroxidation itself is insufficient to inhibit mitochondrial transcription. To determine whether lipid peroxidation played any role in the observed inhibition of mitochondrial transcription, we determined whether lipid peroxidation was induced by ADP/Fe/NADPH, AAPH, AMVN, or HNE. Chemical challenges were performed under identical conditions to those utilized for the transcription assay, and the levels of MDA and HNE produced were determined (Figs. 2A and 2B). Our results showed that AAPH, AMVN, and HNE induced little lipid peroxidation, even at concentrations that inhibited 50% of mitochondrial transcription, whereas ADP/Fe/NADPH, which does not repress transcription, induced high quantities of lipid peroxidation. Specifically, the ADP/Fe/NADPH system generated over 30 times more MDA and four times more HNE than concentrations of AMVN and AAPH that inhibit 50% of mitochondrial transcription. Treatment with HNE did not appear to induce further lipid peroxidation. Together, these results show that the highly reactive HNE can repress transcription, but not at the concentrations generated by AAPH or AMVN treatment. Finally, AAPH does generate some MDA and HNE at concentrations that repress 100% of mitochondrial transcription, and AMVN may also be generating HNE at this point,
*B.S.K./B.P.Y., manuscript in preparation.
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Fig. 2. Quantification of lipid peroxidation byproducts relative to the inhibition they induce. (A) The amount of MDA generated by three concentrations of AAPH, AMVN, or HNE (noted above corresponding column), or by the ADP/Fe/NADPH system, was quantitated by the TBA test and plotted vs. the percentage of transcriptional activity remaining following challenge with that concentration of oxidant. The data in this graph represents four independent experiments. The horizontal line represents MDA levels of untreated mitochondria. ADP/Fe/NADPH values are significant at p < 0.0001; no other values are significant, although 32 mM AAPH approaches significance (p ~ 0.06). (B) The amount of HNE generated by three concentrations of AAPH, and AMVN, or by the ADP/Fe/NADPH system, was quantitated by HPLC and plotted vs. the percentage of transcriptional activity remaining following challenge with that concentration of oxidant. Fifty-fold higher levels of HNE were isolated (from samples treated with concentrations of HNE that repressed 50% of transcription) than were observed in the ADP/Fe/ NADPH-treated samples. To clarify the figure, these data are not graphed. The horizontal line represents HNE levels in untreated mitochondria. The data shown is from a single experiment, but is consistent with the data in Figure 2A and Figure 3, and thus, due to the generally observed approximate correlations between these measurements, it was felt that HNE quantitation need not be repeated.
although, based on the ADP/Fe/NADPH data, the levels induced are not sufficient to explain the inhibition observed. The extent of lipid peroxidation induced was further quantitated by measuring the oxygen uptake by mitochondrial membranes following challenge. Under the conditions used, this measurement is an established index of lipid peroxidation, 24and serves to further confirm the independence of the inhibition of mitochondrial transcription from the process of lipid peroxidation in samples treated with AAPH, AMVN, or ADP/Fe/NADPH. As shown in Figure 3, the ADP/ Fe/NADPH system that generates lipid peroxidation also consumes more oxygen than samples treated with concentrations of AAPH or AMVN that com-
pletely repress transcription. This data reaffirms the MDA and HNE studies and supports the hypothesis that lipid peroxidation does not play a causative role in the inhibition of mitochondrial transcription induced by free radicals generated by AAPH and AMVN. Questions concerning the mechanism of the observed inhibition fall into two classes: "What is the critical target?" and "Why is there a differential sensitivity of different readouts (the induction of lipid peroxidation and the repression of mitochondrial transcription) to different free radical species?" Our mitochondria had been freeze-thawed, and thus do not display coupled respiration, which eliminates the possibility that inhibition of ATP production mediates
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Fig. 3. Quantification of lipid peroxidation by oxygen consumption. Lipid peroxidation was assessed by measuring oxygen consumption by mitochondrial samples treated with AAPH, AMVN, or ADP/Fe/NADPH. Data was obtained using the protocol described elsewhere, in which oxygen consumption is due to lipid peroxidation and not respiration. Lipid peroxidation is expressed as nMoles oxygen/mg protein/rain, with higher levels of oxygen consumption being indicative of higher levels of lipid peroxidation. The horizontal line represents oxygen consumption by untreated mitochondria. Data shown is from two experiments and is consistent with other data that could not be directly compared. Oxygen consumption by ADP/Fe/NADPHtreated mitochondria is significantly different from all others at p < 0.0001, 32 mM AAPH-induced oxygen consumption is significant at p < 0.001 against all other treatments except 10 mM AAPH (p = 0.014). No other comparisons are significant.
this effect and suggests that a component of the transcription complex--either mtDNA or the proteins involved in transcription--is directly attacked by the free radical challenge. Our findings, when compared to those of others, suggest that lipid peroxidation-mediated mtDNA damage is not causally involved in the observed repression ofmitochondrial transcription by AAPH, AMVN, or HNE. This is supported by the observation that even concentrations of AAPH, AMVN, and HNE that are sufficient to completely inhibit transcriptional activity did not consistently alter either the electrophoretic mobility of mtDNA or its quantitative yield, as assayed by Southern blot.* These observations do not, however, eliminate the
*B.S.K./B.P.Y., unpublished data.
possibility that the specific species of free radicals used do oxidatively damage mtDNA, but it must be considered that while many examples of oxidative damage to DNA (such as 8-hydroxy-deoxyguanosine) are known to cause miscoding problems, they are not generally associated with transcription inhibition. One could speculate that the reason for the differential sensitivity observed is that the accessibility of these free radicals to their targets modulates their effects. It is clear, for example, that either the initial chemical, or some signal or damage it generates, must penetrate both the outer and inner mitochondrial membranes to inhibit transcription. The lipophilic nature of both AMVN and HNE may explain why they are over a log-and-a-half more efficient at inhibiting mitochondrial transcription than the hydrophilic AAPH. Similarly, AAPH may be able to inhibit tran-
Mitochondrial transcription scription because at these doses s o m e percentage o f the free radicals generated by A A P H d e c o m p o s i t i o n or the p r o d u c t s generated b y their interaction with the m e m b r a n e can cross the lipid bilayer, whereas those p r o d u c t s generated b y A D P / F e / N A D P H are preferentially m a i n t a i n e d in the m e m b r a n e , thus explaining w h y A D P / F e / N A D P H induces so m u c h lipid peroxidation, yet fails to inhibit m i t o c h o n d r i a l transcription. It also r e m a i n s to be tested h o w other m e a n s o f i n d u c i n g lipid peroxidation w o u l d b e h a v e in these assays. SUMMARY AND CONCLUSIONS T h e results o f this study demonstrate, for the first time, that free radicals can inhibit in vitro m i t o c h o n drial transcription, a n d that this inhibition m a y be free radical species-specific. This is based o n observations that c o n c e n t r a t i o n s o f specific free radical species that inhibit m i t o c h o n d r i a l transcription are insufficient to i n d u c e lipid peroxidation, a n d that treatm e n t with A D P / F e / N A D P H , despite generating high levels o f lipid peroxidation, does n o t inhibit transcription. Together, these results suggest that detectable inhibition o f m i t o c h o n d r i a l transcription a n d the detectable i n d u c t i o n o f m i t o c h o n d r i a l lipid peroxidation can o c c u r independently, a n d that these processes are differentially sensitive to specific free radical species. This suggests the possibility that, d e p e n d i n g on the source o f oxidative challenge, s o m e factor or factors involved in m i t o c h o n d r i a l transcription m a y be a m o n g the first c o m p o n e n t s o f the mitoc h o n d r i a to be affected b y free radicals during periods o f increased oxidative stress. F u r t h e r w o r k c o n c e r n ing the m e c h a n i s m o f the transcriptional inhibition a n d its prevention with antioxidants is in progress. Acknowledgements - - The authors wish to thank Dr. H. Esterbauer
for providing 4-HNE, and Drs. H. Bertrand, J. Herlihy, A. Heydari, P. Hornsby, R. McCarter, J. Nelson, A. Richardson, M. Salih, W. Ward, and the UT Health Science Center (San Antonio) aging group for helpful discussions and comments on the manuscript. This work was supported in part by an American Federation for Aging Grant (AFAR to B.S.K.), training grant (NIA T32 AG00205), the Glenn Foundation, and research grant (NIA AG01188). REFERENCES
1. Harman, D. The biologic clock: The mitochondria? J. Amer Ger Soc. 20:145-147; 1972. 2. Fleming, J. E.; Miquel, J.; Cottell, S. F.; Yengoyan, L. S.; Economos, A. C. Is cell aging caused by respiration dependent injury to the mitochondrial genome? Gerontology 28:44-53; 1982.
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3. Miquel, J. An update on the mitochondrial DNA mutation hypothesis of cell aging. Mut. Res. 275:209-216; 1992. 4. Miquel, J. An integrated theory of aging as the result of mitochondrial DNA mutations in differentiated cells. Arch. Gerontol. Geriatrics 12:99-117; 1991. 5. Wallace, D. C. Mitochondrial genetics: A paradigm for aging and degenerative diseases. Science 256:628-632; 1992. 6. Linnane, A. W.; Marzuki, S.; Ozawa, T.; Tanaka, M. Mitochondrial DNA mutations as an important contributor to aging and degenerative diseases. Lancet i:642; 1989. 7. Esterbauer, H.; Schaur, R. J.; Zollner, H. Chemistry and biochemistry of 4-hydroxynonenal, malondialdehyde and related aldehydes. Free Radic. Biol. Med. 11:81-128; 1991. 8. Weitberg, A. B., Corvese, D. Hydroxy- and hydroperoxy6,8,11,14-eicosatetraenoic acids induce DNA strand breaks in human lymphocytes. Carcinogenesis 10:1029-1031; 1989. 9. Cerutti, P. A.; Emerit, A. P. Membrane mediated chromosomal damage. In: Weinstein, I. B.; Vogel, H. J., eds. Genes and proteins in oncogenesis. New York: Academic Press; 1983:5567. 10. Fraga, C. G.; Tappel, A. L. Damage to DNA concurrent with lipid peroxidation in rat liver slices. Biochem. J. 252:893-896; 1988. 11. Inouye, S. Site specific cleavage of double stranded DNA by hydroperoxide oflinoleic acid. FEBSLett. 172:231-232; 1984. 12. Ueda, K.; Kobayahi, S.; Morita, K. Site-specific DNA damage caused by lipid peroxidation products. BBA 824:341-348; 1985. 13. Fujimoto, K.; Neff, W. E.; Frankel, E. NI The reaction of DNA with lipid oxidation products, metals, and reducing agents. BBA 795:100-107; 1984. 14. Reiss, U.; Tappel, A. Fluorescent product formation and changes in structure of DNA reacted with aeroxidizing arachidonic acid. Lipids 8:199-202; 1972. 15. Akasaka, S. Inactivation of transforming activity of plasmid DNA by lipid peroxidation. BBA 867:201-208; 1986. 16. Park, J.-W.; Floyd, R. A. Lipid peroxidation products mediate the formation of 8-hydroxydeoxyguanosine in DNA. Free Radic. Biol. Med. 12:245-250; 1992. 17. Hruszkewycz, A. M. Lipid peroxidation and mtDNA degeneration: A hypothesis. Mut. Res. 275:243-248; 1992. 18. Hruszkewycz, A. M. Evidence for mitochondrial DNA damage by lipid peroxidation. BBRC 153:19 l- 197; 1988. 19. Hruszkewycz, A. M.; Bergtold, D. S. The 8-hydroxyguanine content of isolated mitochondria increases with lipid peroxidation. Mut. Res. 244:123-128; 1990. 20. Gaines, G.; Attardi, G. Highly efficient RNA-synthesizing system that uses isolated human mitochondria: New initiation events and in vivo-like processing patterns. M C B 4:16051617; 1984. 21. Esterbauer, H.; Cheeseman, K. Determination of aldehydic lipid peroxidation products: Malondialdehyde and 4-hydroxynenal. Methods Enzymol. 186:407-421; 1990. 22, Buege, J. A.; Aust, S. D. Microsomal lipid peroxidation. Methods Enzymol. 52:302-310; 1978. 23. Estabrook, R. W. Mitochondrial respiratory control and the polarographic measurement of ADP:O ratios. Methods Enzytool. 10:41-47; 1967. 24. Yu, B. P.; Suescun, E. A.; Yang, S. Y. Effect of age-related lipid peroxidation on membrane fluidity and phospholipase A2: Modulation by dietary restriction. Mech. Age. Dev. 65:17-33; 1992. 25. Niki, E.; Yamamoto, Y.; Komuro, E.; Sato, K. Membrane damage due to lipid oxidation. Am. ,L Clin Nutr. 53:201S205S; 1991. 26. Fernandez-Silva, P.; Petruzzella, V.; Fracasso, F.; Gadaleta, M. N.; Cantatore, P. Reduced synthesis of mtRNA in isolated mitochondria or senescent rat brain. B B R C 176:645-653, 1991.