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Long-Range Effects of Products of Carbon Tetrachloride-Stimulated Lipid Peroxidation* G. UGAZIO,? M. V. TORRIELLI,? E. BURDINO,? B. C. SAWYERS and T. F. SLATEKS tlstituto de Patologia Generale, Uniuersita di Torino, Corso Raflaello 30, 10125 Torino, Italy, and $Department of Biochemistry, Brunel University, Uxbridge, Middx. UB8 3PH, U.K. Reaction products of lipid peroxidation have been found to affect both erythrocytes (Pfeifer & McCay, 1971) and blood platelets (Silver et al., 1973, 1974). Recent findings in our laboratory (Bini et al., 1975) suggest a possible pathogenic role for lipoperoxidation in producing severe systemic disturbances after a large dose of CCI4. Peroxidation of unsaturated fatty acids gives rise to the formation of various classes of reactive materials, including short-chain aldehydes (Schauenstein, 1967; Cobb & Day, 1965); malonaldehyde, for example, is a well-known derivative of peroxidative decomposition of polyunsaturated fatty acids. Further, prostaglandins are exceptionally reactive products of enzymic or non-enzymic reactions affecting lipoperoxides (Nutgeren et a/., 1966, 1967). The primary aim of the present study was to investigate the possible effects on erythrocytes, blood platelets and capillary permeability of products of lipid peroxidation stimulated by CC14. Male Wistar rats were used in all the experiments. Haemolysis was studied by incubating rat erythrocytes in vitro, prepared by the method of Pfeifer & McCay (1971). Erythrocytes (50pl equivalent) were incubated in the presence of 50mg equiv. of liver microsomal preparation and 1mM-NADPH (Sigma Chemical Co., St. Louis, MO, U.S.A.) in a final volume of 1ml. Where appropriate, the CCI, concentration was 60pI/ml. All the solutions were prepared with 0.05M-phosphate buffer, pH 7.4, containing 0.1 SSM-N~CI. In some instances, the following antioxidants or metabolites of microsomal mixed-function oxidase systems were added at final concentrations ranging between 0.6 and 3mM: NN'-diphenyl-p-phenylenediamine (Schuchardt, Munich, Germany), 2,6-di-t-butyl-pcresol (Fluka A.G., Buchs SG, Switzerland), Santoquin (6-ethoxy-l,2-dihydro-2,2,4trimethylquinoline; Monsanto Co., St. Louis, MO, U.S.A.), propyl gallate (Carlo Erba, Milano, Italy, DL-a-tocopherol (vitamin E; Merck, Darmstadt, Germany), Methyl Viologen (paraquat, 1,l '-dimethyl-4,4'-bipyridinium dichloride; Sigma), ergocalciferol (vitamin Dt; Merck), aminopyrine (4-dimethylamin0-2,3-dimethyl-l -phenyl-3-pyrazolin-5-one; Aldrich-Europe, Beerse, Belgium), codeine (codeine phosphate ; Macfarlane Smith Ltd., U.K.), SKF-525A (2-diethylaminoethyl-2,2-diphenyl-valerate hydrochloride; Smith, Klein and French Laboratories, Philadelphia, PA, U.S.A.), and/or GSH (reduced glutathione; Boehringer, Mannheim, Germany). The mixture was incubated in a Dubnoff apparatus at 37°C for 45min, then diluted with buffered saline to 5ml and centrifuged at 0°C for lOmin at 1OOOOg. Haemolysis was determined on the basis of the E545of the clear supernatnat, and production of malonaldehyde was assayed on the same sample with thiobarbituric acid (Bieri &Anderson, 1960). Where haemolysis was to be related to either lipid peroxidation or the production of halogenated free radicals, the solution was mixed with 0.1 mM-EDTA and incubated under Nz by the procedure of Recknagel et al. (1975). Chloroform formation, detected quantitatively by a g.1.c. apparatus equipped with an electron capture detector (Fractovap, model GV; Carlo Erba), was interpreted as a sign of free-radical reactions occurring in the absence of fully developed lipid peroxidation. Direct haemolysis induced by lipoperoxides was followed by treating rat erythrocytes with linoleic acid hydroperoxide (prepared as described by O'Brien, 1969) as such or as a

* This paper was presented at the 560th Meeting, held at the University ofOxford on I6 and 17 December 1975. Delays in publication have occurred as a result of factors outside the authors' control. Vol. 4

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BIOCHEMICAL SOCIETY TRANSACTIONS

None

A

Aminopyrine (3) Compound SKF-525A (3)

GSH (3) Propyl gdlate (0.6)

Santoquin (0.6) (+) Paraquat (0.6) Dlphenylphenylenediamine (0.6) a-Toco p he r o I (0.6) Di-t-burylcrerol (0.6) Ergocalciferol (0.6)

0, Lipid

v

peroxidation (Es43); M, haemolysis (Es45).

mixture with antioxidants, at concentrations of 6.4 and 1 5 m respectively. ~ The final concentrations of these two compounds in the incubation mixture were 0.48 and 1.13mM respectively. Both short-chain aldehydes and prostaglandins were tested for their direct effect on haemolysis and platelet aggregation. Methylglyoxal, 4-hydroxy-2,3-trans-pent-2-en-l -al, 2-oxopent-2-en-1-a1 or 2-methylpent-2-en-1-a1 were dissolved in 0.9 % NaCl freshly before use at a stock concentration of 3 0 m ~ Malonaldehyde . ( 8 0 m ~was ) prepared by treating 1,1,3,3-tetraethoxypropane(K & K Laboratories, Plainview, NY, U.S.A.) with Dowex 50-WX2 (Serva, Heidelberg, Germany) in water. Prostaglandins A2,B2, E2and F,, (trimetharnine salt) (kindly provided by Dr. J. E. Pike, Upjohn Co., Kalamazoo, . MI, U.S.A.) were dissolved in 0.9 % NaCl at a stock concentration of 3 m ~Haemolysis was studied by treating washed erythrocytes in 0.9 % NaCl with short-chain aldehydes, respectmalonaldehyde or prostaglandins at final concentrations up to 25,40 or 1 . 5 m ~ ively. The mixture was incubated in a Dubnoff apparatus at 37°C for 45min. Rat blood platelets were harvested by the method of Haslam (1964) and treated in oitro at 37°C with either aldehydes or prostaglandins at final concentrations up to 10 or 1mM respectively. Capillary permeability was studied after intradermal injection of linoleic acid hydroperoxide, aldehydes or prostaglandins in rats previously treated with Trypan Blue (BDH, Poole, Dorset, U.K.; 2.5mg in 0.5ml of 0.9% NaCl per lOOg body wt., intravenously). Challenging compounds were given in the final volume of 25p1 at doses between 4nmol and 2pmol respectively for lipoperoxides or aldehydes. Stain diffusion was observed 30min after the intradermal injection. All the antioxidants and the substrates of the mixed-function oxidase system inhibit lipid peroxidation caused by CCl, in the presence of NADPH (Fig. 1). Further, all of them except compound SKF-525A strongly decreased the extent of haemolysis. This compound has a peculiar direct effect on the integrity of erythrocytes, and in fact it by itself was found to induce haemolysis in the absence of lipid peroxidation (results not 1976

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Table 1. Biological effects of products of CCI,-stimuIated lipid peroxidation The smallest concentration or dose active is reported. 'None' means no effect with concentration or dose as follows: '2 7 m ~ ;a ' 4 5 m ~ ; IOmM; '3.0,umol; 1 . 5 m ~ ; 0.75pmol. Platelet Capillary Compound Mol.wt. Haemolysis aggregation permeabilization Linoleic acid 280.4 Linoleic acid hydroperoxide 310.4 4 nmol 5 0 p ~ 240 p~ C'C13 118.4 None 4-Hydroxy-2,3-trans-pentenal 100 Noneb 0.1 5 pmol None" 1.15pmol None None 2-Oxopentenal 100 None None' 2-methyl pent-2-en01 98 None Methy lglyoxal 72 None None None None 2.00pmol Malonaldeh yde 86 None" None' Prostaglandin A2 Noned 334.5 1 mM None 1 mM None Prostaglandin B2 334.5 None 1mM None Prostaglandin E2 352.5 None 1mM None Prostaglandin Flu 475.6 7.1 pmol 5-Hydroxytryptamine 176.2 shown). Ergocalciferol, known as a haemolytic agent (Spirichev & Blazhevich, 1968), did not affect malonaldehyde production and haemolysis under our experimental conditions. Recent findings have shown that codeine affects CCI,-induced haemolysis less than aminopyrine does (results not shown), and this is consistent with the biochemical properties of these two compounds; codeine is devoid of the direct antioxidant effect shown by aminopyrine. Results reported in Table 1 indicate that linoleic acid hydroperoxide is active in modifying all the parameters studied in this work at very small doses or concentrations. Malonaldehyde or short-chain aldehydes, as well as the prostaglandins studied here, did not produce lysis of erythrocytes in vitro other than at high concentrations. Platelet aggregation does not occur in the presence of aldehydes, but takes place when prostaglandins are used. Malonaldehyde and 2-oxopentenal inhibit or delay both haemolysis and platelet aggregation caused by linoleic acid hydroperoxide (results not shown). Capillary-wall permeability was strongly affected by linoleic acid hydroperoxide, although the smallest active dose was much higher than that of 5-hydroxytryptamine (serotonin), namely 4nmol instead of 7.1 pmol. Moreover, 4-hydroxy-2,3-trans-pentenal, 2-oxopentenal and malonaldehyde produced a large increase in the diffusion of Trypan Blue at doses as low as 0.15 and 2,umol respectively. The results of these experiments clearly show that both CC14-stimulated lipid peroxidation and lipoperoxides themselves cause haemolysis and platelet aggregation. Further, lysis of erythrocytes seems to depend on fully developed lipid peroxidation, whereas no haemolysis takes place when only initiation of free-radical reactions occurs (results not shown). Lipoperoxides and some of the short-chain aldehydes affect capillary permeability in uivo, according to the hypothesis reported by Bini et al. (1975). These findings can elucidate, at least partially, the role played by lipoperoxides and the secondary products of lipid peroxidation in the pathogenesis of non-hepatic lethal lesions induced by CCI,. This work was supported by a grant from Consiglio Nazionale delle Ricerche, Roma, Italy (CT-74.OO278.04).

Bieri, J. G . &Anderson, A. A. (1960) Arch. Biochem. Biophys. 90, 105-110 Bini, A. M., Burdino, E., Cessi, C . , Pagnoni, V. M., Ugazio, G. & Vannini, V. (1975) Proc. Int. Meet. Recent Advances in Biochemical Pathology: Toxic Liver Injury, Torino, in the press

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BIOCHEMICAL SOCIETY TRANSACTIONS

Cobb, W. Y . &Day, E. A. (1965) J. Am. Oil Chem. Soc. 42,420-422 Haslam, R. J . (1964) Nature (London) 202, 765-768 Nutgeren, D. H., Beerthuis, R. K. & Van Dorp, D. A. (1966) Recl. Truu. Chim. Puys-Bus 85, 405419 Nutgeren, D. H., Vonkeman, H. & Van Dorp, D. A. (1967) Recl. Truu. Chim. Pays-Bas 86, 1 237-1 245 O’Brien, P. J. (1969) Can. J. Biochem. 47, 485499 Pfeifer, P. M . & McCay, P. B. (1971) J. Biol. Chem. 246, 6401-6408 Recknagel, R. O., Hruszewycz, A. &Glende, E. A., Jr. (1975)Proc.In,. Meet. Recent Adouncesin Biochemical Pathology: Toxic Liuer Injury, Torino, in the press Schauenstein, E. (1967)J. Lipid Res. 8,417428 Silver, M . J., Smith, J. B., Ingerman, C. & Kocsis, J. J. (1973)Prostaglandins 4,863-875 Silver, M. J., Hoch, W., Kocsis, J. J., Ingerman, C. M. &Smith, J. B. (1974) Science 183,10851087 Spirichev, V. B. & Blazhevich, N. V. (1968) Vopr. Med. Khim. 14, 371-375

1976

Long-range effects of products of carbon tetrachloride-stimulated lipid peroxidation.

560th MEETING. OXFORD 353 Long-Range Effects of Products of Carbon Tetrachloride-Stimulated Lipid Peroxidation* G. UGAZIO,? M. V. TORRIELLI,? E. BUR...
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