mitochondria, thus impairing electron transport and therefore impaired A TP production. This would result in the diversion of electrons from their normal ETC recipients and further formation of damaging free radicals (Cassarino and Bennet, 1999). Thus decreased A TP production and increased ROS production is a possible mechanism whereby an impaired ETC would lead to cell death (Beal, 1995; Dykens, 1997; H. Mochizuki el al., 1994; Wu et al., 1990; Wolvetang el al., 1994).
The complex I inhibitors rotenone and N-methylpyridinium ion (Zhang el al., 2001; Smith and Bennett, 1997) generate free radicals in vivo and in vitro (Adams e/ al., 1993; Mochizuki el al., 1994; Wolvetang el al., 1994) thus indicating that generation of free radicals via disruption of the ETC, have harmful effects if not neutralized by the cell. Damage to cells due to these free radicals, i.e. oxidation of cellular constituents including proteins, lipids, and DNA, can be detected through certain measurements for example, malonaldehyde formation, (Esterbaur el ai., 1991; Lovell e/ al.,
1997).
With age, there is oxidative membrane damage to membranes due to mitochondrial ROS production, which parallels the increase in mitochondrial lipid peroxides with aging (Zoratti and Szabo, 1995; Yu et al., 1996; Harman, 1996). Progressive impairment of the ETC activity may also occur with aging due to the accumulation of oxidatively damaged proteins (Bowling and Beal, 1995; Dykens, 1997; Hagen el al., 1997; Gutteridge and Halliwell, 1989; Sohal and Dubey, 1994;
Symonyan and Nalbandyan 1972), leading to further ROS production and oxidative damage. The progressive loss of ATP may therefore result in the loss of NalK-ATPase activity with consequent plasma membrane depolarization, loss of cellular homeostasis and activation of the apoptotic cascade (Beal, 1995; Bowling and Beal, 1995; Dykens, 1997).
Susceptibility to ROS-induced damage is higher in the CNS, than other organs, due to the relative lack of oxidative defenses (Marklund e/ al., 1982; Martilla et al., 1988). Therefore any symptomatic therapy for Alzheimer's disease, an affliction especially in the aged, should not further compromise the functioning of the ETC.
hap!er 4 - Electron Transport Chain and Complex I
4.2 EFFECT OF METRIFONATE ON THE MITOCHONDRIAL CHAIN ACTIVITY
4.2.1 INTRODUCTION
Free radical-mediated cellular damage and impaired ETC have been exhibited in neurodegenerative diseases e.g. Alzheimer's disease and Parkinson's disease. (Beal, 1995; Bowling and Beal 1995;
Harman, 1996; Kish et ai., 1985; Parker et ai., 1989, Parker et aI., 1994; Swerdlow et ai., 1996;
Swerdlow et ai., 1997). Therefore it is important that the agents used in symptomatic treatment of neurodegenerative disorders e.g. Alzheimer's disease not cause further ROS generation or damage.
However, as shown earlier, MET has the potential to induce lipid peroxidation and superoxide generation. Thus it is imperative that the source be determined, i.e. is the cause of increased superoxide generation, due to a disturbance of the ETC resulting in a decreased ETC activity? This chapter aims to investigate the possibility of the disruption of the ETC as a source of free radical production.
4.2.2 MATERIALS AND METHODS
4.2.2.1 Animals
Adult, male, Wistar rats, weighing between 250 and 300g were used for the experiment, and were housed and maintained under the conditions described in section 2.2.2.1.
4.2.2.2 Chemicals and Reagents
All reagents were of the highest quality available. MET, NAD, 2,6 - diclorophenol-indophenol (DPI), and L-Malate were purchased from Sigma Chemical Corporation, St. Louis, MO, U.S.A.
Sucrose was purchased from Saarchem (PTY) Ltd, Krugersdorp, South Africa. All reagents were prepared in O.1M potassium phosphate buffer, pH 7.4.
4.2.2.3 Isolation of Mitochondria from rat brain
The rats were killed by cervical dislocation, and the brains were rapidly excised and homogenized with O.lM potassium phosphate buffer, pH 7.4 to yield a 10% w/v
homogenate. Mitochondrial suspensions were prepared by differential centrifugation according to
Plummer (1971). The brain homogenate was centrifuged at 600g for 10 minutes and
separated into supernatant and pellet. The supernatant was collected and the pellet was re-suspended in half the volume of 0.32M buffered sucrose, and once again centrifuged at 600g for 10 minutes and the supernatant obtained was combined with the previous supernatant. The combined supernatant was centrifuged at 8000g for 10 minutes and the pellet obtained (crude mitochondria) was washed twice in the 0.32M buffered sucrose, and then stored on ice until required.
4.2.2.4 Biological Oxidation Assay
A modified method described by Plummer (J 971) was used. This spectrophotometric technique was employed to determine the 'activity' of the inner mitochondrial membrane electron transport chain of the mitochondrial suspension. The rate of reduction of the synthetic electron acceptor dye, 2,6- dichiorophenol-indophenol in presence of the substrate, L-Malate, is used as an indication of the level of "activity" of the mitochondrial ETC. Homogenate (O.3ml), potassium phosphate buffer (1.6ml) and containing varying concentrations of MET (O.lml) were incubated at 37°C in a water bath for incubation times of 5 and 60 minutes, after which 1 ml of the homogenate mixture was removed and incorporated with NAD (0.5mM), L-Malate (90 JlM), DPI (1.5mM) and potassium phosphate buffer. lbis was inverted once to mix and the decrease in absorbance over a 5-minute period was read at 30 second intervals on a UV NIS spectrophotometer at 600nm. All data is expressed as the rate of change in absorbance per minute at 600nm and corrected for appropriate controls.
Chapter 4 - Electron Transport Chain and Complex I
4.2.3 RESULTS
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