The effect of paraquat on the in vitro activity of cytosol, mitochondrial and microsomal enzyme systems
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Subcellular fractions (mitochondria, microsomes and cytosol) were prepared from the lungs of rabbits and rats to investigate the effects of paraquat (Aldrich Laboratories) on the activity of cytosol and mitochondrial dehydrogenases and on the microsomal respiration and reduced pyridine nucleotide oxidation rate. The normal basal oxygen consumption of rabbit lung microsomes was 1.9 ± 0.3 nmol O2/mg microsomal protein/min, and the oxidation rates of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) and reduced nicotinamide-adenine dinucleotide (NADH) were 4.29 ± 0.53 and 4.0 ± 0.55 nmol/mg microsomal protein/min respectively. One molecule of oxygen can therefore oxidize two molecules of NADPH and NADH, and the generated hydrogen peroxide is probably immediately broken down by the catalase activity of the normal lung microsomal preparation. When Aldrich paraquat (1.0 mM) was added to microsomes metabolizing NADPH (0.5 - 0.75 mM), both the rate of oxygen consumption and the generation of nicotinamide-adenine dinucleotide phosphate (NADP) were significantly (P < 0.001) stimulated over the first 5 minutes, and thereafter returned to within basal limits. When microsomes were preincubated with 1.0 mM paraquat before NADPH was added, the oxygen consumption was substantially lower (10.01 ± 1.01 nmol oxygen/mg microsomal protein/min), while the NADPH oxidation rate was almost similar to the basal rate in the absence of paraquat. This resulted in a striking dissociation in the H/O ratio under these circumstances. The addition of potassium cyanide (KCN) (5.0 mM) prior to paraquat pre-incubation and followed by the addition of NADPH restored the stimulatory effect of paraquat on microsomal respiration and on NADPH oxidation rate. Paraquat (0.01 mM) had no effect on the reaction rates of the following enzyme systems, glucose-6-phosphate dehydrogenase (G-6-PD), glyceraldehyde-3-phosphate dehydrogenase (GAPD), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), and isocitrate dehydrogenase (IDH). However, 0.1 mM paraquat slightly inhibited the mitochondrial IDH system, and 1.0 mM paraquat significantly inhibited all the enzymes tested except for mitochondrial and cytosol MDH. The addition of KCN 5.0 mM led to a total inhibition of the LDH and MDH enzyme systems in vitro, but did not affect the IDH, GAPD and G-6-PD systems. However, when KCN was added before or after the addition of 1.0 mM paraquat to the test systems for IDH, GAPD or G-6-PD the inhibitory effect of paraquat was reversed and the reaction rates returned to normal or almost normal. Paraquat (1.0 mM) had no effect on the nicotinamide-adenine dinucleotide-dependent microsomal respiration, and no basic differences were noted between the responses of rat and rabbit lung microsomes exposed to paraquat in vitro.
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