T. F. Slater

Free Radical Mechanisms in Tissue Injury

Free radicals are molecules or molecular fragments containing a single unpaired electron. In general, free radicals are reactive chemically, some (e.g. HO•) being extremely reactive. However, certain types of free radical, such as nitroxyl-radicals and free radicals stabilized by steric or derealization features, are much less reactive and a few (e.g. diphenyl picryl hydrazyl) are stable enough to be crystallised and stored at temperatures above 0°. Table 1 gives the general structures of free radicals that will be discussed in this short review.

Reactions of the carbon tetrachloride-related peroxy free radical (CCl3O2.) with amino acids : Pulse radiolysis evidence

Abstract Pulse radiolysis studies indicate that the free radical CCl3O2. but not the radical CCl3 reacts rapidly with tryptophan, tyrosine, phenol or promethazine to yield transient products with strong visible absorption spectra.

Cytochrome P-450 distribution in rat liver and the effect of sodium phenobarbitone administration

A microspectrophotometric method for assaying cytochrome P-450 in fresh 24 micrometer unfixed cryostat sections of rat liver has been developed. When used to assay this cytochrome in sections of microsomal preparations it has yielded results equivalent to those obtained by the conventional spectrophotometric assay of the same preparations. Random measurements made throughout sections of liver have given mean values for cytochrome P-450 concentrations which are twice those measured in microsomes prepared from the livers of the same animals (not corrected for the yield in the homogenate). Measurements of the cytochrome P-450 content of liver cells by the microspectrophotometric method show that in liver from male Wistar rats, cells nearer to the central veins contain up to twice as much cytochrome P-450 as those nearer to the portal tract (mean cell concentrations of 26.4 (+/-4.4) mumol/l and 17.5 (+/-3.0) mumol/l respectively). In the livers from similar rats, killed at the same time, but which has received 1 mg/ml sodium phenobarbitone in their drinking water for one week, the cells near the central vein contained up to five times as much cytochrome P-450 as those near the portal tract (mean cell concentrations of 77.3 (+/-25.0) mumol/l and 28.3 (+/-9.6) mumol/l respectively). The results show a selective increase in cytochrome P-450 content by the cells in the centrilobular region after treatment with sodium phenobarbitone and a smaller increase by some of the cells in the periportal region.

Free radical mechanisms in relation to tissue injury

The present paper is a broad-ranging account of free-radical biochemistry in general, and of free-radical mechanisms of tissue injury in particular. Because it is broad-ranging within tight constraints of length it is necessarily lacking in detail on some issues of relevance; the following reviews can be consulted for additional coverage: Slater (1972, 1978, I&), Pryor (1976-84), Mason (1982), Halliwell & Gutteridge (1984). Free radicals can be defined as molecules or molecular fragments containing a single unpaired electron; this unpaired electron usually gives a considerable degree of chemical reactivity to the free radical: in chemical formulas the unpaired electron is conventionally shown as a ‘superscript dot’, as with the hydroxyl free radical OH’. Free radicals can be produced in the cells and tissues of our bodies by various processes and reactions; Table I divides these into two main sections: (I) formation of free radicals as a result of the impact of radiation, and (2) formation by reduction4xidation (redox) reactions involving the transfer of an electron. For discussion of the mechanisms that result in the production of free radicals, see Pryor (1966) and Slater (1972). Table I also indicates major ways by which free-radical intermediates can be converted to non-radical products by the action of free-radical scavengers; discussion of this important aspect of free-radical biochemistry is at the end of this paper. As already mentioned, free radicals are usually reactive chemically although some important examples of stable free radicals are known, such as diphenylpicrylhydrazyl (DPPH’) and Fremy’s salt (potassium nitrosodidphonate). The

Activation of chloroform and related trihalomethanes to free radical intermediates in isolated hepatocytes and in the rat in vivo as detected by the ESR-spin trapping technique

When hepatocytes isolated from phenobarbital-induced rats were incubated with chloroform and the spin trap phenyl-t-butyl nitrone (PBN) under anaerobic conditions, a free radical-spin trap adduct was detectable by ESR spectroscopy. A similar incubation of hepatocytes in the presence of air resulted in an ESR signal that was eight times less intense than that seen under anaerobic conditions; incubation mixtures exposed to pure oxygen had no detectable adduct signal. A significant reduction in the signal intensity was also produced by the addition of cytochrome P-450 inhibitors such as SKF-525A, metyrapone and carbon monoxide, indicating that free radical formation depended upon the reductive metabolism of chloroform mediated by the mixed oxidase system. The origin of the CHCl3-derived free radical has been confirmed by using [13C]CHCl3, while the comparison between the ESR spectra obtained in the presence of deuterated chloroform (CDCl3) and bromodichloro-methane (CHBrCl2) suggests that the free radical derived from CHCl3 may be CHCl2. Free radical intermediates were also detected during the aerobic and anaerobic incubation of isolated hepatocytes with bromoform (CHBr3), and iodoform (CHI3). The intensity of the ESR signal obtained with the various trihalomethanes increases in the order CHCl3 less than CHBrCl2 less than CHBr3 less than CHI3. The formation of PBN-free radical adducts has also been observed in phenobarbital-induced rats in vivo when intoxicated with chloroform, bromoform or iodoform, suggesting that the reductive metabolism of trihalomethanes might be of relevance to their established toxicity in the whole animal.

Studies on the reduction of nitroblue tetrazolium chloride mediated through the action of NADH and phenazine methosulphate.

The general features of the reduction of nitroblue tetrazolium chloride (NBT) by NADH and phenazine methosulphate (PMS) have been studied under aerobic and anaerobic conditions. Under aerobic condition the reduction appears to be mediated through the intermediate formation of the superoxide anion radical O2-.; this reaction is strongly inhibited by superoxide dismutase and by a number of O2-. scavengers such as propyl gallate, (+)-catechin, manganous ions, reduced glutathione and benzoquinone. Cupric ions inhibited the overall reaction by reoxidising reduced PMS. Under anaerobic conditions, superoxide dismutase had only a small inhibitory action and, with the exception of cupric ions, the other substances mentioned above were ineffective as inhibitors. The data presented show that the use of NBT to detect the presence of O2-. is fraught with difficulties due to an equally rapid reduction of NBT by NADH and PMS under anaerobic conditions.

Comparative evaluation of the antioxidant activity of α-tocopherol, α-tocopherol polyethylene glycol 1000 succinate and α-tocopherol succinate in isolated hepatocytes and liver microsomal suspensions

Abstract The antioxidant activity of α-tocopherol polyethylene glycol 1000 succinate (TPGS) and of α-tocopherol succinate (TS) has been examined in isolated hepatocytes and microsomal fractions from rat liver. Both TPGS and TS require esterase activity to yield free α-tocopherol and, hence, antioxidant activity. TPGS and TS consistently exerted a more effective antioxidant protection than an equivalent amount of directly-added free α-tocopherol. The low antioxidant efficiency of directly added free α-tocopherol in such water-based experimental systems as used here seems to be due to its extreme hydrophobicity. TPGS, on the other hand, is an extremely hydrophilic compound that is being examined as a useful source of α-tocopherol in certain clinical situations and is here shown to be a convenient and effective source for experimental studies into lipid peroxidation and antioxidant mechanisms.

Reaction of the trichloromethyl and halothane-derived peroxy radicals with unsaturated fatty acids: A pulse radiolysis study

The absolute rates of reaction of the trichloromethylperoxy radical, CCl3OO., derived from carbon tetrachloride and the halothane peroxy radical, CF3CHClOO., with oleic, linoleic, linolenic and arachidonic acids have been determined using the fast reaction technique of pulse radiolysis. In general, the rates of reaction of the radical derived from carbon tetrachloride are approximately five times greater than those for the halothane related radical. In both cases the rate constant increases with increasing unsaturation of the fatty acid in agreement with the known greater susceptibility of polyunsaturated fatty acids to peroxidative decomposition.

Lipid peroxidation and molecular damage to polyunsaturated fatty acids in rat liver. Recognition of two classes of hydroperoxides formed under conditions in vivo.

The diene conjugates formed during the autoxidation of microsomal lipid extracts, and in endoplasmic reticulum in vivo after exposing rats to CCl4 have been examined by second derivative absorption spectrophotometry. Within a few minutes after administering CCl4 to a rat there is a pronounced signal in microsomal lipid extracts that is ascribed to the cis-trans diene conjugates of microsomal polyunsaturated fatty acids. Somewhat later a second signal becomes evident that is ascribed to trans-trans isomers. The appearance of the trans-trans isomer is very strongly suppressed by prior administration of vitamin E to the rat. It is concluded that the relative contents of cis-trans and trans-trans dienes in lipid extracts of tissue reflect the tissue contents of hydrogen donors as already established for model experiments with polyunsaturated fatty acids in vitro.

Spin trapping of free radical products of CCI4 activation using pulse radiolysis and high energy radiation procedures

Free radicals have been suggested as activated intermediates in many types of tissue injury [ 1,2]. The techniques of electron spin spectroscopy (ESR) and pulse radiolysis are being increasingly used to characterise the free radicals involved, and to obtain quantitative data on their chemical reactivity [3,4]. Carbon tetrachloride is an important and much studied hepatoxin, which is metabolised by the NADPHcytochrome P450 electron-transport chain to an activated intermediate generally considered to be the trichloromethyl radical [5,6]. Attempts [7-91 to detect the Ccl; free radical in whole liver or liver fractions using direct ESR analysis have not been successful, and attention has consequently been directed to the less direct ESR spin trapping techniques [lo-121. Although this procedure is promising for studies both in vitro and in vivo, there are complications in interpretation of the ESR spectra of the free radical species produced in complex biomembrane environments [ 12,131. For this reason, we have studied the interaction of Ccl; with the spin-trap phenylbutylnitrone in a simplified model system where the Ccl; radicals are produced by a pulse of high energy electrons. We have previously obtained kinetic data on the reactivity of Ccl; and its peroxy-derivative CClaO; [14] in this model system. These data, together with ESR analysis with 13CC14 (as suggested in [l]), allow us to unequivocally identify the spintrap adducts of Ccl; and CCl,O;; identical spectra are also reported for microsomal systems, intact hepatocytes and under conditions in vivo.