Arch Toxicol (1997) 71: 513±518
ORGAN TOXICITY AND MECHANISMS
Shunji Ueno á Takashi Suzuki á Nobuyuki Susa
Yoshinori Furukawa á Masayasu Sugiyama
Effect of SKF-525A on liver metabolism and hepatotoxicity
of tri- and dibutyltin compounds in mice
Received: 22 October 1996 / Accepted: 24 February 1997
Abstract The e€ect of pretreatment with SKF-525A,
which inhibits hepatic cytochrome P450 enzymes, on
metabolism and hepatotoxicity was examined in mice
orally administered tributyltin chloride (TBTC) or
dibutyltin dichloride (DBTC) at a dose of 180 lmol/kg.
Analysis of butyltin compounds showed that the main
metabolites in liver of mice treated with TBTC alone
were DBTC (40%) and dibutyl(3-carboxylpropyl)tin
chloride (TCOOH; 12±26%), with the levels of other
butyltin compounds including TBTC comprising <12%
of total butyltin compounds at 3±24 h following treat-
ment. The pretreatment with SKF-525A resulted in
four- to tenfold increased TBTC levels and a signi®cant
decrease of debutylated metabolites, particularly DBTC
(60 and 37% decrease) at both 3 and 6 h in liver of mice
treated with TBTC, leading to complete inhibition of
hepatotoxicity at 24 h. At 24 h after TBTC treatment,
hepatic levels of TBTC and most of the debutylated
metabolites in mice pretreated with SKF-525A did not
di€er signi®cantly when compared to those in unpre-
treated mice, resulting in the induction of hepatotoxicity
at 48 h, although levels of TCOOH decreased even at
24 h. In the case of DBTC treatment, >95% of the
butyltin compounds were detected as DBTC in liver, and
the levels of DBTC inside cells as well as the induction of
DBTC hepatotoxicity were una€ected by pretreatment
with SKF-525A. These results suggest that debutylated
metabolites, in particular DBTC, are the main metabo-
lites of butyltin compounds responsible for the induction
S. Ueno (&) á N. Susa á Y. Furukawa
Department of Veterinary Public Health,
School of Veterinary Medicine and Animal Sciences,
Kitasato University, Higashi 23-35-1, Towada,
Aomori 034, Japan
T. Suzuki
Division of Food, National Institute of Hygienic Sciences,
18-1, Kamiyoga 1-chome, Setagaya-ku, Tokyo 158, Japan
M. Sugiyama
Asunaro Pharmacy, 6±7 Hatsune-cho, Tobata-ku,
Kitakyushu 804, Japan
Ó Springer-Verlag 1997
of hepatotoxicity following in vivo administration of
TBTC. The results also indicate that cytochrome P450
enzymes may play a greater role in the metabolism of
TBTC to form DBTC or butyltin trichloride (MBTC)
than that of DBTC to form MBTC in liver of mice.
Key words Butyltin compounds á Hepatotoxicity á
Cytochrome P450
Introduction
The toxicity of organotin compounds has attracted
special interest owing to expanding utilization and en-
suing pollution of organotin compounds in the form of
plastic stabilizers, catalytic agents, industrial and agri-
cultural biocides, anti-fouling paints and pesticides
(Wilkinson 1984; Snoeiji et al. 1987). Among these or-
ganotin compounds, butyltin compounds have been
recognized as particularly important environmental
pollutants in consideration of their widespread indus-
trial and agricultural applications (Boyer 1989).
Depending upon the number of organic moieties,
butyltin compounds are classi®ed as mono-, di-, tri- or
tetrabutyltins. Among the butyltin compounds, tribu-
tyltin (TBTC) and dibutyltin (DBTC) compounds have
been shown to cause injury to the bile duct and liver in
experimental animals (Andersson et al. 1988; Barnes and
Stoner 1958), while monobutyltin (MBTC) compounds
are reported to be relatively inert (Pelikan and Cerny
1970). Concerning the hepatotoxicity, a recent animal
study has shown that DBTC is more hepatotoxic than
TBTC (Ueno et al. 1994).
The metabolism of the butyltin compounds by cyto-
chrome P450 enzymes has been suggested to play a role
in the induction of biological e€ects: tributyltin was
found to undergo hydroxylation followed by dealkyl-
ation to produce dibutyltin, monobutyltin and inorganic
tin compounds in the presence of microsomes and
NADPH in vitro (Casida et al. 1971; Fish 1984; Fish
et al. 1976; Kimmel et al. 1977). Moreover, recent
514
Table 1 List of tri- and dibutyltin compounds and their
abbreviations
Standard Abbrev.
Tri-n-butyltin chloride TBTC
Di-n-butyl (3-hydroxybutyl) tin chloride T3OH
Di-n-butyl (4-hydroxybutyl) tin chloride T4OH
Di-n-butyl (3-oxybutyl) tin chloride T3CO
Di-n-butyl (3-carboxypropyl) tin chloride TCOOH
Di-butyltin dichloride DBTC
n-Butyl (3-hydroxybutyl) tin dichloride D3OH
n-Butyl (4-hydroxybutyl) tin dichloride D4OH
n-Butyl (3-oxobutyl) tin dichloride D3CO
n-Butyl (3-carboxybutyl) tin chloride DCOOH
n-Butyltin trichloride MBTC
studies using the gas chromatography/helium atmo-
spheric pressure microwave-induced plasma/atomic de-
tection system (GC/MIP/AED) for analysis of organotin
compounds, have shown a variety of metabolites in rat
liver formed during the metabolism of TBTC in vivo
(Table 1, Fig. 1; Matsuda et al. 1993). Regarding the
relation between the metabolism and hepatotoxicity of
tributyltin compound in vivo, we have previously shown
that inhibition of cytochrome P450 enzymes in the liver
of mice pretreated with SKF-525A prevents the he-
patotoxicity caused by TBTC compounds (Ueno et al.
1995). Thus, hepatic metabolism of TBTC by cyto-
chrome P450 enzymes may be associated with the in-
duction of hepatotoxicity. However, the active
metabolites responsible for the induction of hepatotox-
icity remained obscure.
In the present study, we have extended these prelim-
inary ®ndings by examining the e€ects of SKF-525A on
the hepatic levels of metabolites in mice treated with
TBTC and DBTC in vivo. Levels of the metabolites of
butyltin compounds in the livers of mice were estimated
by means of GC/MIP/AED (Suzuki et al. 1994). Our
results demonstrate that hepatic levels of DBTC play an
Fig. 1 Hypothetical pathway of
metabolism of tributyltin chloride
(TBTC). (from Matsuda et al.
1993)
important role in the induction of hepatotoxicity by
TBTC.
Materials and methods
Animals
Male albino mice of the same colony (ddY strain) weighing 30 g
at 6 weeks of age, were used throughout the study. The mice were
housed in groups in plastic cages and maintained on standard
laboratory diet (rat chow MF; Oriental Yeast Co., Ltd.) and tap
water. A lighting regime was imposed of 12 h light (7.00 a.m. to
7.00 p.m.) and 12 h dark (7.00 p.m. to 7.00 a.m.). Ambient tem-
perature during the study was maintained at 21 °C.
Treatment
Tributyltin chloride and dibutyltin dichloride (Tokyo Kasei Kogyo
Co. Ltd.) were dissolved in corn oil (Sigma) and administered
orally (180 lmol/kg, 10 ml/kg). N,N-diethylaminoethyl-2,2-diphe-
nylvalerate hydrochloride (SKF-525A, Research Biochemicals) was
dissolved in normal physiological saline. A dose of 50 mg/kg SKF-
525A, an inhibitor of cytochrome P450, was administered by i.p.
injection 15 min before oral administration of the butyltin com-
pounds (Shan et al. 1976).
Extraction of butyltin compounds from liver
Following administration of the compounds, the mice were anes-
thetized with ether, and blood was collected by cardiac puncture.
The animals were then sacri®ced, the liver was quickly excised and
washed with ice-cold physiological saline. Brie¯y, the butyltin
compounds in liver were extracted by the method of Suzuki et al.
(1994). The homogenized sample (<5 g wet liver in 10 ml of 0.9%
saline) was acidi®ed with 12 ml of HCl (36%) in a 50 ml centrifuge
tube with a screw cap, and the mixture was shaken vigorously, and
allowed to stand for 10 min. After the addition of 20 ml ether and
2 g NaCl, the mixture was shaken for 10 min on a KM shaker
(Iwaki Co., Ltd.), followed by centrifugation at 3000 rpm for
5 min. This extraction procedure was repeated twice.
The ether extract was evaporated in vacuo at 35 °C, and the
residue dissolved in n-hexane/ethyl acetate (2:1, v/v; 5 ml). The
resulting solution was transferred to a 1 cm i.d. chromatographic

Fig. 2 E€ects of pretreatment with SKF-525A on ornithine carbamyl
transferase (OCT) activity in serum of mice orally administered
tributyltin chloride (TBTC; A) or dibutyltin chloride (DBTC; B).
SKF-525A at a dose of 50 mg/kg was administered by i.p. injection 15
min before TBTC or DBTC (180 lmol/kg). Each value is of
mean ‹ SD (n ˆ 4). **P < 0.01 compared with the control group;
aa
P < 0.01 compared with the group treated with TBTC alone
column containing 5 g of HCl-treated silica gel (Wakogel C-100;
Wako Chemical Industries), prepared with n-hexane, and with
1 cm of anhydrous Na2SO4 on the top. The column was eluted with
a mixture of n-hexane/ethyl acetate (2:1, v/v; 50 ml). The eluate was
evaporated in vacuo, the residue was dissolved in ether (3 ml), and
transferred to a screw-capped centrifuge tube (50 ml). Methyl-
magnesium bromide (Tokyo Kasei Kogyo, c. 3 M in ether, 2 ml)
was added carefully to the solution described above, mixed gently,
screw-capped, and allowed to stand for 1 h in a water bath at
40 °C. Ten milliliters of distilled water were added drop by drop to
the solution in an ice bath until violent bubbling ceased with the
addition of water. After gentle mixing, anhydrous Na2SO3 (0.2 g)
and saturated NH4Cl (4 ml) were added to the solution, which was
shaken vigorously. This reaction mixture was extracted twice with
n-hexane (10 ml), the combined n-hexane extract was dried over
anhydrous Na2SO4, and concentrated exactly to 2 ml.
Measurement of butyltin compounds in the samples
The levels of each of the butyltin compound in the samples were
determined by the method of Suzuki et al. (1994). An HP model
5890 series II gas chromatograph (Hewlett-Packard; Avonbale, Pa.,
USA) equipped with a split/splitless injection port interfaced to an
HP model 5921A atomic emission detector equipped with a turbo
makeup gas valve was used. Two capillary columns were used: a
cross-linked cyanopropyl phenyl methyl silicone (DB-225) [J&W
Scienti®c, Folsom, Calif., USA; 0.25 mm (i.d.) ´ 30 m ´ 0.25 lm
(®lm thickness)] and a cross-linked 5% phenyl methyl silicone (DB-
5) [J&W Scienti®c; 0.25 mm (i.d.) ´ 30 m ´ 0.25 lm (®lm thick-
ness)].
Operating conditions for DB-225 were as follows: column oven,
programmed from 35 °C (held 2 min) at a rate of 30 °C/min to
220 °C (held 15 min); injection port (splitless), 220 °C; AED sol-
vent vent o€ time, 3.6 min; AED cavity temperature, 220 °C; AED
cavity pressure, 1.5 psi; AED cavity scavenger gases, 3.5 kg/cm2
(H2), 1.4 kg/cm2 (O2); AED spectrometer purge ¯ow (N2), 2 l/min;
wavelengths for measurement, 303.435 nm. Operating conditions
for DB-5 were as follows: column oven, programmed from 35 °C
(held 2 min) at a rate of 30 °C/min to 200 °C (held 0 min), followed
by 15 °C/min to 280 °C (held 1 min); injection port, 250 °C; AED
solvent vent o€ time, 4 min; AED cavity temperature, 280 °C;
other conditions were the same as for DB-225. The gas ¯ow rates
515
(head pressure) for He carrier gas were 173 kPa. Quantitative de-
termination was performed with internal standards using tri-n-
propyl ethyltin. All standards were purchased or prepared as re-
ported previously (Suzuki et al. 1994). The chemical names and
their abbreviations are shown in Table 1, and the speculated
pathway of TBTC metabolism is shown in Fig. 1 (Matsuda et al.
1993).
Evaluation of liver injury
In order to evaluate the liver injury induced by butyltin com-
pounds, the activity of ornithine carbamyl transferase (OCT) in
serum was measured as previously described (Ueno et al. 1995).
The activity de®ned as 1 IU/l was the amount necessary to catalyze
the formation of 1 lmol citrulline/min per liter serum.
Statistical analysis
Data from studies with only two groups were analyzed by the
Student t test for equal variance, or by the Welch t-test for unequal
variance, except for the serum OCT activity, after analysis of
variance by Bartlett's test. The serum OCT activity was not nor-
mally distributed; therefore, the statistical di€erences were evalu-
ated by the Mann-Whitney U test.
Results
Hepatotoxicity
Figure 2 shows the e€ect of SKF-525A on the hepato-
toxicity of TBTC and DBTC, as evaluated by serum
OCT activity. The median value of this enzymatic ac-
tivity in mice treated with corn oil alone was 6.7 IU/l
(n ˆ 20), and pretreatment with SKF-525A did not af-
fect the activity of OCT in serum during the experi-
mental period (data not shown). As shown in Fig. 2,
hepatotoxicity of TBTC and DBTC appeared at 24 h
(median of 200.8 IU/l, n ˆ 10) and 12 h (median of
50.7 IU/l, n ˆ 10) respectively after administration.
When mice were pretreated with SKF-525A, there was a
complete inhibition of the hepatotoxicity induced by
TBTC at 24 h (median of 3.8 IU/l, n ˆ 10); however,
this inhibitory e€ect disappeared at 48 h (median of
100.5 IU/l, n ˆ 10) following the oral administration of
516

Fig. 3 E€ects of pretreatment with SKF-525A on the total butyltin
contents in liver of mice orally administered 180 lmol/kg TBTC (A)
or DBTC (B). Each value is of mean ‹ SD (n ˆ 4). *P < 0.05,
**P < 0.01 compared with hepatic levels of total butyltin in
unpretreated TBTC or DBTC treated mice
after TBTC treatment. In the case of DBTC adminis-
tration (Fig. 3B), similar pretreatment with SKF-525A
caused a signi®cant decrease of total butyltin in liver
only at 3 h after administration.

TBTC. On the other hand, similar pretreatment had no
e€ect on the hepatotoxicity induced by DBTC at 12 h
(median of 76.4 IU/l, n ˆ 10), 24 h (unpretreated, me-
dian of 225.0 IU/l, n ˆ 10; SKF-525A, median of
219.5 IU/l, n ˆ 10), and 48 h (unpretreated, median of
113.9 IU/l, n ˆ 10; SKF-525A, median of 151.5 IU/l,
n ˆ 10). Thus, SKF-525A pretreatment can suppress the
hepatotoxicity caused by TBTC, but not that by DBTC.
Total hepatic levels of butyltin
Figure 3 shows the e€ects of the pretreatment with
SKF-525A on the contents of total butyltin in the liver
of mice at 3, 6, and 24 h after oral administration of
TBTC and DBTC, respectively. As shown in Fig. 3A,
when compared to unpretreated TBTC-treated mice, the
pretreatment with SKF-525A resulted in a signi®cant
decrease of the total liver butyltin liver at 3 and 24 h
Hepatic levels of metabolites
The data of Table 2 show the e€ects of pretreatment
with SKF-525A on the hepatic levels of each butyltin
metabolite in mice at 3, 6, and 24 h after oral adminis-
tration of TBTC and DBTC, respectively. Since pre-
treatment with SKF-525A caused alteration of total
butyltin levels in liver (Fig. 3), the levels of each me-
tabolite were expressed as a percentage of the total bu-
tyltin content. The hepatic levels of each butyltin
metabolite during 24 h after oral administration of
TBTC indicate that DBTC and TCOOH were the main
form of butyltin compound occurring in the liver after
administration of TBTC. The levels of D3OH and
T3OH decreased while the levels of TCOOH increased
proportionately with time.
When the mice were pretreated with SKF-525A, he-
patic levels of TBTC and T3OH increased at both 3 and
6 h while those of T3CO increased only at 6 h. In con-

Table 2 Percentage of each

metabolite of TBTC in liver of Pretreatment Unpretreated SKF-525A
mice pretreated with or without
SKF-525A 3h 6h 24 h 3h 6h 24 h

TBTC 7.2 ‹ 2.0 11.7 ‹ 0.6 8.0 ‹ 1.7 72.1 ‹ 5.2aa 43.9 ‹ 4.2aa 22.9 ‹ 10.1
T3OH 2.4 ‹ 0.8 2.2 ‹ 0.5 1.0 ‹ 0.5 7.6 ‹ 2.0aa 13.4 ‹ 1.8aa 1.4 ‹ 0.6
T4OH 0.4 ‹ 0.2 0.6 ‹ 0.1 0.3 ‹ 0.1 0.2 ‹ 0.1 0.2 ‹ 0.0** ND
T3CO 0.3 ‹ 0.1 0.3 ‹ 0.0 0.3 ‹ 0.0 0.4 ‹ 0.1 1.0 ‹ 0.1aa 0.3 ‹ 0.1
TCOOH 12.0 ‹ 2.2 17.6 ‹ 1.6 25.6 ‹ 7.8 ND 0.6 ‹ 0.2** 5.9 ‹ 3.2**
**
DBTC 40.6 ‹ 3.9 39.6 ‹ 5.1 36.4 ‹ 3.5 17.1 ‹ 4.4 25.0 ‹ 1.0** 45.4 ‹ 12.0
D3OH 12.1 ‹ 1.4 9.7 ‹ 1.3 6.5 ‹ 1.0 1.5 ‹ 0.6** 9.3 ‹ 2.2 7.2 ‹ 2.9
D4OH 1.6 ‹ 0.3 1.1 ‹ 0.2 1.1 ‹ 0.2 ND 0.3 ‹ 0.1** 0.4 ‹ 0.1**
D3CO 7.3 ‹ 1.1 5.7 ‹ 0.9 5.6 ‹ 2.6 0.3 ‹ 0.2** 3.1 ‹ 1.1* 4.9 ‹ 2.1
DCOOH 8.3 ‹ 2.1 8.6 ‹ 2.2 8.8 ‹ 2.6 ND 0.2 ‹ 0.1** 6.8 ‹ 1.4
MBTC 7.9 ‹ 0.9 3.1 ‹ 1.2 6.4 ‹ 2.4 0.9 ‹ 0.2** 3.2 ‹ 0.8 4.8 ‹ 1.6

The mice were orally administered TBTC (180 lmol/kg) with or without pretreatment with SKF-525A.
Each value is of mean ‹ SD (n = 4) . (ND Not detected; all metabolite abbreviations are given in
Table 1) *P<0.05, **P<0.01 compared with hepatic levels of butyltin in unpretreated TBTC-treated
mice a P<0.05, aa P<0.01 compared with hepatic levels of butyltin in unpretreated TBTC-treated mice

trast the levels of T4OH and TCOOH decreased, at 6 h
and at both 3 and 6 h respectively after TBTC treatment.
In particular, the levels of TBTC at 3 and 6 h increased
tenfold and fourfold compared with the unpretreated
TBTC-treated mice. However at 24 h after TBTC
treatment, hepatic levels of TBTC, T3OH, and T3CO
did not di€er signi®cantly when compared to those of
unpretreated mice; the levels of T4OH and TCOOH
were either not detectable or decreased relative to un-
pretreated animals. In the case of debutylated butyltin
metabolites, the levels of DBTC, D4OH, D3CO, and
DCOOH decreased at both 3 and 6 h, while the levels of
D3OH and MBTC decreased only at 3 h after TBTC
treatment. In particular, the levels of DBTC at 3 and 6 h
decreased 60 and 37% compared with the unpretreated
TBTC-treated mice at 3 and 6 h, respectively.
In the case of the DBTC treatment, >94% of the
butyltin compounds in the liver was DBTC [98.5 ‹ 0.2,
96.7 ‹ 1.8 and 94.8 ‹ 1.0 (S.D.)% of total butyltin
level at 3, 6, and 24 h, respectively, n ˆ 4] and the levels
of DBTC were not a€ected by pretreatment with SKF-
525A [99.5 ‹ 0.3, 99.2 ‹ 0.2 and 95.5 ‹ 0.85 (S.D.)%
of total butyltin level at 3, 6, and 24 h, respectively,
n ˆ 4]. The levels of metabolites such as D3OH, D4OH,
D3CO, and MBTC were <3% of total butyltin level,
and were not a€ected by SKF-525A treatment (data not
shown).
Discussion
The present study showed that SKF-525A pretreatment
resulted in complete inhibition of hepatotoxicity by
TBTC only up to 24 h after TBTC administration to
mice. In contrast, SKF-525A pretreatment did not a€ect
the induction of hepatotoxicity by DBTC. The e€ects of
SKF-525A against TBTC hepatotoxicity are consistent
with the ®ndings of previous study (Ueno et al. 1995).
Although SKF-525A pretreatment had no e€ect on he-
patotoxicity at 48 h after TBTC treatment, these results
demonstrated that SKF-525A suppressed the hepato-
toxicity caused by TBTC.
Regarding the total levels of butyltin compounds in
liver, the pretreatment with SKF-525A resulted in a
signi®cant decrease in the total butyltin contents in liver
at 3 and 24 h after TBTC treatment. Similarly, this
pretreatment decreased hepatic levels of total butyltin at
3 h in DBTC-treated mice. It should be noted that SKF-
525A as well as any other pharmacological agent may
have e€ects on cells in addition to those for which the
agent has been targeted. Thus, the protection of SKF-
525A toward hepatotoxicity caused by TBTC may be at
least partially attributed to decreased metal uptake by
liver. However, the pretreatment with SKF-525A had no
e€ect on total butyltin contents in liver at 6 h after
TBTC treatment, nor at 6 and 24 h after DBTC treat-
ment. This suggests that the results of metal uptake
cannot totally explain the observed e€ect of SKF-525A
on the hepatotoxicity caused by TBTC and DBTC.
517
A previous study using atomic absorption spectro-
photometry showed that the exposure of rat to TBTC
compounds resulted in a transient increase of the TBTC
content in liver, followed by a rapid decrease and leading
to an increase of DBTC and MBTC (Iwai et al. 1981). In
addition to the ®ndings of these butyltin compounds
inside cells, recent studies have identi®ed a variety of
metabolites of TBTC in organs by means of GC/AED
(Table 1, Fig. 3; Matsuda et al. 1993; Suzuki et al. 1994).
In the liver of rats orally administered TBTC com-
pounds, DCOOH compounds have been shown to be
the main products (Matsuda et al. 1993). In the present
study, similar metabolites of TBTC were observed in the
liver of mice in vivo. However, the main products of
butyltin were DBTC (>36%) and TCOOH (12±26%)
for 24 h following TBTC treatment (Table 2), indicating
di€erences in hepatic metabolism of TBTC between rats
and mice.
Hepatic metabolism of TBTC was clearly suppressed
by pretreatment with SKF-525A, because hepatic levels
of TBTC at 3 and 6 h were dramatically increased and
hepatic levels of most metabolites were decreased at 3
and 6 h after TBTC treatment (Table 2). SKF-525A
pretreatment resulted in complete inhibition of the he-
patotoxicity by TBTC at 24 h (Fig. 3A). Therefore these
results suggest that the suppressive e€ect of SKF-525A
against TBTC hepatotoxicity may be closely related to
the suppression of TBTC metabolism in liver, and that
the formation of metabolic products of TBTC in liver is
associated with the induction of hepatotoxicity by
TBTC.
It should be noted that pretreatment with SKF-525A
caused a decrease of TBTC levels in liver upto 6 h,
leading to the suppression of hepatotoxicity at 24 h after
oral administration. Also, hepatic levels of TBTC at
24 h were not a€ected signi®cantly by SKF-525A, re-
sulting in the appearance of hepatotoxicity at 48 h. This
®nding may be explained by the fact that hepatotoxicity
was evaluated in terms of serum OCT activity. Because
the release of this hepatic enzyme was used as an index
of hepatotoxicity, there may have been a time delay
before the expression of liver injury.
The hypothetical metabolic pathway of TBTC by
cytochrome P450 is as shown in Fig. 1. The biological
oxidation at positions 3 and 4 of a butyl group of TBTC
will give T3OH or T3CO and T4OH, respectively, and
the oxidation of CH2OH in T4OH to COOH will yield
TCOOH. However, these non-debutylated metabolites
may not be related to the induction of hepatotoxicity,
because pretreatment with SKF-525A increased the
levels of T3OH and T3CO at 3 and/or 6 h, while the
levels of T4OH and TCOOH at 24 h were not detectable
and decreased, respectively, compared with those of
unpretreated mice (Table 2).
The debutylation of TBTC, T3CO, T3OH, T4OH,
and TCOOH will give DBTC, D3CO, D3OH or DBTC,
DBTC or D4OH, and DCOOH, respectively, and the
hydroxylation of T3OH and T4OH will also give
DCOOH, possibly via 3,4-diol and then a hypothetical
518
intermediate shown in brackets in Fig. 1. On the other
hand, removal of a 4-hydroxylbutyl group from 3,4-diol
will yield D3OH. Hepatic levels of these debutylated
metabolites except for D3OH were signi®cantly sup-
pressed by SKF-525A at both 3 and 6 h, and their levels
except for D4OH were similar to those of unpretreated
controls at 24 h after TBTC administration (Table 2).
Therefore, it is possible that the formation of debu-
tylated products such as DBTC, D3CO and DCOOH
may be partially associated with the induction of TBTC
hepatotoxicity. In particular, >36% of the butyltin
compound in liver of mice treated with TBTC was in the
form of DBTC (Table 2). Additionally, >94% of total
butyltin compounds in liver of mice treated with DBTC
was in the form of DBTC and the pretreatment with
SKF-525A had no e€ect on hepatotoxicity and hepatic
levels of DBTC, suggesting DBTC was very stable inside
cells. Moreover, our previous (Ueno et al. 1994) and
present study both showed that DBTC is more hepato-
toxic than TBTC in vivo. Although debutylation of
these metabolites such as DBTC, D3OH, D3CO, and
D4OH will yield MBTC, MBTC has been reported to be
relatively inert in liver (Pellikan and Cerny 1970; Ueno
et al. 1994).
Thus, these results suggest that DBTC inside cells
might be the critical form which is responsible for the
liver injury caused by TBTC. The pretreatment with
SKF-525A had some e€ect on DBTC and MBTC in
mice treated with TBTC but had no e€ect on the levels
in mice treated with DBTC. Therefore, the results also
suggest that cytochrome P450 enzymes might be more
related to the metabolism of TBTC to form DBTC or
MBTC than that of DBTC to form MBTC in the liver of
mice. Further studies are necessary to con®rm this hy-
pothesis. In conclusion, the present study showed that
the suppressive e€ect of SKF-525A, an inhibitor of cy-
tochrome P450, against TBTC hepatotoxicity was as-
sociated with inhibition of TBTC metabolism in liver,
based upon the analysis of metabolites of TBTC using
GC/MIP/AED. The biotransformation of TBTC by
cytochrome P450 enzymes plays an important role in the
induction of hepatotoxicity in vivo.
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