Cytochrome free radical intermediates

No information is available on the adverse health effects of hexachloroethane in humans. Animal studies revealed that hexachloroethane primarily causes liver and kidney toxicity. Effects on the nervous system and lungs have also been reported. The mechanism by which these effects are mediated is not well characterized. Reductive metabolism by cytochrome P-450 and production of a free radical intermediate have been suggested as factors in hexachloroethane-induced hepatotoxicity (Nastainczyk et al. 1982a Thompson et al. 1984 Town and Leibman 1984). Accordingly, one possible approach may be to reduce free radical injury. To that end, oral administration of N-acetylcysteine can be used as a means of reducing free radical injury. Also, oral administration of vitamin E and vitamin C may be of value since they are free radical scavengers. 

In the course of the reaction a number of substrates, termed redogenic , were capable of reducing a number of electron acceptors, such as oxygen, methylene blue, and cytochrome c, and the reducing agent was the free radical intermediate. This same peroxidase system was found to be capable of generating a free radical from chlorpromazine [66], which was capable of reducing the enzyme intermediate peroxidase compound II to peroxidase a result not obtained with the other substrates. 

Of the plant peroxidases, which are found in abundance in the peroxisomes, the 40-kDa monomeric horseradish peroxidase has been studied the most. " It occurs in over 30 isoforms and has an extracellular role in generating free radical intermediates for polymerization and crosslinking of plant cell wall components. Secreted fungal peroxidases, e.g., such as those from Coprinus ° and Arthromyces form a second class of peroxidases with related struc-tues. A third class is represented by ascorbate peroxidase from the cytosol of the pea " and by the small 34-kDa cytochrome c peroxidase from yeast mitochondria (Fig. 16-11). The latter has a strong preference for reduced cytochrome c as a substrate (Eq. 16-9). " ... 

Several studies have shown that the reactive binding species generated by 1,1-dimethylhydrazine metabolism may be free radical intermediates. Rat liver microsomes and rat hepatocytes are capable of metabolizing 1,1-dimethylhydrazine to form methyl radical intermediates (Albano et al. 1989 Tomasi et al. 1987). The formation of tliese radicals was inhibited by the addition of inhibitors of cytochrome P-450 (SKF 525A, metyrapone, and carbon monoxide) and inhibitors of the flavin-containing monooxygenase system (methimazole). The formation of free radicals could also be supported nonenzymatically by the presence of copper ion (Tomasi et al. 1987). These data indicate that at least two independent enzyme systems and one nonenzymatic pathway may be involved in the metabolism of... 

In the enzymatic reactions themselves, there is also strong evidence to support a stepwise mechanism involving free-radical intermediates. For example, cytochrome P-450cam gives hydroxylation of c -camphor only in the 5-exo position, but deuterium-labeling studies show that either the 5-exo or the 5-endo hydrogen is lost (Reaction 5.78). ... 

Fig. 11 Biochemical pathways for the formation, detoxification, and cellular effects of xenobiotic free radical intermediates and reactive oxygen species (ROS). Fe iron, G-6-P glucose-6-phos-phate, GSH glutathione, GSSG glutathione disulfide, H2O2 hydrogen peroxide, FIO hydroxyl radical, LPO lipoxygenase, NADP nicotinamide adenine dinucleotide phosphate, O2 superoxide, P450 cytochromes P450, PHS prostaglandin H synthase, SOD superoxide dismutase. (Modified from Wells et al. 1997)...

I. Mechanism of toxicity. Carbon tetrachloride and chloroform are CNS depressants and potent hepatic and renal toxins. They may also increase the sensitivity of the myocardium to arrhythmogenic effects of catecholamines. The mechanism of hepatic and renal toxicity is thought to be a result of a toxic free-radical intermediate of cytochrome P-450 metabolism. (Bioactivation of CCI4 has become a model for chemical toxicity induced by free radicals.) Chronic use of metabolic enzyme inducers such as phenobarbital and ethanol increases the toxicity of carbon tetrachloride. Carbon tetrachloride is a known animal and suspected human carcinogen. Chloroform is embryotoxic and an animal carcincogen. 

Metabolism of the c/s and trans isomers of chlordane by humans and laboratory animals appears to be qualitatively similar (Kutz et al. 1976, 1979), although monkeys may be less efficient than rats (Khasawinah 1989), and rats may metabolize frans-nonachlor more efficiently than humans do (Tashiro and Matsumura 1978). Metabolism appears to be largely oxidative, involving hepatic microsomal cytochrome P-450 (Kawano et al. 1989). Epoxide hydrolase is probably the predominant enzyme involved in further degradation of oxychlordane, but the process appears to be slow in animals and humans. In addition, reductive dehalogenation, probably resulting in the formation of reactive free radical intermediates, may be important in the toxicity of chlordane (Brimfield and Street 1981 Kawano et al. 1989). 

Owing to the wide range of reactions in which flavin coenzymes are involved, a unitary mechanism of action appears unlikely. It seems clear that in enzymes where the fully reduced form is reacting with a one-electron acceptor (such as NADH-cytochrome b-reductase, NADPH-cytochrome c-reductase, and ferridoxin NADP reductase), the mechanism involves flavin semiquinones as intermediates. These are free-radical intermediates and can be detected by electron spin resonance (ESR). Flavin semiquinones can be stabilized because of multiple resonance forms, the odd electron being shared throughout the entire isoalloxazine ring. The free-radical intermediate may be further oxidized or reduced, one electron at a time. A number of enzjmies requir-... 

Nitroblue tetrazolium (NBT, 3,3 -(3,3,-dimethoxy-l,l,-biphenyl-4,4 -diyl)bis-2-(4-nitrophe-nyl)-5-phenyl-2H-tetrazolium dichloride) is reduced by superoxide to formazan as a final product, which can be measured spectrophotometrically [73]. Although the rate constant for NBT reduction by superoxide is moderately high 5.88+0.12x 104 1 mol 1 s 1 [74], the formation of formazan is not a simple one-electron transfer process, and the final product is formed as a result of disproportionation of intermediate free radicals. Similar to cytochrome c, NBT is easily reduced by the other reductants that confines its application for superoxide detection. Moreover, similar to epinephrine, NBT free radical is apparently... 

The metabolism of carbon tetrachloride proceeds via cytochrome P-450-dependent dehalogenation (Sipes et al. 1977). The first step involves cleavage of one carbon-chlorine bond to yield Cl- and a trichloromethyl free radical, which is then oxidized to the unstable intermediate trichloromethanol, the precursor of phosgene. Hydrolytic dechlorination of phosgene yields C02 and HC1 (Shah et al. 1979). Although there are similarities in the metabolism of chloroform and carbon tetrachloride, metabolic activation of chloroform produces primarily phosgene, whereas the level of phosgene production from... 

Thus, the susceptibility is the result of accumulation of the drug in the target organ to reach concentrations not achieved in other tissues. This is then followed by what is probably a combination of events such as formation of a reactive intermediate, possibly a free radical, stimulation of lipid peroxidation and depletion of GSH, and then peroxidative damage to cell membranes and mitochondria. Whether metabolic activation by cytochromes P-450, or chemical rearrangement, or reductive activation, or all the three are involved is not currently clear. 

Mitochondria are also involved in the cell s response to oxidative stress. As we have seen, several steps in the path of oxygen reduction in mitochondria have the potential to produce highly reactive free radicals that can damage cells. The passage of electrons from QH2 to cytochrome bL through Complex III, and passage of electrons from Complex I to QH2, involve the radical Q as an intermediate. The Q can, with a low probability, pass an electron to 02 in the reaction... 

On the contrary, in cytochrome c peroxidase, the intermediate is a (porphy-rin)Fe(IV)=0 complex with a free radical derived from the one-electron oxidation of an amino acid residue in the vicinity of the heme [10], The second step of the HRP catalytic cycle is the one-electron oxidation by compound I of HRP substrates that are generally electron-rich aromatic compounds. This leads to the second intermediate, called compound II, of the catalytic cycle, which is a (porphyrin) Fe(IV)=0 complex. The one-electron reduction of compound II by HRP substrates regenerates HRP in its resting iron(III) state (Figure 5a). 

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