Archives of
Arch Toxicol (1992) 66: 182- 187
Toxicolog 9 Spfinger-Verlag 1992
Studies on the hepatotoxicity induced by bis (tributyltin) oxide Mitsuaki Yoshizuka l, Kazuo Hara l, Nobuya Haramaki3, Mitsuru Yokoyama 2, Naoki Moril, Yoshiaki Doi 1, Akio Kawahara l, and Sunao Fujimoto 1 l Department of Anatomy, and 2 Laboratory of Electron Microscopy, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807, Japan 3 Department of Medical Biochemistry, Kurume University School of Medicine, Kurume 830, Japan Received 20 August 1991/Accepted 29 October 1991
Abstract. The toxic effects of bis (tributyltin) oxide (TBTO) on the rat liver were studied with an electron microscope and the accumulation sites of tin were determined with an X-ray microanalyzer. The activities of serum enzymes and the concentration of serum bilirubin were also analyzed. Male Wistar rats received an intramuscular injection of 0.5 ml/kg of TBTO. Marked swelling of the mitochondria appeared in the hepatocytes 4 h after injection of TBTO. Cytoplasmic vacuoles, which contained degenerated mitochondria, gradually increased in number in these hepatocytes. This in turn may have caused a decrease in the volume of hepatic cell cords and an enlargement of sinusoids in the entire hepatic lobule. However, fine structures of intrahepatic bile ducts were not altered. By X-ray microanalysis, tin peaks were preferentially obtained from swollen mitochondria of the hepatocytes. By polarographic analysis of the respiratory responses of mitochondria, it was demonstrated that rates of state 4 respiration and respiratory control ratio were significantly disturbed in TBTO-treated rats in comparison with those of controls. The activities of AST (aspartate aminotransferase) and ALT (alanine aminotransferase) were significantly increased after TBTO treatment, but those of ALP (alkaline phosphatase), LAP (leucine aminopeptidase) and total bilirubin were not changed. These results indicated that parenterally administered TBTO accumulated in the liver cell mitochondria and disturbed oxidative phosphorylation. Mitochondrial dysfunction might induce severe damage of the hepatocytes. Four days after injection of TBTO, hepatic structures and chemical indices were almost restored by the regeneration of hepatocytes.
Introduction Tributyltin compounds have been widely used as molluscicides, as antifoulants on ships, fish nets, and cages, as wood preservatives, and as biocides for leather processing. Recently, the biological effects of tributyltin compounds were summarized (WHO 1990; Henschler 1991). In regard to the toxic effect of tributyltin compounds on the liver,a few reports have been published (Krajnc et al. 1984; Davis et al. 1987). They indicated that orally administered bis (tributyltin) oxide was accumulated in rat and mouse livers, and induced structural changes of the bile canalic~i and the common bile ducts. However, they did not mention the ultrastructural changes of the hepatocytes. Therefore, we decided to investigate detailed morph0logical alterations of rat liver after parenteral administration of bis (tributyltin) oxide (TBTO) using an electron microscope. The accumulation sites of TBTO in the liver tissue were examined with an X-ray microanalyzer. Several in vitro studies have indicated that organotin compounds act as inhibitors of oxidative phosphorylati0n in the mitochondria (Aldridge 1958; Aldridge et al. 1977: Cain et al. 1977). Therefore, we carded out a polarographic study to elucidate the effect on the respiratory responses of liver cell mitochondria. Furthermore, we determined the serum enzyme activities and the concentration of serum bilirubin.
Materials and methods
Key words: Bis(tributyltin) oxide - Liver - Electron microscopy - X-ray microanalysis - Biochemistry
Animals. Adult male Wistar UOEH rats were housed in individual stainless-steel cages and maintained with a 12-h light-dark cycle. Exposure to light was from 6 to 18 hours. The rats had been fed laboratory chow and water ad libitum until sacrificed between 11 and 12 hours.
Offprint requests to: Mitsuaki Yoshizuka, Department of Anatomy, University of Occupational and Environmental Health, School of Medicine, Kitakyushu 807, Japan
Light and electron microscopy. In our preliminary study, we found thata single intramuscular injection of 2 ml/kg body weight of undiluted TBTO was the LDs0 (lethal dose for 50% survival group) in the male Wistar rats. A quarter of the LDs0 was chosen in the present study. Thirty-nine adult male Wistar rats (weighing 2 5 0 - 300 g) received an intramuscular injection of 0.5 ml/kg body weight of bis (tributyltin)
183
Fig. 1. Depletion of glycogen particles in the cytoplasm of the hepatocytesand an enlargement of sinusoids are noted in the centrilobular zone 4h after TBTO treatment. CV: central vein • 200
Fig. 3. An electron micrograph showing the extremely swollen mitochondria, which are often seen in the hepatocytes in 4 h after TBTO treatment. • 16000
Fig. 2. The cytoplasmic vacuoles in the hepatocyte arise from dilated cistemae of the rough surfaced endoplasmic reticulum 4 h after TBTO treatment. Remnants of degenerated cytoplasmic membranes are found inthese vacuoles. Deformed mitochondria and increasing cisternae of the smooth surfaced endoplasmic reticulum surround the vacuoles, x 12000
Fig. 4. Various sizes of cytoplasmic vacuoles are found in the sinusoidal region of hepatocytes 8 h after TBTO treatment, x 3200
oxide (Aldrich Chemical Co. Inc. WI, USA, purity; 96%) and after time intervals of 1, 2, 4, 6, 8, 12, 16, 20, 24 h and 2, 3, 4, and 7 days after injection, the animals were deeply anesthetized with an injection of pentobarbital and the livers were isolated. Four untreated male rats served as controls. Specimens were fixed in 2% paraformaldehyde and 1% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at 4~ for 2 h, postfixed in 2% osmium tetroxide in the same buffer at 4~ for 2 h, dehydrated in a graded series of ethanol and embedded in epoxy resin. For light microscopy, semithin sections were made with a Huxley ultramicrotome and stained with buffered toluidine blue. For electron microscopy, ultrathin sections were stained by uranyl acetate and lead citrate and observed with a JEM 100CX electron microscope.
250-300 g) received an intramuscular injection of the same dose of TBTO as described above, and after time intervals of 4 and 8 h, and 4 days after injection, the animals were deeply anesthetized and the livers were isolated. Three untreated male rats served as controls. Mitochondria were prepared from the liver of all animals according to the procedure described by Ogura et al. (1978). Mitochondrial protein was determined by the biuret method. Oxygen utilization of mitochondria was determined polarographically with a Clark type oxygen electrode. The reaction medium contained mitochondria (3 mg protein/ml), 210 mM mannitol, 10 mM TRIS-HCI buffer (pH 7.4), 10 mM phosphate buffer (pH 7.4), 10 mM KCI, 2 mM MgCI2 and 0.2 mM EDTA, and glutamate and malate were used as substrates of respiration. ADP-stimulated respiration and ADP-limited respiration were designated as state 3 and state 4 respiration, respectively. The respiration control ratio (state 3/state 4) was calculated from the respiratory response shown on the oxygen consumption chart (Estabrook 1967).
X-ray microanalysis. Nonstained ultrathin sections were analyzed for detection of the accumulation sites of TBTO in the liver tissue with a JEM 2000EX electron microscope equipped with a LINK QX 200J energy dispersive X-ray microanalyzer.
Analyses of the activi~ of serum enzymes: Twelve male Wistar rats Oxygen consumption of mitochondria. To determine the oxygen consumption of the liver cell rnitochondria, nine male Wistar rats (weighing
(weighing 2 5 0 - 300 g) received an intramuscular injection of the same dose of TBTO as described above, and 4, 8 and 12 h, and 4 days after the
184 Table 1. The effect of TBTO on the respiratory responses of liver cell mitochondriaa Oxygen consumption b State 3 Control (n = 3)
State 4
Respiratory control ratio
97.27 +6.35
17.72 +2.43
5.75 ___0.77
TBTO 4 h (n = 3)
101.06 +3.00
36.10* __.5.27
2.69* +0.61
TBTO 8 h (n = 3)
90.00 ___8.25
35.34* ___4.10
2.55* ___0.16
TBTO 4 d (n = 3)
88.20 +6.82
16.21 ___2.79
5.38 +0.12
a All values expressed as mean -L-_SD Statistical analyses were carded out by a Student t-test * Significantly different from the control values (p 17 c t s
q.gtt0
< -.2 FS=255 MEMI :
Fig. 8. A An energy dispersive X-ray spectrum from one of the swollen
mit0chondria in the hepatocytes shows a Sn L- ct peak at 3.44 keV. B An X-ray spectrum from mitochondria of the control rat hepatocyte. No tin peaks are obtained. In these spectra, an osmium peak arises from the
eh
KeU 257=
10. ! > 16
et.s
fixative, chloride peaks arise from epoxy resin, copper peaks arise from a specimen holder of the electron microscope and molybdenum peaks arise from the molybdenum grid
Table 2. The activities of serum enzymes and the concentration of serum bilirubin a Control (n = 5)
TBTO 4 h (n = 3)
TBTO 8 h (n = 3)
TBTO 12 h (n = 3)
TBTO 4 d (n = 3)
AST (IU/l)
99.30 _ 17.61
198" +_21.50
249** + 15.67
373** +-30.21
112 _+ 28.23
ALT (IU/1)
39.30 -4- 1.15
41.50 +- 3.65
53.25* ___ 2.14
61.38" +- 4.26
41.00 +- 2.48
ALP (IU/I)
471.35 + 127.00
442.75 +- 88.00
389.50 _+ 137.25
480.75 __+98.65
520.35 +- 140.00
LAP (IUh)
61.00 ___ 9.16
57.35 +_ 7.52
65.35 +- 10.76
60.00 +15.29
55.56 _ 8.60
T.Bil. (mg/dl)
0.10 4- 0.02
0.10 4- 0.05
0.13 +_ 0.07
0.13 +- 0.07
0.10 +- 0.05
a All values expressed as mean _ SD Statistical analyses were carried out by a Student t-test Significantly different from the control values (p
Arch Toxicol (1992) 66: 182- 187
Toxicolog 9 Spfinger-Verlag 1992
Studies on the hepatotoxicity induced by bis (tributyltin) oxide Mitsuaki Yoshizuka l, Kazuo Hara l, Nobuya Haramaki3, Mitsuru Yokoyama 2, Naoki Moril, Yoshiaki Doi 1, Akio Kawahara l, and Sunao Fujimoto 1 l Department of Anatomy, and 2 Laboratory of Electron Microscopy, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807, Japan 3 Department of Medical Biochemistry, Kurume University School of Medicine, Kurume 830, Japan Received 20 August 1991/Accepted 29 October 1991
Abstract. The toxic effects of bis (tributyltin) oxide (TBTO) on the rat liver were studied with an electron microscope and the accumulation sites of tin were determined with an X-ray microanalyzer. The activities of serum enzymes and the concentration of serum bilirubin were also analyzed. Male Wistar rats received an intramuscular injection of 0.5 ml/kg of TBTO. Marked swelling of the mitochondria appeared in the hepatocytes 4 h after injection of TBTO. Cytoplasmic vacuoles, which contained degenerated mitochondria, gradually increased in number in these hepatocytes. This in turn may have caused a decrease in the volume of hepatic cell cords and an enlargement of sinusoids in the entire hepatic lobule. However, fine structures of intrahepatic bile ducts were not altered. By X-ray microanalysis, tin peaks were preferentially obtained from swollen mitochondria of the hepatocytes. By polarographic analysis of the respiratory responses of mitochondria, it was demonstrated that rates of state 4 respiration and respiratory control ratio were significantly disturbed in TBTO-treated rats in comparison with those of controls. The activities of AST (aspartate aminotransferase) and ALT (alanine aminotransferase) were significantly increased after TBTO treatment, but those of ALP (alkaline phosphatase), LAP (leucine aminopeptidase) and total bilirubin were not changed. These results indicated that parenterally administered TBTO accumulated in the liver cell mitochondria and disturbed oxidative phosphorylation. Mitochondrial dysfunction might induce severe damage of the hepatocytes. Four days after injection of TBTO, hepatic structures and chemical indices were almost restored by the regeneration of hepatocytes.
Introduction Tributyltin compounds have been widely used as molluscicides, as antifoulants on ships, fish nets, and cages, as wood preservatives, and as biocides for leather processing. Recently, the biological effects of tributyltin compounds were summarized (WHO 1990; Henschler 1991). In regard to the toxic effect of tributyltin compounds on the liver,a few reports have been published (Krajnc et al. 1984; Davis et al. 1987). They indicated that orally administered bis (tributyltin) oxide was accumulated in rat and mouse livers, and induced structural changes of the bile canalic~i and the common bile ducts. However, they did not mention the ultrastructural changes of the hepatocytes. Therefore, we decided to investigate detailed morph0logical alterations of rat liver after parenteral administration of bis (tributyltin) oxide (TBTO) using an electron microscope. The accumulation sites of TBTO in the liver tissue were examined with an X-ray microanalyzer. Several in vitro studies have indicated that organotin compounds act as inhibitors of oxidative phosphorylati0n in the mitochondria (Aldridge 1958; Aldridge et al. 1977: Cain et al. 1977). Therefore, we carded out a polarographic study to elucidate the effect on the respiratory responses of liver cell mitochondria. Furthermore, we determined the serum enzyme activities and the concentration of serum bilirubin.
Materials and methods
Key words: Bis(tributyltin) oxide - Liver - Electron microscopy - X-ray microanalysis - Biochemistry
Animals. Adult male Wistar UOEH rats were housed in individual stainless-steel cages and maintained with a 12-h light-dark cycle. Exposure to light was from 6 to 18 hours. The rats had been fed laboratory chow and water ad libitum until sacrificed between 11 and 12 hours.
Offprint requests to: Mitsuaki Yoshizuka, Department of Anatomy, University of Occupational and Environmental Health, School of Medicine, Kitakyushu 807, Japan
Light and electron microscopy. In our preliminary study, we found thata single intramuscular injection of 2 ml/kg body weight of undiluted TBTO was the LDs0 (lethal dose for 50% survival group) in the male Wistar rats. A quarter of the LDs0 was chosen in the present study. Thirty-nine adult male Wistar rats (weighing 2 5 0 - 300 g) received an intramuscular injection of 0.5 ml/kg body weight of bis (tributyltin)
183
Fig. 1. Depletion of glycogen particles in the cytoplasm of the hepatocytesand an enlargement of sinusoids are noted in the centrilobular zone 4h after TBTO treatment. CV: central vein • 200
Fig. 3. An electron micrograph showing the extremely swollen mitochondria, which are often seen in the hepatocytes in 4 h after TBTO treatment. • 16000
Fig. 2. The cytoplasmic vacuoles in the hepatocyte arise from dilated cistemae of the rough surfaced endoplasmic reticulum 4 h after TBTO treatment. Remnants of degenerated cytoplasmic membranes are found inthese vacuoles. Deformed mitochondria and increasing cisternae of the smooth surfaced endoplasmic reticulum surround the vacuoles, x 12000
Fig. 4. Various sizes of cytoplasmic vacuoles are found in the sinusoidal region of hepatocytes 8 h after TBTO treatment, x 3200
oxide (Aldrich Chemical Co. Inc. WI, USA, purity; 96%) and after time intervals of 1, 2, 4, 6, 8, 12, 16, 20, 24 h and 2, 3, 4, and 7 days after injection, the animals were deeply anesthetized with an injection of pentobarbital and the livers were isolated. Four untreated male rats served as controls. Specimens were fixed in 2% paraformaldehyde and 1% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at 4~ for 2 h, postfixed in 2% osmium tetroxide in the same buffer at 4~ for 2 h, dehydrated in a graded series of ethanol and embedded in epoxy resin. For light microscopy, semithin sections were made with a Huxley ultramicrotome and stained with buffered toluidine blue. For electron microscopy, ultrathin sections were stained by uranyl acetate and lead citrate and observed with a JEM 100CX electron microscope.
250-300 g) received an intramuscular injection of the same dose of TBTO as described above, and after time intervals of 4 and 8 h, and 4 days after injection, the animals were deeply anesthetized and the livers were isolated. Three untreated male rats served as controls. Mitochondria were prepared from the liver of all animals according to the procedure described by Ogura et al. (1978). Mitochondrial protein was determined by the biuret method. Oxygen utilization of mitochondria was determined polarographically with a Clark type oxygen electrode. The reaction medium contained mitochondria (3 mg protein/ml), 210 mM mannitol, 10 mM TRIS-HCI buffer (pH 7.4), 10 mM phosphate buffer (pH 7.4), 10 mM KCI, 2 mM MgCI2 and 0.2 mM EDTA, and glutamate and malate were used as substrates of respiration. ADP-stimulated respiration and ADP-limited respiration were designated as state 3 and state 4 respiration, respectively. The respiration control ratio (state 3/state 4) was calculated from the respiratory response shown on the oxygen consumption chart (Estabrook 1967).
X-ray microanalysis. Nonstained ultrathin sections were analyzed for detection of the accumulation sites of TBTO in the liver tissue with a JEM 2000EX electron microscope equipped with a LINK QX 200J energy dispersive X-ray microanalyzer.
Analyses of the activi~ of serum enzymes: Twelve male Wistar rats Oxygen consumption of mitochondria. To determine the oxygen consumption of the liver cell rnitochondria, nine male Wistar rats (weighing
(weighing 2 5 0 - 300 g) received an intramuscular injection of the same dose of TBTO as described above, and 4, 8 and 12 h, and 4 days after the
184 Table 1. The effect of TBTO on the respiratory responses of liver cell mitochondriaa Oxygen consumption b State 3 Control (n = 3)
State 4
Respiratory control ratio
97.27 +6.35
17.72 +2.43
5.75 ___0.77
TBTO 4 h (n = 3)
101.06 +3.00
36.10* __.5.27
2.69* +0.61
TBTO 8 h (n = 3)
90.00 ___8.25
35.34* ___4.10
2.55* ___0.16
TBTO 4 d (n = 3)
88.20 +6.82
16.21 ___2.79
5.38 +0.12
a All values expressed as mean -L-_SD Statistical analyses were carded out by a Student t-test * Significantly different from the control values (p 17 c t s
q.gtt0
< -.2 FS=255 MEMI :
Fig. 8. A An energy dispersive X-ray spectrum from one of the swollen
mit0chondria in the hepatocytes shows a Sn L- ct peak at 3.44 keV. B An X-ray spectrum from mitochondria of the control rat hepatocyte. No tin peaks are obtained. In these spectra, an osmium peak arises from the
eh
KeU 257=
10. ! > 16
et.s
fixative, chloride peaks arise from epoxy resin, copper peaks arise from a specimen holder of the electron microscope and molybdenum peaks arise from the molybdenum grid
Table 2. The activities of serum enzymes and the concentration of serum bilirubin a Control (n = 5)
TBTO 4 h (n = 3)
TBTO 8 h (n = 3)
TBTO 12 h (n = 3)
TBTO 4 d (n = 3)
AST (IU/l)
99.30 _ 17.61
198" +_21.50
249** + 15.67
373** +-30.21
112 _+ 28.23
ALT (IU/1)
39.30 -4- 1.15
41.50 +- 3.65
53.25* ___ 2.14
61.38" +- 4.26
41.00 +- 2.48
ALP (IU/I)
471.35 + 127.00
442.75 +- 88.00
389.50 _+ 137.25
480.75 __+98.65
520.35 +- 140.00
LAP (IUh)
61.00 ___ 9.16
57.35 +_ 7.52
65.35 +- 10.76
60.00 +15.29
55.56 _ 8.60
T.Bil. (mg/dl)
0.10 4- 0.02
0.10 4- 0.05
0.13 +_ 0.07
0.13 +- 0.07
0.10 +- 0.05
a All values expressed as mean _ SD Statistical analyses were carried out by a Student t-test Significantly different from the control values (p
