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Hepatotoxicity of heated and oxygenated corn oil
Exp ToxicPathol 1992; 44: 255-258 Gustav Fischer Verlag lena
Short communication
Department of Pathology, Osaka Medical College, Takatsuki City, Osaka, Japan
Hepatotoxicity of heated and oxygenated corn oil Y. SHIBA YAMA With 4 figures and 1 table Received: April 19, 1991; Accepted: August 14, 1991 Address for correspondence: Dr. YURO SHIBA YAMA, Department of Pathology, Osaka Medical College, Daigaku-Cho, Takatsuki City, Osaka, Japan. Key words: Hepatotoxicity; Liver cell damage; Corn oil; Lipid peroxides; Oxygen radicals; Peroxides.
Summary
Materials and methods
The present study was undertaken in rats to examine the hepatotoxicity of ingested heated and oxygenated corn oil. Activities of serum glutamic pyruvic transaminase markedly increased in the rats given heated and oxygenated corn oil for two weeks as compared with that of the control group. In the rats given heated and oxygenated corn oil, small fat droplets were found in the liver cells, but liver cell necrosis was not seen. Ultrastructurally, there was no conspicuous change in the liver cells, except for depletion of glycogen and dilatation of cisternae of rough and smooth surfaced endoplasmic reticulum. These experimental data show that ingestion of heated and oxygenated corn oil induces hepatic injury, and that the development of hepatic injury may relate to liver cell membrane damage due to active oxygen radicals contained in heated and oxygenated corn oil.
Sixty-three male Wistar rats weighing about 200 g were used. These animals were maintained under controlled temperature (24°C) and were fed standard rat chow and water ad libitum. The rats were given 1 ml of physiological saline solution, nontreated corn oil or heated and oxygenated corn oil per 100 g body weight by stomach tube twice a day for 14 days. The heated and oxygenated corn oil was produced by bubbling oxygen to corn oil heated to 180°C for 8 h. The rats were then placed under ether anesthesis, and blood was withdrawn from the inferior vena cava. Activities of serum glutamic pyruvic transaminase and serum levels of lipid peroxides, total cholesterol, triglycerides and free fatty acids were determined. Lipid peroxides were expressed in terms of nmol malondialdehyde per ml serum (Y AGl 1976). The liver was excised and weighed. Small pieces of the liver were fixed with 2 % glutaraldehyde solution buffered with phosphate for 2 h, refixed with 1 % buffered osmium tetroxide for 1.5 hand embedded in Epon-812. Ultra-thin sections were stained with uranyl acetate and lead acetate and examined with a Hitachi H500 electron microscope. The remaining liver was fixed in 10 % neutral formalin for three days for light microscopic examination. The sections embedded in paraffin were stained with hematoxylin and eosin. Frozen sections were also prepared and stained with Oil-Red O. Results are expressed as means ± S.D. Statistical significance (P < 0.05) was determined using analysis of variance.
Introduction It is thought that excessive ingestion of heated and oxygenated oil induces hepatic injury, although the mechanism is not clear. Lipid peroxides may play an important role in the development of the hepatic injury, because the heated and oxygenated oil contains a large amount of lipid peroxides (KIMURA et a1. 1985). Lipid peroxides are metabolized in the liver cells and the liver cells may be injured by cytotoxicity of lipid peroxides and the breakdown products (CZAJA et a1. 1975; HRUSZKEWYCZ et a1. 1978; BENEDETTI et a1. 1979; NISHIGAKI et a1. 1980; UEDA et a1. 1985). However, only very few morphological and biochemical findings are available for elucidation of the mechanism for the liver cell damage. In the present study, hepatic injury following ingestion of heated and oxygenated com oil was examined in rats, morphologically and biochemically.
Results Body weight, liver weight, actiVIties of serum glutamic pyruvic transaminase as well as serum levels of lipid peroxides, total cholesterol, triglycerides and free fatty acids in the control, the non-treated com oil and the heated and oxygenated com oil groups are shown in Table 1. In the heated and oxygenated com oil group, the liver weight increased, Exp Toxic Pathol 44 (1992) 5
255
Table 1. Body weight, liver weight and glutamic pyruvic transaminase activity, lipid peroxides, total cholesterol, triglycerides and free fatty acids in serum. A. Control
B. Non-treated C. Heated and oxycorn oil genated corn oil
N
21
21
21
Body weight (g)
243 ± 36
227 ± 33
183 ± 36ab
Liver weight (g)
9.0 ± 1.1
8.5 ± 1.2
10.6 ± 1.5 ab
Serum glutamic pyruvic transaminase (Log U/l)
1.84 ± 0.14
1.78±0.13
2.17 ± 0.16 ab
Serum lipid peroxides (nmollml)
5.5 ± 1.3
6.9 ± 2.3
5.9 ± 2.2
(mg/dl)
75 ± 9
70 ± 12
94 ± 20 ab
Serum triglycerides (Log mg/dl)
1.71±0.18
1.65±0.17
1.83 ± 0.42
Serum free fatty acids (Log [lEqll)
2.75±0.1O
2.99 ± O.13 a
3.16 ± 0.28 ab
Serum total cholesterol
Values are means ± S. D.; N: Number of rats; a: P<0.05, compared to Group A; b: P<0.05, compared to Group B.
250
~
... ~
• Control • Corn oil • Peroxidized oorn oil
150
.. ..
'E
= ~ V>
-
<->
.~ 100 C1 I
<->
••
'E
..'.
=
~
b4 E
~
Co'")
50
~
.'
.. ...
:
,.
... . ..
..
o~----------~-------o 5 10 15 lipid peroxides (nmol/mll
Fig. 1. The relationship between activity of serum glutamic pyruvic transaminase and serum level of lipid peroxides. 256
Exp Toxic Pathol 44 (1992) 5
Fig. 2. The liver of a rat given heated and oxygenated corn oil (H & E, X 400). Small fat droplets are seen in the liver cells.
Fig. 3. Electron micrograph of the liver of a rat given heated and oxygenated corn oil (x 5,250). Depletion of glycogen and accumulation of small fat droplets are seen in the liver cell.
ex
Fig. 4. Electron micrograph of the liver of a rat given heated and oxygenated corn oil 12,450). Dilatation of cisternae of rough and smooth surfaced endoplasmic reticulum and lamellar configurations in mitochondria are seen. Neither a conspicuous change in the other organelles of the liver cells nor alteration of the liver cell membrane is found.
although the body weight decreased, as compared with the control and the non-treated corn oil group. The activity of serum glutamic pyruvic transaminase and the serum levels of free fatty acids and total cholesterol increased as compared with those of the control and the non-treated corn oil group, but the serum levels of lipid peroxides and triglycerides did
not increase. There was no relationship between the activity of serum glutamic pyruvic transaminase and the serum level of lipid peroxides (fig. 1). There was no significant change in the livers of the control and the non-treated corn oil group, light- and electronmicroscopically. Small fat droplets in the liver cells were Exp Toxic Pathol 44 (1992) 5
257
found in the. heated and oxygenated com oil group (fig. 2). Liver cell necrosis was not seen. Depletion of glycogen, dilatation of cisternae of rough and smooth surfaced endoplasmic reticulum, small fat droplets, and lamellar configurations in mitochondria were found ultrastructurally in the liver cells (fig. 3 and 4).
control or the non-treated com oil group. Moreover, there was no relationship between the activities of serum glutamic pyruvic transaminase activity and the serum levels of lipid p((roxides. These experimental data indicate that the serum level of lipid peroxides can not be used as an index of the hepatic injury due to lipid peroxides.
Discussion
References
It is thought that ingested lipid peroxides are absorbed from the gut, transported to the liver through the portal venous system and metabolized in the liver cells. The lipid peroxides and their breakdown products may injure the liver cells due to peroxidation of the cell membrane by active oxygen radicals (HRUSZKEWYCZ et al. 1978; BENEDETTI et al. 1979; VEDA et al. 1985). In fact, it has been reported that repeated oral administration of organic peroxides induces loss of body weight as well as depletion of glycogen and fatty metamorphosis in the liver cells in rats (FLOYD and STOKINGER 1958). In man, ingestion of a solution of methyl ethyl ketone peroxides has caused fatal hepatic necrosis (KARHUNEN et al. 1990). The present study clearly demonstrates that the administration of heated and oxygenated com oil, which contains a large amount of lipid peroxides, induces loss of body weight, an increase in liver weight as well as accumulation of small fat droplets and~depletion of glycogen in the liver cells. These morphological findings agree with those of the previous report (FLOYD and STOKINGER 1958). Thus, the hepatic injury induced by heated and oxygenated com oil may be due to lipid peroxides. It is possible that the hepatic injury is liver cell membrane damage due to peroxidation by active oxygen radicals, since the level of serum glutamic pyruvic transaminase activity is elevated without hepatocellular necrosis. In the present study, however, the serum level of lipid peroxides did no elevate as compared with that of the
BENEDETTI A, CASINI AF, FERRALl M, et al: Effects of diffusible products of peroxidation of rat liver microsomal lipid. Biochem J 1979, 180: 303-312. CZAJA AJ, RIZZO TA, SMITH WR Jr, et al: Acute liver disease after cutaneous thermal injury. J Trauma 1975, 15: 887-894. FLOYD EP, STOKINGER HE: Toxicity studies of certain organic peroxides and hydroperoxides. Am Ind Hyg Assoc J 1958, 19: 205-212. HRUSZKEWYCZ AM, GLEN DE EA, RECKNAGEL RO: Destruction of microscomal cytochrome PA50 and glucose-6-phosphatase by lipid extracted from peroxidized microsomes. Toxicol Appl Pharmacol 1978,46: 695-702. KARHUNEN PJ, OJANPERA I, LALU K, et al: Peripheral zonal hepatic necrosis caused by accidental ingestion of methyl ethyl ketone peroxide. Hum Exp Toxicol1990, 9: 197-200. KIMURA Y, OKUDA H, OKUDA T, et al: Studies on the activities of tannins and related compounds from medicinal plants and drugs. VII. Effects of extracts of leaves of artemisia species, and caffeic acid and chi orogenic acid on lipid metabolic injury in rats fed peroxidized oil. Chern Pharm Bull 1985, 33: 2028-2034. NISHIGAKI I, HAGIHARA M, HIRAMATSU M, et al: Effect of thermal injury on lipid peroxide levels of rat. Biochem Med 1980, 24: 185-189. UEDA K, KOBAYASHI S, MORITA J, et al: Site-specific damage caused by lipid peroxidation products. Biochem Biophys Acta 1985, 824: 341-348. YAGI K: A simple fluorometric assay for lipoperoxide in blood plasma. Biochem Med 1976, 12: 212-216.
258
Exp Toxic Patho] 44 (1992) 5
Short communication
Department of Pathology, Osaka Medical College, Takatsuki City, Osaka, Japan
Hepatotoxicity of heated and oxygenated corn oil Y. SHIBA YAMA With 4 figures and 1 table Received: April 19, 1991; Accepted: August 14, 1991 Address for correspondence: Dr. YURO SHIBA YAMA, Department of Pathology, Osaka Medical College, Daigaku-Cho, Takatsuki City, Osaka, Japan. Key words: Hepatotoxicity; Liver cell damage; Corn oil; Lipid peroxides; Oxygen radicals; Peroxides.
Summary
Materials and methods
The present study was undertaken in rats to examine the hepatotoxicity of ingested heated and oxygenated corn oil. Activities of serum glutamic pyruvic transaminase markedly increased in the rats given heated and oxygenated corn oil for two weeks as compared with that of the control group. In the rats given heated and oxygenated corn oil, small fat droplets were found in the liver cells, but liver cell necrosis was not seen. Ultrastructurally, there was no conspicuous change in the liver cells, except for depletion of glycogen and dilatation of cisternae of rough and smooth surfaced endoplasmic reticulum. These experimental data show that ingestion of heated and oxygenated corn oil induces hepatic injury, and that the development of hepatic injury may relate to liver cell membrane damage due to active oxygen radicals contained in heated and oxygenated corn oil.
Sixty-three male Wistar rats weighing about 200 g were used. These animals were maintained under controlled temperature (24°C) and were fed standard rat chow and water ad libitum. The rats were given 1 ml of physiological saline solution, nontreated corn oil or heated and oxygenated corn oil per 100 g body weight by stomach tube twice a day for 14 days. The heated and oxygenated corn oil was produced by bubbling oxygen to corn oil heated to 180°C for 8 h. The rats were then placed under ether anesthesis, and blood was withdrawn from the inferior vena cava. Activities of serum glutamic pyruvic transaminase and serum levels of lipid peroxides, total cholesterol, triglycerides and free fatty acids were determined. Lipid peroxides were expressed in terms of nmol malondialdehyde per ml serum (Y AGl 1976). The liver was excised and weighed. Small pieces of the liver were fixed with 2 % glutaraldehyde solution buffered with phosphate for 2 h, refixed with 1 % buffered osmium tetroxide for 1.5 hand embedded in Epon-812. Ultra-thin sections were stained with uranyl acetate and lead acetate and examined with a Hitachi H500 electron microscope. The remaining liver was fixed in 10 % neutral formalin for three days for light microscopic examination. The sections embedded in paraffin were stained with hematoxylin and eosin. Frozen sections were also prepared and stained with Oil-Red O. Results are expressed as means ± S.D. Statistical significance (P < 0.05) was determined using analysis of variance.
Introduction It is thought that excessive ingestion of heated and oxygenated oil induces hepatic injury, although the mechanism is not clear. Lipid peroxides may play an important role in the development of the hepatic injury, because the heated and oxygenated oil contains a large amount of lipid peroxides (KIMURA et a1. 1985). Lipid peroxides are metabolized in the liver cells and the liver cells may be injured by cytotoxicity of lipid peroxides and the breakdown products (CZAJA et a1. 1975; HRUSZKEWYCZ et a1. 1978; BENEDETTI et a1. 1979; NISHIGAKI et a1. 1980; UEDA et a1. 1985). However, only very few morphological and biochemical findings are available for elucidation of the mechanism for the liver cell damage. In the present study, hepatic injury following ingestion of heated and oxygenated com oil was examined in rats, morphologically and biochemically.
Results Body weight, liver weight, actiVIties of serum glutamic pyruvic transaminase as well as serum levels of lipid peroxides, total cholesterol, triglycerides and free fatty acids in the control, the non-treated com oil and the heated and oxygenated com oil groups are shown in Table 1. In the heated and oxygenated com oil group, the liver weight increased, Exp Toxic Pathol 44 (1992) 5
255
Table 1. Body weight, liver weight and glutamic pyruvic transaminase activity, lipid peroxides, total cholesterol, triglycerides and free fatty acids in serum. A. Control
B. Non-treated C. Heated and oxycorn oil genated corn oil
N
21
21
21
Body weight (g)
243 ± 36
227 ± 33
183 ± 36ab
Liver weight (g)
9.0 ± 1.1
8.5 ± 1.2
10.6 ± 1.5 ab
Serum glutamic pyruvic transaminase (Log U/l)
1.84 ± 0.14
1.78±0.13
2.17 ± 0.16 ab
Serum lipid peroxides (nmollml)
5.5 ± 1.3
6.9 ± 2.3
5.9 ± 2.2
(mg/dl)
75 ± 9
70 ± 12
94 ± 20 ab
Serum triglycerides (Log mg/dl)
1.71±0.18
1.65±0.17
1.83 ± 0.42
Serum free fatty acids (Log [lEqll)
2.75±0.1O
2.99 ± O.13 a
3.16 ± 0.28 ab
Serum total cholesterol
Values are means ± S. D.; N: Number of rats; a: P<0.05, compared to Group A; b: P<0.05, compared to Group B.
250
~
... ~
• Control • Corn oil • Peroxidized oorn oil
150
.. ..
'E
= ~ V>
-
<->
.~ 100 C1 I
<->
••
'E
..'.
=
~
b4 E
~
Co'")
50
~
.'
.. ...
:
,.
... . ..
..
o~----------~-------o 5 10 15 lipid peroxides (nmol/mll
Fig. 1. The relationship between activity of serum glutamic pyruvic transaminase and serum level of lipid peroxides. 256
Exp Toxic Pathol 44 (1992) 5
Fig. 2. The liver of a rat given heated and oxygenated corn oil (H & E, X 400). Small fat droplets are seen in the liver cells.
Fig. 3. Electron micrograph of the liver of a rat given heated and oxygenated corn oil (x 5,250). Depletion of glycogen and accumulation of small fat droplets are seen in the liver cell.
ex
Fig. 4. Electron micrograph of the liver of a rat given heated and oxygenated corn oil 12,450). Dilatation of cisternae of rough and smooth surfaced endoplasmic reticulum and lamellar configurations in mitochondria are seen. Neither a conspicuous change in the other organelles of the liver cells nor alteration of the liver cell membrane is found.
although the body weight decreased, as compared with the control and the non-treated corn oil group. The activity of serum glutamic pyruvic transaminase and the serum levels of free fatty acids and total cholesterol increased as compared with those of the control and the non-treated corn oil group, but the serum levels of lipid peroxides and triglycerides did
not increase. There was no relationship between the activity of serum glutamic pyruvic transaminase and the serum level of lipid peroxides (fig. 1). There was no significant change in the livers of the control and the non-treated corn oil group, light- and electronmicroscopically. Small fat droplets in the liver cells were Exp Toxic Pathol 44 (1992) 5
257
found in the. heated and oxygenated com oil group (fig. 2). Liver cell necrosis was not seen. Depletion of glycogen, dilatation of cisternae of rough and smooth surfaced endoplasmic reticulum, small fat droplets, and lamellar configurations in mitochondria were found ultrastructurally in the liver cells (fig. 3 and 4).
control or the non-treated com oil group. Moreover, there was no relationship between the activities of serum glutamic pyruvic transaminase activity and the serum levels of lipid p((roxides. These experimental data indicate that the serum level of lipid peroxides can not be used as an index of the hepatic injury due to lipid peroxides.
Discussion
References
It is thought that ingested lipid peroxides are absorbed from the gut, transported to the liver through the portal venous system and metabolized in the liver cells. The lipid peroxides and their breakdown products may injure the liver cells due to peroxidation of the cell membrane by active oxygen radicals (HRUSZKEWYCZ et al. 1978; BENEDETTI et al. 1979; VEDA et al. 1985). In fact, it has been reported that repeated oral administration of organic peroxides induces loss of body weight as well as depletion of glycogen and fatty metamorphosis in the liver cells in rats (FLOYD and STOKINGER 1958). In man, ingestion of a solution of methyl ethyl ketone peroxides has caused fatal hepatic necrosis (KARHUNEN et al. 1990). The present study clearly demonstrates that the administration of heated and oxygenated com oil, which contains a large amount of lipid peroxides, induces loss of body weight, an increase in liver weight as well as accumulation of small fat droplets and~depletion of glycogen in the liver cells. These morphological findings agree with those of the previous report (FLOYD and STOKINGER 1958). Thus, the hepatic injury induced by heated and oxygenated com oil may be due to lipid peroxides. It is possible that the hepatic injury is liver cell membrane damage due to peroxidation by active oxygen radicals, since the level of serum glutamic pyruvic transaminase activity is elevated without hepatocellular necrosis. In the present study, however, the serum level of lipid peroxides did no elevate as compared with that of the
BENEDETTI A, CASINI AF, FERRALl M, et al: Effects of diffusible products of peroxidation of rat liver microsomal lipid. Biochem J 1979, 180: 303-312. CZAJA AJ, RIZZO TA, SMITH WR Jr, et al: Acute liver disease after cutaneous thermal injury. J Trauma 1975, 15: 887-894. FLOYD EP, STOKINGER HE: Toxicity studies of certain organic peroxides and hydroperoxides. Am Ind Hyg Assoc J 1958, 19: 205-212. HRUSZKEWYCZ AM, GLEN DE EA, RECKNAGEL RO: Destruction of microscomal cytochrome PA50 and glucose-6-phosphatase by lipid extracted from peroxidized microsomes. Toxicol Appl Pharmacol 1978,46: 695-702. KARHUNEN PJ, OJANPERA I, LALU K, et al: Peripheral zonal hepatic necrosis caused by accidental ingestion of methyl ethyl ketone peroxide. Hum Exp Toxicol1990, 9: 197-200. KIMURA Y, OKUDA H, OKUDA T, et al: Studies on the activities of tannins and related compounds from medicinal plants and drugs. VII. Effects of extracts of leaves of artemisia species, and caffeic acid and chi orogenic acid on lipid metabolic injury in rats fed peroxidized oil. Chern Pharm Bull 1985, 33: 2028-2034. NISHIGAKI I, HAGIHARA M, HIRAMATSU M, et al: Effect of thermal injury on lipid peroxide levels of rat. Biochem Med 1980, 24: 185-189. UEDA K, KOBAYASHI S, MORITA J, et al: Site-specific damage caused by lipid peroxidation products. Biochem Biophys Acta 1985, 824: 341-348. YAGI K: A simple fluorometric assay for lipoperoxide in blood plasma. Biochem Med 1976, 12: 212-216.
258
Exp Toxic Patho] 44 (1992) 5