1. The general features of the reaction by which carbon tetrachloride stimulates lipid peroxidation have been elucidated in rat liver microsomal suspensions and in mixtures of microsomes plus cell sap. The production of lipid peroxides has been correlated with malonaldehyde production in the systems used. 2. The stimulation of malonaldehyde production by carbon tetrachloride requires a source of reduced NADP(+) and is dependent on the extent of the endogenous peroxidation of the microsomal membranes: if extensive endogenous peroxidation occurs during incubation then no stimulation by carbon tetrachloride is apparent. 3. The stimulation of malonaldehyde production by carbon tetrachloride has been shown to be proportional to the square root of the carbon tetrachloride concentration in the incubation mixture. It is concluded that the stimulation of malonaldehyde production by carbon tetrachloride results from an initiation process that is itself dependent on the homolytic dissociation of carbon tetrachloride to free-radical products. 4. The increased production of malonaldehyde due to carbon tetrachloride is accompanied by a decreased activity of glucose 6-phosphatase in rat liver microsomal suspensions. 5. The relative activities of bromotrichloromethane, fluorotrichloromethane and chloroform have been evaluated in comparison with the effects of carbon tetrachloride in increasing malonaldehyde production and in decreasing glucose 6-phosphatase activity. Bromotrichloromethane was more effective, and fluorotrichloromethane and chloroform were less effective, than carbon tetrachloride in producing these two effects. It is concluded that homolytic bond fission of the halogenomethanes is a requisite for the occurrence of the two effects observed in the endoplasmic reticulum.

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1. The effects of a number of free-radical scavengers and other agents on the stimulation of malonaldehyde production due to low concentrations of carbon tetrachloride have been studied in rat liver microsome suspensions. 2. Promethazine, propyl gallate and NN'-diphenyl-p-phenylenediamine were extremely active in inhibiting the stimulation of malonaldehyde production due to carbon tetrachloride; inhibitory effects were demonstrable with these agents at 0.1mum. 3. Low concentrations (1-100nm) of vitamin E-polyethylene glycol 1000-succinate increased the stimulation of malonaldehyde production due to carbon tetrachloride, but higher concentrations of the vitamin E preparation decreased both the stimulation due to carbon tetrachloride and the endogenous peroxidation that occurs in the absence of carbon tetrachloride. 4. Other agents tested that were effective in the range 1-20mum in decreasing the stimulation of malonaldehyde production due to carbon tetrachloride were inosine, desferrioxamine and EDTA. Agents tested that were not effective, except at very high concentrations (100mum or greater), were Nupercaine, Cetab and sodium phenobarbitone. 5. The results are discussed in terms of the mechanisms responsible for the observed inhibitions of malonaldehyde production, and of the relevance of the in vitro system to the liver damage produced by carbon tetrachloride in vivo.

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Cultured normal mammalian melanocytes exposed to a variety of antioxidants in the presence of millimolar concentrations of 4-hydroxyanisole exhibit dose-dependent modifications of cytotoxicity. While some antioxidants reduced the extent of damage produced by 4-hydroxyanisole, others appeared to increase it. Similar effects were found in a model system using lysis of human erythrocytes as an index of cell damage. Estimations on rat liver microsomes in the presence of tyrosinase and 4-hydroxyanisole showed increased peroxidation only at low substrate concentrations.

A. S. HALLSWORTH 1964 3. The c-amino group availabilities of the 640A, 220A' and structureless fibrils were also identical (69-5 %) on dinitrophenylation at higher ionic strengths.4. When dinitrophenylated in water, the proportions of reactive c-amino groups in the 640A and the two long-spacing aggregates were considerably less (approx. 44 %) than those of the same aggregates in 1 % sodium chloride.5. As the ionic strength of the aqueous dinitrophenylation medium was decreased, the molar ratio of lysyl to hydroxylysyl c-amino groups available to fluorodinitrobenzene in the 640A and the two long-spacing aggregates also decreased.6. Most samples of the fibrous long-spacing aggregate contained about 10 % of the glycoprotein used in their preparation.7. The significance of these findings is discussed in relation to the calcification of collagen fibrils in vitro.

1. The actions of various inhibitors of the microsomal NADPH-cytochrome P-450 electron-transport chain have been studied on the stimulatory effect of carbon tetrachloride on malonaldehyde production. 2. Carbon monoxide, pchloromercuribenzoate, ,-diethylaminoethyl-3,3'-diphenylpropy1 acetate (SKF 525A) and nicotinamide did not decrease the stimulatory action of carbon tetrachloride on malonaldehyde production when present in concentrations shown to be capable of strongly inhibiting the demethylation of aminopyrine. 3. In contrast with the effects of the substances mentioned above, low concentrations of cytochrome cstrongly depressedthe stimulatory action ofcarbon tetrachloride on malonaldehyde production while increasing the endogenous rate of peroxidation. 4. Aging the microsomal suspensions at 0°C caused a rapid decrease in aminopyrine demethylation activity and in lipid peroxidation catalysed by ADP and Fe2+. The stimulation of malonaldehyde production by carbon tetrachloride was relatively unaffected, however, by aging the microsomes at 0°C for 3 days; during this period cytochrome P-450 decreased by more than 30%. 5. The conclusion is reached that the interaction between carbon tetrachloride and the NADPH-cytochrome P-450 electron-transport chain necessary for the stimulation of malonaldehyde production involves a locus near to if not identical with the NADPH-cytochrome c reductase flavoprotein.General features of the enzyme reaction responsible for the stimulation of malonaldehyde production by carbon tetrachloride in suspensions of rat liver endoplasmic reticulum were described in the preceding paper (Slater & Sawyer, 1971a).The components of the endoplasmic reticulum required for the stimulation of malonaldehyde production by carbon tetrachloride have been studied and evidence is presented here that the major interaction occurs in the vicinity of the NADPH-cytochrome c reductase flavoprotein. Differences in the pathways involved in the stimulatory action of carbon tetrachloride on lipid peroxidation or in the endogenous peroxidation that is dependent on NADPH in the absence of carbon tetrachloride have also been studied; it has been found possible to affect selectively either of these two routes independently of the other. Some of these results have been reported in abbreviated form (Slater, 1967a,b).