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Effect of interval CCl4 administration on levels of hydroperoxide and antioxidants in rat
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Lipid Damage and Repair EFFECT OF INTERVAL CCI 4 ADMINISTRATION ON LEVELS OF HYDROPEROXIDE AND ANTIOXIDANTS IN RAT Naoki Takahashi, Masako Katsurada, Yuji Ohori Etsuo Niki*, Yorihiro Yamamoto* Mitsubishi Kasei Corp., Toxicology Lab.,Research Center, i000 Kamoshida-cho, Midori-ku, Yokohama 227, Japan *Department of Reaction Chemistry, Faculty of Engineering, University of Tokyo, Hongo, Bunkyoku, Tokyo 113, Japan To find a role of oxidative stress in cirrhosis induced by CCI 4 administration, change of liver levels of antioxidants and phosphatidylcholine hydroperoxide(PC-OOH) were studied. CCl4(Iml/kg) in liquid paraffin was given to 4 weeks-old male SD rats at intervals of three to four days for 6 weeks. Liquid paraffin were given to control subjects. Significant lower body weights than control subjects were observed in CCI 4 after 2 weeks, while liver weights increased after i week. In CCI,4 administrated subjects, significant morphologic changes of liver were observed after 3 weeks and levels of -tocopherol and ascorbic acid were decreased after 2 weeks. PC-OOH levels were determined by HPLC with isoluminol chemiluminescence detection system and those in CCI 4 administrated subjects were about 3 times higher than those in control subjects. However, PC-OOH level was decreased to control level after 3 weeks. These results indicated that the increment of oxidative stress by interval CCI 4 administration and involvement of it in cirrhosis.
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EFFECTf f DIETARYFAT CN LIPID PEROXIDATIONSTATUSOF MDUSE BRAINTISSUE Junji Terao, Akihiko Nagao, Hiramitsu Suzuki, and ~L=gumi Yamazaki, National FoodResearch Institute/MAFF, Tsukuba 305 Japan, *lwate Pref. Brewing and Food Research Institute, lwate CLIO, Japan Role of dietary fats and oils in the physiological and pathological conditions has attracted nuch attention in recent , ~ . Dietary fats and oils seemto be more or less Fesponsible for the ccmposition of m~nbranelipids in all tissues, ll~erefore, long-term intake of scme edible oils may alter the sensitivity to lipid peroxidation leading to oxidative damage. It is therefore of ~uch interest to know whether the intake of highly oxidizable oils affects the sensitivity towards lipid peroxidation in brain tissue, which is rich in perexidation-sensitive polyunsaturated fatty acids such as docosahexaenoicacid. W~ designed a long -term feeding trial using mice (ICR strain, male and female 5 weeks after born), which were divided to a conventional diet (Oriental Yeast Co. Ltd. Japan) and 6%olard, rapeseed oil, or perilla oil diet groups. Micewere fed the diet ad libitum for 15 manths. Fatty acid composition of brain lipids from each group reflected that of dietary fat. Perilla oil was found to be highly oxidizable because of high concentration of alpha-linolenic acid (n-3). Fbwever, the concentration of vitamin E and the molar ratio of vitamin E to doubly allylic hydrogen in brain lipids were higher in the perilla oil-feeding group. The results of TBA assay and oxygen absorption rate of brain tissue homogenate also suggested that perilla oil did not accelerate lipid paroxidation status in mousebrain lipids. Therefore, we conclude that lipid perexidation status in brain tissue does not reflect directly the oxidizability of dietary lipids.
PHOSPHATIDYLCIIOLINE BYDROPEROXIDE ACCUMULATION IN THE LIV~ AND BRAIN OF AGING RATS ?eruo Miyazawa~, Toshihide Suzuki ~, Kenshiro Fujimoto a and Takashi Kaneda~ ~Departaent of Food Chemistry, Thoku University, Sendal 98 1, Japan; ~Kobriyama Women's College, Kohriyama 963, Japan
A NEGATIVE CORRELATION BETWEEN LIPID PEROXIDA'II(f~ AND HEMOLYSIS Jeroen J.M. van den Bergl, Nanneke J. de Fouw2, Ben Roelofsea:; Udo M.T. Houtsmuller2 and Jos A.F. Op den Kamp3 1Children's Hospital Oakland Research Institute, Oakland, CA 94609, U.S.A., 2Unilever Research Laboratorium, Vlaardingen, The Netherlands, 3C.B.L.E., State University of Utrecht, The Netherlands.
Lipid hydroperoxide formation has been received much attention with i t s possible contribution to the aging, but for which no direct evidence has been observed. The purpose of this study is to estimate the in vivo formation and accumulation of phosphatidylcholine hydroperoxide (I~OH) in various tissues of the aging rat. Phosphatidylcholine(PO is one of the most important functional lipids in biomenbranes, and P~OH is a primary peroxidatiun product of PC. The tissue PCOOH concentration was determined by chemiluminescence detection-high performance liquid chromatography methed(T. Miyazawa; Free Rad. Biol. Med. 7, 209-217, 1989). For the aging studies, Sprague-Dawley male rats were bred on standard p e l l e t rations for 1,7,12 and 18 months. Me-dependent PCOOH accumulation was clearly observed for the l i v e r and brain. Liver PCOOHconcentration was 274-+37(mean-+SE), 674_+10, 1983_+174 and 2558_+156 psol/g liver for 1,7,12 and 18 month r a t s , respectively. Brain PCOOH concentration was 131_+21, 168_+20, 474_+31 and 573_+59 pmol/g brain, respectively. PCOOHcontents in heart and lung were not changed following the aging. Large decreases in PC content in total lipid of the liver and brain were observed. This may r e f l e c t the ttydroperoxidatioos and suggests the functional change of subcellular membranes following the aging. From the r e s u l t s , we propose significant levels of P(~)OH accumulation are really occurred in the liver and brain depending on the aging of animals.
Rabbit red blood cells (RBC) enriched in n-3 polyunsaturated fatly acids (PUFAs) were obtained from rabbits on a fish oil diet. Relative to ceils from control rabbits, these RBCs showed large increases in 20:5, 22:5, 22:6, and a decrease in 18:1 and 18:2 fatty acid concentrations. Control and PUFA-enriched RBC (10%) were incubated with 1.0/2.0 mM cumene hydroperoxide (cumOOH) and 0.5/1.0 laJVlheroin. The extent of lipid peroxidation (LP), quantitated by GC fatty acid analysis, was always higher for the PUFA-enriched RBC, but the extent of peroxidation of arachidonic acid (20:4), the concentration of which is equal in control and PUFA-enricbed RBC, was always higher in control cells. Similar observations as for 20:4 were made using the fluorescent PUFA, parinaric acid (PnA), as a peroxidation probe in RBC under cumOOH-induced oxidative stress. PnA peroxidation was slightly decreased in PUFA-em'iched RBC (29.5 + 1.4 % (n---4)of PnA oxidized after 10 rain incubation with 250 I.tM cumOOH) compared to control cells (32.9 _+ 1.6 % (n--4)). These results can be explained by a competition mechanism. In PUFA-enriched RBC, the probability of a radical encountering a PUFA is higher, which is expressed in a higher extent of LP. Peroxidation of 20:4 and PnA in PUFA-em'iched cells is lower than in control cells, because radicals that would attack 20:4 in control ceils encounter other PUFAs in PUFA-enriched cells. Enhanced LP in PUFA-enriched RBC was accompanied by a drastically lower extent of hemolysis. Thus, there appears to be a negative correlation between LP and hemolysis in our experiments. Although LP in itself may lead to hemolysis, this is apparently not the (only) important factor here. CumOOH-indueed oxidation of RBC is known to affect both lipids and proteins. As primary radical generation will be the same in both cases, the increased PUFA content is suggested to have a protective effect for the cell, scavenging radicals that would otherwise have damaged membrane proteins, which may lead to hemolysis.
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Lipid Damage and Repair EFFECT OF INTERVAL CCI 4 ADMINISTRATION ON LEVELS OF HYDROPEROXIDE AND ANTIOXIDANTS IN RAT Naoki Takahashi, Masako Katsurada, Yuji Ohori Etsuo Niki*, Yorihiro Yamamoto* Mitsubishi Kasei Corp., Toxicology Lab.,Research Center, i000 Kamoshida-cho, Midori-ku, Yokohama 227, Japan *Department of Reaction Chemistry, Faculty of Engineering, University of Tokyo, Hongo, Bunkyoku, Tokyo 113, Japan To find a role of oxidative stress in cirrhosis induced by CCI 4 administration, change of liver levels of antioxidants and phosphatidylcholine hydroperoxide(PC-OOH) were studied. CCl4(Iml/kg) in liquid paraffin was given to 4 weeks-old male SD rats at intervals of three to four days for 6 weeks. Liquid paraffin were given to control subjects. Significant lower body weights than control subjects were observed in CCI 4 after 2 weeks, while liver weights increased after i week. In CCI,4 administrated subjects, significant morphologic changes of liver were observed after 3 weeks and levels of -tocopherol and ascorbic acid were decreased after 2 weeks. PC-OOH levels were determined by HPLC with isoluminol chemiluminescence detection system and those in CCI 4 administrated subjects were about 3 times higher than those in control subjects. However, PC-OOH level was decreased to control level after 3 weeks. These results indicated that the increment of oxidative stress by interval CCI 4 administration and involvement of it in cirrhosis.
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EFFECTf f DIETARYFAT CN LIPID PEROXIDATIONSTATUSOF MDUSE BRAINTISSUE Junji Terao, Akihiko Nagao, Hiramitsu Suzuki, and ~L=gumi Yamazaki, National FoodResearch Institute/MAFF, Tsukuba 305 Japan, *lwate Pref. Brewing and Food Research Institute, lwate CLIO, Japan Role of dietary fats and oils in the physiological and pathological conditions has attracted nuch attention in recent , ~ . Dietary fats and oils seemto be more or less Fesponsible for the ccmposition of m~nbranelipids in all tissues, ll~erefore, long-term intake of scme edible oils may alter the sensitivity to lipid peroxidation leading to oxidative damage. It is therefore of ~uch interest to know whether the intake of highly oxidizable oils affects the sensitivity towards lipid peroxidation in brain tissue, which is rich in perexidation-sensitive polyunsaturated fatty acids such as docosahexaenoicacid. W~ designed a long -term feeding trial using mice (ICR strain, male and female 5 weeks after born), which were divided to a conventional diet (Oriental Yeast Co. Ltd. Japan) and 6%olard, rapeseed oil, or perilla oil diet groups. Micewere fed the diet ad libitum for 15 manths. Fatty acid composition of brain lipids from each group reflected that of dietary fat. Perilla oil was found to be highly oxidizable because of high concentration of alpha-linolenic acid (n-3). Fbwever, the concentration of vitamin E and the molar ratio of vitamin E to doubly allylic hydrogen in brain lipids were higher in the perilla oil-feeding group. The results of TBA assay and oxygen absorption rate of brain tissue homogenate also suggested that perilla oil did not accelerate lipid paroxidation status in mousebrain lipids. Therefore, we conclude that lipid perexidation status in brain tissue does not reflect directly the oxidizability of dietary lipids.
PHOSPHATIDYLCIIOLINE BYDROPEROXIDE ACCUMULATION IN THE LIV~ AND BRAIN OF AGING RATS ?eruo Miyazawa~, Toshihide Suzuki ~, Kenshiro Fujimoto a and Takashi Kaneda~ ~Departaent of Food Chemistry, Thoku University, Sendal 98 1, Japan; ~Kobriyama Women's College, Kohriyama 963, Japan
A NEGATIVE CORRELATION BETWEEN LIPID PEROXIDA'II(f~ AND HEMOLYSIS Jeroen J.M. van den Bergl, Nanneke J. de Fouw2, Ben Roelofsea:; Udo M.T. Houtsmuller2 and Jos A.F. Op den Kamp3 1Children's Hospital Oakland Research Institute, Oakland, CA 94609, U.S.A., 2Unilever Research Laboratorium, Vlaardingen, The Netherlands, 3C.B.L.E., State University of Utrecht, The Netherlands.
Lipid hydroperoxide formation has been received much attention with i t s possible contribution to the aging, but for which no direct evidence has been observed. The purpose of this study is to estimate the in vivo formation and accumulation of phosphatidylcholine hydroperoxide (I~OH) in various tissues of the aging rat. Phosphatidylcholine(PO is one of the most important functional lipids in biomenbranes, and P~OH is a primary peroxidatiun product of PC. The tissue PCOOH concentration was determined by chemiluminescence detection-high performance liquid chromatography methed(T. Miyazawa; Free Rad. Biol. Med. 7, 209-217, 1989). For the aging studies, Sprague-Dawley male rats were bred on standard p e l l e t rations for 1,7,12 and 18 months. Me-dependent PCOOH accumulation was clearly observed for the l i v e r and brain. Liver PCOOHconcentration was 274-+37(mean-+SE), 674_+10, 1983_+174 and 2558_+156 psol/g liver for 1,7,12 and 18 month r a t s , respectively. Brain PCOOH concentration was 131_+21, 168_+20, 474_+31 and 573_+59 pmol/g brain, respectively. PCOOHcontents in heart and lung were not changed following the aging. Large decreases in PC content in total lipid of the liver and brain were observed. This may r e f l e c t the ttydroperoxidatioos and suggests the functional change of subcellular membranes following the aging. From the r e s u l t s , we propose significant levels of P(~)OH accumulation are really occurred in the liver and brain depending on the aging of animals.
Rabbit red blood cells (RBC) enriched in n-3 polyunsaturated fatly acids (PUFAs) were obtained from rabbits on a fish oil diet. Relative to ceils from control rabbits, these RBCs showed large increases in 20:5, 22:5, 22:6, and a decrease in 18:1 and 18:2 fatty acid concentrations. Control and PUFA-enriched RBC (10%) were incubated with 1.0/2.0 mM cumene hydroperoxide (cumOOH) and 0.5/1.0 laJVlheroin. The extent of lipid peroxidation (LP), quantitated by GC fatty acid analysis, was always higher for the PUFA-enriched RBC, but the extent of peroxidation of arachidonic acid (20:4), the concentration of which is equal in control and PUFA-enricbed RBC, was always higher in control cells. Similar observations as for 20:4 were made using the fluorescent PUFA, parinaric acid (PnA), as a peroxidation probe in RBC under cumOOH-induced oxidative stress. PnA peroxidation was slightly decreased in PUFA-em'iched RBC (29.5 + 1.4 % (n---4)of PnA oxidized after 10 rain incubation with 250 I.tM cumOOH) compared to control cells (32.9 _+ 1.6 % (n--4)). These results can be explained by a competition mechanism. In PUFA-enriched RBC, the probability of a radical encountering a PUFA is higher, which is expressed in a higher extent of LP. Peroxidation of 20:4 and PnA in PUFA-em'iched cells is lower than in control cells, because radicals that would attack 20:4 in control ceils encounter other PUFAs in PUFA-enriched cells. Enhanced LP in PUFA-enriched RBC was accompanied by a drastically lower extent of hemolysis. Thus, there appears to be a negative correlation between LP and hemolysis in our experiments. Although LP in itself may lead to hemolysis, this is apparently not the (only) important factor here. CumOOH-indueed oxidation of RBC is known to affect both lipids and proteins. As primary radical generation will be the same in both cases, the increased PUFA content is suggested to have a protective effect for the cell, scavenging radicals that would otherwise have damaged membrane proteins, which may lead to hemolysis.
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