* Laboratory of Veterinary Public Health;
Laboratory of Veterinary Anatomy;
Laboratory of Veterinary Internal Medicine;
Laboratory of Biochemistry, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada-shi, Aomori 034-8628, Japan;
¶ Department of Veterinary Medicine, Faculty of Agriculture, Iwate University, Moriaka-shi, Iwate 020-8550, Japan;
|| Laboratory of Environmental Health and Toxicology, Kyoto Prefectural University, Sakyo-ku, Kyoto 606-5822, Japan; and
||| Sugiyama Pharmacy, Tamagawa-cho, Yamaguchi 759-3112, Japan
Received February 17, 2003; accepted May 9, 2003
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ABSTRACT |
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Key Words: butyltin; hepatotoxicity; metabolism; mitochondria; mice; guinea pig.
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INTRODUCTION |
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Metabolism of butyltin compounds by cytochrome P450 enzymes has been suggested to play an important role in the induction of biological effects: Tributyltin was found to undergo hydroxylation followed by dealkylation to produce dibutyltin, monobutyltin, and inorganic compounds in the presence of microsomes and nicotinamide adenine dinucleotide phosphate (NADPH) in vitro (Casida et al., 1971; Fish, 1984
; Fish et al., 1976
; Kimmel et al., 1977
). Moreover, several studies have shown a variety of metabolites in rat (Matsuda et al., 1993
) and mouse liver (Ueno et al., 1997
) formed during the metabolism of TBTC in vivo. Regarding the relation between the metabolism and hepatotoxicity of tributyltin compound in vivo, we have reported that inhibition of cytochrome P450 enzymes in the liver of mice prevents the hepatotoxicity caused by TBTC compounds (Ueno et al., 1995
, 1997
). Thus, hepatic metabolism of TBTC may be associated with the induction of hepatotoxicity by these butyltin compounds.
It has been well known that the toxicity of organotin differs greatly among experimental animal species. For instance, previous studies have shown that TBTC induced hepatotoxicity in rats and mice but not in guinea pigs and rabbits (Boyer, 1989) and that DBTC did not induce any toxicity in guinea pigs (Barnes and Magee, 1958
). However, the reason for the difference in the toxicity between these experimental animals remains unclear. We have recently shown differences in the effect of TBTC and DBTC on the induction of hepatotoxicity between mice, rats, and guinea pigs, and reported that these differences were in part related to differences in hepatic metabolism and distribution of butyltin compounds within cell organelles because remarkable morphological changes were induced in the mitochondria of hepatocytes by TBTC and DBTC in mice, whereas no such changes were observed in the mitochondria of rats and guinea pigs (Ueno et al., 2003
). In this study, these preliminary observations have been extended by examining the effects of TBTC and DBTC on the mitochondrial respiration in the hepatocytes of mice and guinea pigs in vitro and in vivo, using polarography. In addition, the levels of butyltin metabolites in hepatic mitochondria in vivo and the affinity of DBTC for hepatic mitochondrial fractions in vitro were also investigated to elucidate the mechanism of mitochondrial damages caused by butyltin compounds in these animals. Our results illustrated that the differences in hepatotoxicity of TBTC or DBTC between mice and guinea pigs might be associated with the differences in the depression of mitochondrial respiration, possibly due to the higher affinity of DBTC for sulfhydryl groups in hepatic mitochondria of mice.
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MATERIALS AND METHODS |
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Treatment.
TBTC and DBTC (Tokyo Kasei Kogyo Co., Ltd., Tokyo, Japan) were dissolved in corn oil (SIGMA, St. Louis, MO) and administered orally (10 ml/kg for mice, 1 ml/kg for guinea pigs). The dosage of TBTC or DBTC in our experiments was 360 µmol/kg (117.2 mg/kg) or 120 µmol/kg (36.6 mg/kg), respectively. These dosages of butyltins are equivalent to twice the minimum dosage that induced hepatotoxicity in mice (Ueno et al., 1994).
Serological evaluation of liver injury.
To evaluate the liver injury induced by the butyltin compounds, the activities of ornithine carbamyl transferase (OCT; EC: 2.1.3.3), aspartic acid aminotransferase (AST; EC: 2.6.1.1), and alanine aminotransferase (ALT; EC: 2.6.1.2) in serum were observed at 24 h after administration. OCT activity was measured as previously described (Ueno et al., 1994) and was defined as 1 IU/l, the amount necessary to catalyze the formation of 1 µmol citrulline/min/l serum. The activities of AST and ALT were determined by blood chemistry autoanalyzer AU-550 (Olympus Company, Tokyo, Japan) using a kit (Boehringer Mannheim Co. Ltd., Mannheim, Germany) based on UV method.
Preparation of mitochondrial fraction.
The animals were killed by decapitation, and livers were removed immediately. One gram of the livers was homogenized in 9 ml of 10-mM TrisHCl buffer (pH 7.4) containing 0.21 M mannitol, 0.07 M sucrose, and 0.1 mM EDTA. The homogenate was centrifuged at 600 x g for 10 min at 4°C, and the supernatant was centrifuged at 9,000 x g for 10 min at 4°C. After the supernatant was removed by decantation, the mitochondria-rich pellet was gently resuspended in 9 ml of the buffer, and the centrifugation procedure was repeated. The mitochondrial fraction obtained was suspended in 1 ml of 10-mM TrisHCl buffer (pH 7.4) containing 225 mM mannitol, 75 mM sucrose, 10 mM KCl, and 5 mM KH2PO4 (Trounce et al., 1996).
Polarography.
According to the method of Morikawa et al. (1996), mitochondrial respiration was measured at 25°C with an oxygen monitor using a Clark-type oxygen electrode in the same medium used to suspend the mitochondria. Respiratory substrates were used at final concentrations of 5 mM succinate (plus a 0.33 µM rotenone). Mitochondria (1 mg protein) and 125 nmol of adenosine-5'-diphosphate (ADP) were added to initiate phosphorylating oxygen consumption (State 3). Because the respiratory control index (RCI; State 3/State 4) reflects the degree of coupling of respiration with adenosine-5'-triphosphate (ATP) production, the RCI was calculated by dividing the rate of oxygen consumption in the presence of ADP by that in the absence of ADP. In the in vitro study, after State 3 respiration was measured, butyltin compounds dissolved in dimethyl sulfoxide (DMSO; 5 µl) were added to the reaction mixture (1 ml) at State 4 respiration, and after a few minutes, the same amount of ADP was added again to measure State 3 respiration. The IC50 values (the concentration that decreases the oxygen consumption rates in State 3 respiration) were calculated graphically. After it had been confirmed that the addition of DMSO to the incubation mixture did not affect State 3 respiration, different concentrations of TBTC or DBTC were tested. Nonlinear regression analysis was carried out to obtain the concentration-response curves for the oxygen consumption rates in the presence or absence of the butyltin compounds using data analysis software (SigmaPlot 2001: Sigmoid, 3 parameter) from SPSS Science software products (Chicago, IL).
Measurement of butyltin metabolites in the mitochondrial fractions of hepatocytes.
The butyltin compounds were extracted and purified from the mitochondrial fractions by the method of Suzuki et al. (1994) The levels of each butyltin compound in the samples were determined by the method of Suzuki et al. (1994)
, using an HP Model 5890 Series II gas chromatograph (Hewlett-Packard, Avondale, PA) equipped with a split/splitless injection port interfaced to an HP Model 5921A atomic emission detector equipped with a turbo makeup gas valve. All standards were purchased or prepared as reported previously (Suzuki et al., 1994
). The chemical names, their abbreviations, and detection limits are shown in Table 1
.
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Affinity of DBTC for the mitochondrial fractions isolated from hepatocytes.
To compare the affinity of DBTC for mitochondrial fractions, the purified mitochondrial fractions (510 mg protein/ml) were exposed to DBTC at 50 µg/ml for 5 min, then the contents of DBTC were measured as above. Furthermore, we also observed the effects of iodoacetamide, a sulfhydryl blocker, on the affinity of DBTC for these fractions. After the mitochondrial fractions were pretreated with 1 mM of iodoacetamide for 5 min, the fractions were exposed to the same conditions of DBTC, and the contents of DBTC were measured.
Statistical analysis.
Because the serum enzyme activities were not normally distributed, the statistical differences in these data were evaluated by the Mann-Whitney U test. The data obtained from polarography in vivo were analyzed using one-way ANOVA, and a p value less than 0.05 was considered statistically significant. Differences between treatment and control groups were compared with Dunnett test. Data from studies with only two groups were analyzed by the Students t test for equal variance or the Welch t test for unequal variance after Bartletts test.
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RESULTS |
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Effects of Butyltin Compounds on Mitochondrial Respiration in Vivo
Table 3 shows the respiratory responses of mitochondria from the livers of the experimental animals treated with TBTC or DBTC at 24 h after oral administration. The treatment of mice with TBTC or DBTC resulted in a significant decrease of the succinate-linked State 3 respiration, as well as of RCI, although no significant influence was observed in guinea pigs. Thus, the in vivo treatment of TBTC or DBTC in mice specifically inhibited the mitochondrial State 3 respiration of liver.
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In the case of DBTC, as shown in Table 5, the total butyltin levels in hepatic mitochondria were also greater in the mice than in the guinea pigs. With respect to metabolites in hepatic mitochondria, DBTC showed very little degradation in these animals, even at 24 h after the administration, because about 90% of butyltin compounds were distributed as DBTC in the mitochondrial fraction of both mice and guinea pigs. As was the case with TBTC, the levels of DBTC in the fraction were about three times greater in the mice than in the guinea pigs.
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DISCUSSION |
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This and our previous studies also showed that mice were more sensitive to hepatotoxicity than guinea pigs and that guinea pigs were insensitive to hepatotoxicity caused by butyltin compounds, because the administration of TBTC or DBTC resulted in a significant increase in the activity of OCT, ALT, and AST enzymes in mice, whereas these enzyme activities were not affected in guinea pigs (Table 2). Under the same experimental conditions, although the total butyltin levels in liver have been shown to be greater in guinea pigs, compared with mice (Ueno et al., 2003
), these serological results were consistent with the histopathological results in which swelling and collapse of mitochondria were observed in mice livers but not in livers of guinea pigs (data not shown). In addition, this study suggested that the swelling of mitochondria induced by TBTC or DBTC in mice might lead to an uncoupling oxidative phosphorylation by means of the electron transport system, because the administration of TBTC (180 µmol/kg) or DBTC (60 µmol/kg) showed a statistically significant facilitation of State 4 respiration at 48 h in mice liver in vivo (succinate as substrate: control, 27.6 ± 2.1; TBTC, 39.3 ± 5.5; DBTC, 43.3 ± 4.0 nano atoms O/min/mg protein). Thus, these results strongly indicated that the influences of TBTC and DBTC on the mitochondria in liver might play a critical role in the induction of hepatotoxicity by these organotin compounds in vivo.
Previous studies have reported inhibitory effects on isolated mitochondria respiration by TBTC and DBTC in vitro (Aldridge, 1976; 1977
). Similarly, our in vitro study also showed that TBTC and DBTC suppressed State 3 respiration linked by succinate as the substrates (Table 4
). It is interesting to note that the in vivo inhibition against hepatic mitochondrial respiration by TBTC and DBTC was observed only in mice (Table 3
), whereas there was no significant difference between mice and guinea pigs in the IC50 of TBTC and DBTC on State 3 respiration in vitro (Table 4
). Furthermore, the inhibitory effects of TBTC on State 3 respiration of the isolated mitochondria were 10 times or more as strong as those of DBTC in vitro (Table 4
), although there was no significant difference between the treatment of mice with TBTC (360 µmol/kg) and DBTC (120 µmol/kg) in the inhibition of the hepatic mitochondrial respiration in vivo. It is possible that the differences between in vivo and in vitro experiments may result from the differences in the levels of TBTC and DBTC in hepatic mitochondria of mice and guinea pigs, because our previous studies showed that the hepatotoxicity of TBTC was closely related to its metabolism in the liver in vivo (Ueno et al., 1995
; 1997
). The present analysis of metabolites of TBTC in the mitochondrial fraction revealed that the main metabolite of TBTC was DBTC in both mice and guinea pigs in vivo (Table 5
). With respect to DBTC treatment, DBTC showed very little degradation in the mitochondrial fractions of both species. Interestingly, the mitochondrial levels of DBTC in mice treated with either TBTC or DBTC were three times greater than those in guinea pigs. On the other hand, the mitochondrial levels of TBTC in liver of the TBTC-treated guinea pigs was about 10 times greater than those in mice; nevertheless, no mitochondrial injuries were observed in guinea pigs in vivo. Although the critical metabolites that are responsible for mitochondrial damages caused by these butyltin compounds are not clear, these results strongly indicated that the mitochondrial damages induced by TBTC or DBTC in vivo may be closely associated with the levels of DBTC but not TBTC in this fraction of hepatocytes.
Our previous analysis of cellular distributions of DBTC in the liver after the metal administration in vivo showed that the levels of DBTC in mitochondrial fractions in mice hepatocytes were greater than those in guinea pigs (Ueno et al., 2003). As expected, higher levels of organotin compounds in the mitochondrial fractions of hepatocytes were also observed in the mice than in the guinea pigs at 24 h after administration of either TBTC or DBTC (Table 5
). Therefore, the butyltin compounds appear to become more concentrated in the mitochondrial fractions of mice hepatocytes than in those of guinea pigs. Because sulfhydryl groups (reduced form), which organize dithiol structure, have been shown to have a high affinity for DBTC (Aldridge, 1977
; Merkord et al., 2000
), we compared the contents of sulfhydryl groups in mitochondrial fractions of livers between these animals (Table 6
) to understand the difference in distribution of DBTC in the mitochondrial fractions. When the mitochondrial fractions were denatured by SDS, there was no difference between mice and guinea pigs in the content of total sulfhydryl groups (reduced and oxidized form). On the other hand, the concentrations of sulfhydryl groups (reduced form) in the mitochondrial fractions without the denaturation were about two times higher in mice than in guinea pigs. Thus, the levels of sulfhydryl groups (reduced form) may be higher in the mitochondrial fractions of mice than in those of guinea pigs. Moreover, the affinity of DBTC for the mitochondria of mice hepatocytes was significantly higher than that in guinea pigs, and the pretreatment of the isolated mitochondria with a sulfhydryl blocker such as iodoacetamide reduced the affinity of DBTC only in mice (Table 7
). These results indicated that the differences in the mitochondrial levels of DBTC between mice and guinea pigs in vivo might be due to the differences in the levels of sulfhydryl groups in the respective mitochondria. Our preliminary in vitro experiments showed that the amount of sulfhydryl groups of glutathione, which could be detected with DTNB, were not affected by the treatment with DBTC, whereas the amount of sulfhydryl groups of dimercapto-propanol, which organize dithiol structure, was reduced by DBTC (data not shown). Thus, it is possible that the mitochondrial levels of sulfhydryl groups, in particular dithiol, in mice liver may be higher than those in guinea pigs.
In conclusion, this study indicated that the difference in susceptibility to butyltin compounds between mice and guinea pigs may be closely associated with the inhibition of mitochondrial respiration, possibly due to the higher affinity of butyltin compounds, in particular DBTC, for hepatic mitochondria in mice. The results also suggest that DBTC in the mitochondrial fractions might be one of the main critical forms responsible for mitochondrial damages caused by TBTC and DBTC in vivo. Recently, Stridh et al. (1999) reported that low concentrations of TBTC triggered an immediate depletion of intercellular ATP followed by necrotic death in Jurket cells and showed that the mode of cell death was typically apoptotic when ATP levels were maintained by the addition of glucose. Moreover, depending on the situations of the cells, such as resting cells or CD3-stimulated cells, they also reported that TBTC could induce apoptosis or necrosis in human peripheral blood lymphocytes (Stridh et al., 2001
). Thus, the effects of butyltin compounds on mitochondrial function may play an important role in both cell necrosis and apoptosis in vitro. Our present results should provide useful clues for future research concerned with the toxicity of butyltin compounds in vivo.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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