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Growth-related lipid peroxidation in tumour microsomal membranes and mitochondria
537 Biochimica et Biophysics Acta, 574 (1979) 537-541 o Elsevier/North-Holland Biomedical Press
BBA Report BBA 51257
GROWTH-RELATED LIPID PEROXIDATION IN TUMOUR MICROSOMAL MEMBRANES AND MITOCHONDRIA
G.M. BARTOLI and T. GALEOTTI* Institute of General Pathology, Catholic University, S. Cuore,, Via Pineta Sacchetti 644, 00168 Roma (Italy) (Received April 2nd, 1979) Key words: Lipid peroxidation;
Tumor growth; (Microsomal membrane,
Mitochondria)
Summary
Microsomes and mitochondria isolated from Morris hepatomas 3924A (fast-growing) and 44 (slow-growing) and Ehrlich ascites tumour cells exhibit a NADPHdependent peroxidation of endogenous lipids lower than that of the corresponding fractions from rat liver. Moreover, the 0, and ascorbate-dependent lipid peroxidations are decreased in microsomes from the two Morris hepatomas. The peroxidative activity appears to be inversely related to the growth rate of the tumours. It is suggested that the low susceptibility of tumour membranes to peroxidative agents may be a factor responsible for the high mitotic activity of this tissue.
Early studies have indicated that the non-enzymic lipid peroxidation induced by ascorbic acid is very low in tumour mitochondria [1,2]. Recently, Player et al. [3] also observed a low activity of the NADPHdependent lipid peroxidation in mitochondria isolated from hepatoma D30. However, no data are yet available on the non-enzymic or enzymic lipid peroxidation in tumour microsomal membranes. Such additional information appears of particular interest in the light of the hypothesis that peroxides generated from peroxidation of polyunsaturated fatty acids inhibit cell division [ 4-71. Moreover, the possibility to work with membranes isolated from tumours of different growth rates may enable a more definite examination of the correlation between peroxide production and mitotic activity in transformed cells. In this paper, we report results of measurements of NADPH-dependent *To whom all correspondence should be sent.
538
lipid peroxidation in microsomes [ 81 and mitochondria [ 91 from normal and cancerous rat liver with a large difference in growth rate (Morris hepatomas 3924A and 44), as well as from Ehrlich ascites tumour cells. Data are also presented of the lipid peroxidation induced by superoxide redicals (0; ) and ascorbate in microsomes from rat liver, Morris hepatomas 3924A and 44. The results support the view that cell proliferation in tumours is correlated with the peroxides generated during the turnover of membrane lipids. A preliminary account of part of this work has been given [lo]. Morris hepatoma 3924A and 44 [ll] were transplanted into both hind legs of inbred rats of the ACI/T (subcutaneous) and Buffalo (intramuscular) strains, respectively. Some experiments were performed using hepatoma 44-bearing rats inoculated in H.P. Morris laboratory and shipped by air. The tumours were excised 3-4 weeks (hepatoma 3924A) and 4-6 months (hepatoma 44) after tr~spl~tation. Ehrlich hyperdiploid ascites tumour cells of the Lettre’ strain (ELD) were maintained by weekly intraperitoneal transfer of ascitic fluid in albino Swiss mice and harvested 6-S days after inoculation, Other details concerning the isolation of hepatomas and preparation of ascites cells are reported elsewhere [12,13]. Normal male rats of the ACI/T strain, weighing 150-200 g, were used for rat liver preparations. Mito~hondria were isolated in 0.25 M sucrose, 0.5 mM EGTA, 5 mM Hepes (pH 7.4) by the conventional procedure, except for ELD cells (which were treated with bacterial protease before homogenization [ 141). The mitochondria were washed once with the isolation medium and twice with 0.125 M KCl, 20 mM Tris-HCl (pH 7.4) and suspended in the latter medium. Liver and hepatoma microsomes were prepared from the postmitochondrial supe~atant after cent~fugation at 18 000 X g for 10 min and 105 000 X g for 60 min. ELD cells were suspended in the sucrose/EGTA/Hepes medium and microsomes prepared from the homogenate obtained by Ultra-Turrax (Janke and Kunkel KG) disruption of the cells at 0°C for 5 s [ 151. Centrifugation was performed at 8000 and 18 000 X g for 10 min and 105 000 X g for 60 min. The microsomal fractions were washed once in 0.15 M KCl, 50 mM Tris-HCI (pH 7.5). Lipid peroxidation was measured at 25°C as malonaldehyde production in the presence of 4 mM ADP, 0.05 mM FeCl, and, when indicated, 0.33 mM xanthine. The reaction was started either by 0.4 mM NADPH or by 50 Mg/ml xanthine oxidase or by 0.25 mM sodium ascorbate. Malonaldehyde was determined at 535 nm by the thiobarbituric acid assay [ 161. NADPH~ytochrome c reductase was measured by the method of Jones and Wakil [17]. Proteins were estimated by the biuret method ]18]. Cytochrome c , ADP and NADPH were obtained from Boehringer, Mannheim. Ascorbic acid (sodium salt), xanthine oxidase (Grade I), N-2hydroxyethylpiperazine-N’-2-ethanesulfonic acid (Hepes), thiobarbituric acid and subtilisin (bacterial protease, Type VII) were purchased from Sigma Chemical Co. (St. Louis). EGTA was obtained from Koch-Light Laboratories (UK). All other chemicals were products of E. Merck (Darmstadt) except for NaCN (J.T. Baker Chemical Co., Deventer, The Netherlands). The enzymic lipid peroxidation induced by NADPH in the microsomal fraction isolated from rat liver and three different tumours has been measured (Fig. 1). It is evident that the extent of malonaldehyde production
539
Fig. 1. NADPH-dependent lipid peroxidation of microsomal fractions from rat liver (a), Morris hepatoma 44 (A) and 3924A (0) and Ehrlich ascites tumour cells (X). 0.2 mg/ml (rat liver and hepatoma 44) and 1 mg/ml (hepatoma 3924A and ELD) of microsomal proteins were suspended in an oxygensaturated medium of the following composition: 0.15 M KCl. 50 mM Tris-HCl (pH 7.5). 4 mM ADP and 0.05 mM FeCl, . The suspensions were incubated at 25’C in a Dubnoff metabolic shaker. Each point represents the mean of 4-6 experiments; the vertical lines correspond to twice the S.E.
during 40-min incubation is lower in tumour microsomes with respect to liver, being 20% in the fraction isolated from the well-differentiated Morris hepatoma 44 and nearly 10% in that isolated from the poorly-differentiated Morris hepatoma 3942A. A behaviour similar to that of the hepatoma 3942A is shown by the rapidly growing ELD cells. Thus, it appears that tumour transformation induces a substantial decrease in the rate of NADPHdependent lipid peroxidation and that such change is more pronounced in tumour cells with a rapid growth rate, i.e. hepatoma 3942A and ELD cells. Obviously it is not easy to establish whether the behaviour of lipid peroxidation in tumour microsomes is the cause or the consequence of the different growth rate. However, if the hypothesis that lipid peroxides are able to control cell division is true, on the basis of our data we are inclined to believe that the higher the degree of transformation is, the less the ability of the microsomal NADPHdependent lipid peroxidation is to inhibit tumour growth. Tumour transformation may have induced in microsomes changes either in the activity of the enzyme system involved in lipid peroxidation, i.e. NADPH-cytochrome c reductase [ 191, or in the membrane lipid composition and assembly. Both these possibilities have been tested. The activity of the flavoprotein follows a pattern similar to that of lipid peroxidation, being lower in the membranes from the two fast-growing tumours (Morris hepatoma 3942A: 17.8 + 1.2 (5) nmol - min-’ - mg-’ protein; Ehrlich ascites: 26.1 + 2.0 (5) nmol - min-’ - mg-’ protein) than in the slow-growing hepatoma 44 (42.4 + 1.1 (5) nmol - min-’ mg-’ protein). Such values correspond to 20-30 and 50% of the liver, respectively. It is known that microsomal lipid peroxidation may be also promoted by superoxide radicals [20] l
540
and Pederson and Aust [21] have suggested that the reaction mechanism involved in this system is different from that of the NA~PH-dependent lipid peroxidation. Fig. 2A shows that the rate of lipid peroxidation, induced by xanthine and xanthine oxidase (to generate superoxide radicals), has a behaviour which inversely follows that of the growth ability of the transformed tissue. Indeed, the activity is more markedly depressed in the microsomal fraction isolated from Morris hepatoma 3942A than 44. Similarly, lipid peroxides induced by ascorbate accumulate in a smaller amount in the more rapidly growing tumour (Fig. 2B). These results suggest that, besides
Fig, 2. Lipid peroxidation induced by supzoxide ‘iacikals (Af and ascorbate (3) of microsomd fractions from rat liver I*). Morris hepatoma 44 f&) and Motis he~atoma 3924A
the low activity of NADPH-cytochrome c reductase, changes in the structure andfor composition of tumour microsamal membranes make the fatty acids less vulnerable to peroxidative agents, such as free radicals generated both within the membrane [22,23] and in the soluble fraction of the cell, It has been reported, for instance, that, in normal tissues, the lipid peroxidation is dependent on the content of polyunsaturated fatty acids [24} and Reitz et al. [ZS] have shown that tumour microsomal membranes isolated from the fast growing Morris hepatoma 7777 contain about 50% less phospholipid than the controls. Moreover, in microsomes and mitochondria of this tumour, the polyenoic fatty acids decrease and, concomitantly, the monoenoic increase. In addition, Feo et al. [26,27] have reported that in microsomes and mitochondria isolated from the rapidly growing Morris hepatomas 5123 and 39248, although no changes in the distribution of the phospholip~d classes occur, the molar ratio cholesterol: phospholipids increases significantly, These data could also be relevant in relation to the observation that cholesterol feeding in rats reduces the liver microsomal NADPH-dependent lipid peroxidation [28]. Finally, it should be mentioned that differences
541
were observed in the ultrastructure of the endoplasmic reticulum of Morris hepatomas of different growth rate with respect to rat liver [29]. The NADPHdependent lipid peroxidation has been also measured in mitochondria of rat liver and tumours of different growth rate (rat liver: 12.3 f 1.8 (3) nmol/40 min per mg protein; hepatoma 44: 8.5 (2) nmol/40 min per mg protein; hepatoma 3924A: 6.1 * 0.8 (6) nmol/40 min per mg protein; Ehrlich ascites: 5.9 t 0.4 (4) nmol/40 min per mg protein). Again, it appears that the peroxidative activity is inversely related to the mitotic activity of the tumours. Our data on ELD cells and hepatoma ,3924A are consistent with previous findings made with ascorbate- and NADPHinduced lipid peroxidation of mitochondria from fast-growing tumours [ 2,3] . In conclusion it can be stated that, in analogy to other tissues which undergo very rapid cell division, such as testis and intestinal epithelium [ 301, tumours are somewhat resistant to lipid peroxidation and their growth rate appears to be correlated with the amount of lipid peroxides generated from the peroxidation of the polyunsaturated fatty acids of the membrane phospholipids. We thank Miss Monica Masucci for her help during some phases of this work. This work was in part supported by a Grant from Minister0 Pubblica Istruzione 1977. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Thiele, E.H. and Huff, J.W. (1960) Arch. Biochem. Biophys. 88, 208-211 Utsumi, K., Yamamoto, G. and Inaba, K. (1965) Biochim. Biophys. Acta 105.368-371 Player, T.J., Mills, D.J. and Horton, A.A. (1977) Biochem. Sot. Trans. 5, 1506-1508 Wolfson, N., Wilbur. K.M. and Bemheim. F. (1956) EXP. Ceil Res. 10,556-558 Cole, B.T. (1956) Proc. Sot. EXP. Biol. Med. 93, 290-294 Glushchenko, N.N.. Shestakova. S.V. and DaniIov, V.S. (1975) Biol. Nauki (Moscow) 18. 51-53 Player, T.J., Mills, D.J. and Horton, A.A. (1977) Biochem. Biophys. Res. Commun. 78.1397-1402 Hochstein, P. and Em&r, L. (1963) Biochem. Biophys. Res. Commun. 12, 388-394 Pfeifer. P.M. and McCay. P.B. (1972) J. Biol. Chem. 247, 6763-6769 BartoIi. G.M., Palombini, G. and Galeotti. T. (1978) 12th FEBS Meeting, Dresden. Abstract 2424 Monks. H.P. and Wagner. B.P. (1968) Methods Cancer Res. 4, 125-152 Russo, M.A., Galeotti, T. and van Rossum, G.D.V. (1976) Cancer Res. 36.41604174 Galeotti. T., Azzi. A. and Chance, B. (1970) Biochim. Biophys. Acta 197, 11-24 Kobayashi. S., Hagibara. B.. Masuzumi, M. and Okunuki, K. (1966) Biochim. Biophys. Acta 113. 421437 BartoIi, G.M.. Dani, A., Galeotti, T., Russo, M. and Terranova, T. (1975) Z. Krebsforsch. 83, 223-231 Hunter, F.E.. Gebicki, J.M.. Hoffsten, P.E., Weinstein, J. and Scott, A. (1963) J. Biol. Chem. 238.828-835 Jones, P.D. and WakiI. S.J. (1967) J. Biol. Chem. 242, 5267-5273 Layne, E. (1957) Methods Enzymol. 3.450451 Hrycay, E.G. and O’Brien. P.J. (1973) Arch. Biochem. Biophys. 157, 7-22 Pederson, T.C. and Aust. S.D. (1973) Biochem. Biophys. Res. Commun. 52.1071-1078 Pederson. T.C. and Aust. S.D. (1975) Biochim. Biophys. Acta 385, 232-241 Aust, S.D., Roerig. D.L. and Pederson, T.C. (1972) Biochem. Biophys. Res. Commun.. 47. 1133-1137 BartoIi, G.M., Galeotti. T., Palombini, G., Parisi, G. and Azzi, A. (1977) Arch. Biochem. Biophys. 184,276-281 Barber, A.A. (1966) Lipids 1.146-151 Reitz, R.C., Thompson, J.A. and Morris, H.P. (1977) Cancer Res. 37. 561-567 Fee. F., Canuto, R.A.. Bertone, G.. Garcea. R. and Pani, P. (1973) FEBS Lett. 33. 229-232 Fee, F., Canuto. R.A.. Garcea. R. and Gabriel, L. (1975) Biochim. Biophys. Acta 413, 116-134 Tsai. A.C.. Thie. G.M. and Lin, C.R-s (1977) J. Nutr. 107. 31(t319 Hruban, Z., Machizuki. Y., Morris, H.P. and Slessers, A. (1972) Lab. Invest. 26, 86-99 Mead, J.F. (1976) in Free Radicals in Biology (Pryor, W.A.. ed.). Vol. I. PP. 51-68, Academic Press, New York, NY
BBA Report BBA 51257
GROWTH-RELATED LIPID PEROXIDATION IN TUMOUR MICROSOMAL MEMBRANES AND MITOCHONDRIA
G.M. BARTOLI and T. GALEOTTI* Institute of General Pathology, Catholic University, S. Cuore,, Via Pineta Sacchetti 644, 00168 Roma (Italy) (Received April 2nd, 1979) Key words: Lipid peroxidation;
Tumor growth; (Microsomal membrane,
Mitochondria)
Summary
Microsomes and mitochondria isolated from Morris hepatomas 3924A (fast-growing) and 44 (slow-growing) and Ehrlich ascites tumour cells exhibit a NADPHdependent peroxidation of endogenous lipids lower than that of the corresponding fractions from rat liver. Moreover, the 0, and ascorbate-dependent lipid peroxidations are decreased in microsomes from the two Morris hepatomas. The peroxidative activity appears to be inversely related to the growth rate of the tumours. It is suggested that the low susceptibility of tumour membranes to peroxidative agents may be a factor responsible for the high mitotic activity of this tissue.
Early studies have indicated that the non-enzymic lipid peroxidation induced by ascorbic acid is very low in tumour mitochondria [1,2]. Recently, Player et al. [3] also observed a low activity of the NADPHdependent lipid peroxidation in mitochondria isolated from hepatoma D30. However, no data are yet available on the non-enzymic or enzymic lipid peroxidation in tumour microsomal membranes. Such additional information appears of particular interest in the light of the hypothesis that peroxides generated from peroxidation of polyunsaturated fatty acids inhibit cell division [ 4-71. Moreover, the possibility to work with membranes isolated from tumours of different growth rates may enable a more definite examination of the correlation between peroxide production and mitotic activity in transformed cells. In this paper, we report results of measurements of NADPH-dependent *To whom all correspondence should be sent.
538
lipid peroxidation in microsomes [ 81 and mitochondria [ 91 from normal and cancerous rat liver with a large difference in growth rate (Morris hepatomas 3924A and 44), as well as from Ehrlich ascites tumour cells. Data are also presented of the lipid peroxidation induced by superoxide redicals (0; ) and ascorbate in microsomes from rat liver, Morris hepatomas 3924A and 44. The results support the view that cell proliferation in tumours is correlated with the peroxides generated during the turnover of membrane lipids. A preliminary account of part of this work has been given [lo]. Morris hepatoma 3924A and 44 [ll] were transplanted into both hind legs of inbred rats of the ACI/T (subcutaneous) and Buffalo (intramuscular) strains, respectively. Some experiments were performed using hepatoma 44-bearing rats inoculated in H.P. Morris laboratory and shipped by air. The tumours were excised 3-4 weeks (hepatoma 3924A) and 4-6 months (hepatoma 44) after tr~spl~tation. Ehrlich hyperdiploid ascites tumour cells of the Lettre’ strain (ELD) were maintained by weekly intraperitoneal transfer of ascitic fluid in albino Swiss mice and harvested 6-S days after inoculation, Other details concerning the isolation of hepatomas and preparation of ascites cells are reported elsewhere [12,13]. Normal male rats of the ACI/T strain, weighing 150-200 g, were used for rat liver preparations. Mito~hondria were isolated in 0.25 M sucrose, 0.5 mM EGTA, 5 mM Hepes (pH 7.4) by the conventional procedure, except for ELD cells (which were treated with bacterial protease before homogenization [ 141). The mitochondria were washed once with the isolation medium and twice with 0.125 M KCl, 20 mM Tris-HCl (pH 7.4) and suspended in the latter medium. Liver and hepatoma microsomes were prepared from the postmitochondrial supe~atant after cent~fugation at 18 000 X g for 10 min and 105 000 X g for 60 min. ELD cells were suspended in the sucrose/EGTA/Hepes medium and microsomes prepared from the homogenate obtained by Ultra-Turrax (Janke and Kunkel KG) disruption of the cells at 0°C for 5 s [ 151. Centrifugation was performed at 8000 and 18 000 X g for 10 min and 105 000 X g for 60 min. The microsomal fractions were washed once in 0.15 M KCl, 50 mM Tris-HCI (pH 7.5). Lipid peroxidation was measured at 25°C as malonaldehyde production in the presence of 4 mM ADP, 0.05 mM FeCl, and, when indicated, 0.33 mM xanthine. The reaction was started either by 0.4 mM NADPH or by 50 Mg/ml xanthine oxidase or by 0.25 mM sodium ascorbate. Malonaldehyde was determined at 535 nm by the thiobarbituric acid assay [ 161. NADPH~ytochrome c reductase was measured by the method of Jones and Wakil [17]. Proteins were estimated by the biuret method ]18]. Cytochrome c , ADP and NADPH were obtained from Boehringer, Mannheim. Ascorbic acid (sodium salt), xanthine oxidase (Grade I), N-2hydroxyethylpiperazine-N’-2-ethanesulfonic acid (Hepes), thiobarbituric acid and subtilisin (bacterial protease, Type VII) were purchased from Sigma Chemical Co. (St. Louis). EGTA was obtained from Koch-Light Laboratories (UK). All other chemicals were products of E. Merck (Darmstadt) except for NaCN (J.T. Baker Chemical Co., Deventer, The Netherlands). The enzymic lipid peroxidation induced by NADPH in the microsomal fraction isolated from rat liver and three different tumours has been measured (Fig. 1). It is evident that the extent of malonaldehyde production
539
Fig. 1. NADPH-dependent lipid peroxidation of microsomal fractions from rat liver (a), Morris hepatoma 44 (A) and 3924A (0) and Ehrlich ascites tumour cells (X). 0.2 mg/ml (rat liver and hepatoma 44) and 1 mg/ml (hepatoma 3924A and ELD) of microsomal proteins were suspended in an oxygensaturated medium of the following composition: 0.15 M KCl. 50 mM Tris-HCl (pH 7.5). 4 mM ADP and 0.05 mM FeCl, . The suspensions were incubated at 25’C in a Dubnoff metabolic shaker. Each point represents the mean of 4-6 experiments; the vertical lines correspond to twice the S.E.
during 40-min incubation is lower in tumour microsomes with respect to liver, being 20% in the fraction isolated from the well-differentiated Morris hepatoma 44 and nearly 10% in that isolated from the poorly-differentiated Morris hepatoma 3942A. A behaviour similar to that of the hepatoma 3942A is shown by the rapidly growing ELD cells. Thus, it appears that tumour transformation induces a substantial decrease in the rate of NADPHdependent lipid peroxidation and that such change is more pronounced in tumour cells with a rapid growth rate, i.e. hepatoma 3942A and ELD cells. Obviously it is not easy to establish whether the behaviour of lipid peroxidation in tumour microsomes is the cause or the consequence of the different growth rate. However, if the hypothesis that lipid peroxides are able to control cell division is true, on the basis of our data we are inclined to believe that the higher the degree of transformation is, the less the ability of the microsomal NADPHdependent lipid peroxidation is to inhibit tumour growth. Tumour transformation may have induced in microsomes changes either in the activity of the enzyme system involved in lipid peroxidation, i.e. NADPH-cytochrome c reductase [ 191, or in the membrane lipid composition and assembly. Both these possibilities have been tested. The activity of the flavoprotein follows a pattern similar to that of lipid peroxidation, being lower in the membranes from the two fast-growing tumours (Morris hepatoma 3942A: 17.8 + 1.2 (5) nmol - min-’ - mg-’ protein; Ehrlich ascites: 26.1 + 2.0 (5) nmol - min-’ - mg-’ protein) than in the slow-growing hepatoma 44 (42.4 + 1.1 (5) nmol - min-’ mg-’ protein). Such values correspond to 20-30 and 50% of the liver, respectively. It is known that microsomal lipid peroxidation may be also promoted by superoxide radicals [20] l
540
and Pederson and Aust [21] have suggested that the reaction mechanism involved in this system is different from that of the NA~PH-dependent lipid peroxidation. Fig. 2A shows that the rate of lipid peroxidation, induced by xanthine and xanthine oxidase (to generate superoxide radicals), has a behaviour which inversely follows that of the growth ability of the transformed tissue. Indeed, the activity is more markedly depressed in the microsomal fraction isolated from Morris hepatoma 3942A than 44. Similarly, lipid peroxides induced by ascorbate accumulate in a smaller amount in the more rapidly growing tumour (Fig. 2B). These results suggest that, besides
Fig, 2. Lipid peroxidation induced by supzoxide ‘iacikals (Af and ascorbate (3) of microsomd fractions from rat liver I*). Morris hepatoma 44 f&) and Motis he~atoma 3924A
the low activity of NADPH-cytochrome c reductase, changes in the structure andfor composition of tumour microsamal membranes make the fatty acids less vulnerable to peroxidative agents, such as free radicals generated both within the membrane [22,23] and in the soluble fraction of the cell, It has been reported, for instance, that, in normal tissues, the lipid peroxidation is dependent on the content of polyunsaturated fatty acids [24} and Reitz et al. [ZS] have shown that tumour microsomal membranes isolated from the fast growing Morris hepatoma 7777 contain about 50% less phospholipid than the controls. Moreover, in microsomes and mitochondria of this tumour, the polyenoic fatty acids decrease and, concomitantly, the monoenoic increase. In addition, Feo et al. [26,27] have reported that in microsomes and mitochondria isolated from the rapidly growing Morris hepatomas 5123 and 39248, although no changes in the distribution of the phospholip~d classes occur, the molar ratio cholesterol: phospholipids increases significantly, These data could also be relevant in relation to the observation that cholesterol feeding in rats reduces the liver microsomal NADPH-dependent lipid peroxidation [28]. Finally, it should be mentioned that differences
541
were observed in the ultrastructure of the endoplasmic reticulum of Morris hepatomas of different growth rate with respect to rat liver [29]. The NADPHdependent lipid peroxidation has been also measured in mitochondria of rat liver and tumours of different growth rate (rat liver: 12.3 f 1.8 (3) nmol/40 min per mg protein; hepatoma 44: 8.5 (2) nmol/40 min per mg protein; hepatoma 3924A: 6.1 * 0.8 (6) nmol/40 min per mg protein; Ehrlich ascites: 5.9 t 0.4 (4) nmol/40 min per mg protein). Again, it appears that the peroxidative activity is inversely related to the mitotic activity of the tumours. Our data on ELD cells and hepatoma ,3924A are consistent with previous findings made with ascorbate- and NADPHinduced lipid peroxidation of mitochondria from fast-growing tumours [ 2,3] . In conclusion it can be stated that, in analogy to other tissues which undergo very rapid cell division, such as testis and intestinal epithelium [ 301, tumours are somewhat resistant to lipid peroxidation and their growth rate appears to be correlated with the amount of lipid peroxides generated from the peroxidation of the polyunsaturated fatty acids of the membrane phospholipids. We thank Miss Monica Masucci for her help during some phases of this work. This work was in part supported by a Grant from Minister0 Pubblica Istruzione 1977. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Thiele, E.H. and Huff, J.W. (1960) Arch. Biochem. Biophys. 88, 208-211 Utsumi, K., Yamamoto, G. and Inaba, K. (1965) Biochim. Biophys. Acta 105.368-371 Player, T.J., Mills, D.J. and Horton, A.A. (1977) Biochem. Sot. Trans. 5, 1506-1508 Wolfson, N., Wilbur. K.M. and Bemheim. F. (1956) EXP. Ceil Res. 10,556-558 Cole, B.T. (1956) Proc. Sot. EXP. Biol. Med. 93, 290-294 Glushchenko, N.N.. Shestakova. S.V. and DaniIov, V.S. (1975) Biol. Nauki (Moscow) 18. 51-53 Player, T.J., Mills, D.J. and Horton, A.A. (1977) Biochem. Biophys. Res. Commun. 78.1397-1402 Hochstein, P. and Em&r, L. (1963) Biochem. Biophys. Res. Commun. 12, 388-394 Pfeifer. P.M. and McCay. P.B. (1972) J. Biol. Chem. 247, 6763-6769 BartoIi. G.M., Palombini, G. and Galeotti. T. (1978) 12th FEBS Meeting, Dresden. Abstract 2424 Monks. H.P. and Wagner. B.P. (1968) Methods Cancer Res. 4, 125-152 Russo, M.A., Galeotti, T. and van Rossum, G.D.V. (1976) Cancer Res. 36.41604174 Galeotti. T., Azzi. A. and Chance, B. (1970) Biochim. Biophys. Acta 197, 11-24 Kobayashi. S., Hagibara. B.. Masuzumi, M. and Okunuki, K. (1966) Biochim. Biophys. Acta 113. 421437 BartoIi, G.M.. Dani, A., Galeotti, T., Russo, M. and Terranova, T. (1975) Z. Krebsforsch. 83, 223-231 Hunter, F.E.. Gebicki, J.M.. Hoffsten, P.E., Weinstein, J. and Scott, A. (1963) J. Biol. Chem. 238.828-835 Jones, P.D. and WakiI. S.J. (1967) J. Biol. Chem. 242, 5267-5273 Layne, E. (1957) Methods Enzymol. 3.450451 Hrycay, E.G. and O’Brien. P.J. (1973) Arch. Biochem. Biophys. 157, 7-22 Pederson, T.C. and Aust. S.D. (1973) Biochem. Biophys. Res. Commun. 52.1071-1078 Pederson. T.C. and Aust. S.D. (1975) Biochim. Biophys. Acta 385, 232-241 Aust, S.D., Roerig. D.L. and Pederson, T.C. (1972) Biochem. Biophys. Res. Commun.. 47. 1133-1137 BartoIi, G.M., Galeotti. T., Palombini, G., Parisi, G. and Azzi, A. (1977) Arch. Biochem. Biophys. 184,276-281 Barber, A.A. (1966) Lipids 1.146-151 Reitz, R.C., Thompson, J.A. and Morris, H.P. (1977) Cancer Res. 37. 561-567 Fee. F., Canuto, R.A.. Bertone, G.. Garcea. R. and Pani, P. (1973) FEBS Lett. 33. 229-232 Fee, F., Canuto. R.A.. Garcea. R. and Gabriel, L. (1975) Biochim. Biophys. Acta 413, 116-134 Tsai. A.C.. Thie. G.M. and Lin, C.R-s (1977) J. Nutr. 107. 31(t319 Hruban, Z., Machizuki. Y., Morris, H.P. and Slessers, A. (1972) Lab. Invest. 26, 86-99 Mead, J.F. (1976) in Free Radicals in Biology (Pryor, W.A.. ed.). Vol. I. PP. 51-68, Academic Press, New York, NY