Trace Nutrients Research 24 : 90 − 97（2007）

Mechanism on the Cell Death of T-lymphocytes Induced by Organotin in Vitro
Akiko YAMAGUCHI1）, Kenichi TOMIYAMA1）, Yurie SAYAMA1）, Takao KURIYAMA1）,
Harunobu NAKASHIMA2）, Yoshikazu MATSUDA3）and Yasuaki ARAKAWA1）
1）

Department of Hygiene & Preventive Medicine, Faculty of Health Sciences, The University of Shizuoka＊,

2）

Osaka Prefectural Institute of Public Health＊＊, 3）Central Research Institute, Japan Clinic Co., Ltd ＊＊＊Summary
Organotin compounds are toxic to the immune system. Alkyltin compounds such as dibutyltin (DBT) and tributyltin (TBT) cause severe thymus atrophy. At histopathological examination, there is marked depletion of lymphocytes and the depletion is due to the cell death. In this study, the mechanism on the cell death induced by DBT orTBT was investigated from the angle of apoptosis in T-lymphocytes.
The results revealed that DBT induced caspase-independent cell death (necrosis), whereas TBT induced caspasedependent cell death (apoptosis). Furthermore, we demonstrated the mechanism on TBT-induced apoptosis. Bidcleavage by activation of caspase-8 produced tBid and released cytochrome c from mitochondria. Release ofcytochrome c induced the activation of caspase-3 through caspase-9. CAD activated by caspase-3 induced DNA fragmentation. That is to say, it was elucidated that TBT induced caspase-dependent and mitochondria-mediated celldeath.

Organotin compounds exert powerful toxic action on the immune system, brain nervous system and endocrine
system. Organotin compounds such as dibutyltin (DBT), dioctyltin (DOT) and tributyltin (TBT) compounds caused
immunodeficiency of cell-mediated immunity and T cell-dependent humoral immunity due to severe thymus
atrophy1−8）. This atrophy depends on marked depletion of lymphocytes and the depletion is due to the cell death.

Moreover, under the long-term exposure from 5 weeks to 8 weeks, dibutyltin-induced atrophy is reversible, whereas
tributyltin-induced atrophy is irreversible. These results suggest that there might be different mechanism between
the cell death induced by dibutyltin and tributyltin

1−8）

.

On the other hand, there are morphologically and biochemically two distinct forms in the mode of cell death,referred to as apoptosis and necrosis. Apoptotic cell death is morphologically characterized by cell shrinkage, theblebbing of plasma membranes, nuclear breakdown and DNA fragmentation. The biochemical hallmark of apoptosis
is the cleavage of chromosomal DNA into nucleosomal units, which appears to be the final blow in the cell deathprocess. Apoptosis is mediated by the activation of a family of cysteine proteases with specificity for aspartic acidresidues, referred to as caspases.
Caspases are synthesized as precursor forms, and an apoptotic signal converts the precursors to mature enzymes,
which subsequently cleave other caspases that are downstream in the cascade. Caspases activated by apoptotic signals cleave various cellular substrates which may be responsible for the morphological changes that occur in thecells.
＊＊＊Address：52−1, Yada, Suruga-ku, Shizuoka 422−8526, Japan＊＊＊Address：1−3−69, Nakamichi, Higashinari-ku, Osaka 537−0025, Japan＊＊＊Address：10−1, Kainichi-cho, Uzumasa, Ukyo-ku, Kyoto 616−8555, Japan― 90 ―Fas ligand is expressed in activated T cells, and induces apoptosis in target cells by binding to Fas, its receptor.
The Fas engagement activates a cascade of caspase proteases, as found in most apoptotic processes. Activation ofcell surface receptor Fas leads to rapid inactivation of the electron transfer activity of cytochrome c and subsequent
release of cytochrome c from mitochondria. In addition, the activation of cell surface death receptor leads to rapid
activation of caspase-8, the apical caspase in the Fas-induced apoptotic pathway. The activation of caspase-8 initiates
two pathway leading to the activation of downstream caspases. One is the activation of downstream caspases likecaspase-3, caspase-6 and caspase-7 by directly cleaving them, the other is the activation of these downstream caspases indirectly by causing cytochrome c release from mitochondria that triggers caspase activation through Apaf-1.
The release of cytochrome c triggers the interaction of Apaf-1 and caspase-9, which in turn results in the activation of caspase-9. Activated caspase-9 then cleaves and activates procaspase-3, an event that leads to the cleavage of9）other death substrates, cellular and nuclear morphological changes, and ultimately, cell death .

Bid, a BH3 domain-containing proapoptotic Bcl-2 family member, is a specific proximal substrate of caspase-8 during Fas-induced apoptosis that functions upstream of BclXL10）. That is to say, Bid acts downstream of caspase-8 andupstream of BclXL in Fas-induced apoptosis. The cleavage of Bid by caspase-8 mediates the mitochondrial damage in11）
the Fas pathway of apoptosis . While full-length Bid is localized in cytosol, truncated Bid (tBid) translocates to mito-chondria and thus transduces apoptotic signals from cytoplasmic membrane to mitochondria. tBid induces first theclustering of mitochondria around the nuclei and release of cytochrome c independent of caspase activity, and thenthe loss of mitochondrial membrane potential, cell shrinkage, and nuclear condensation in a caspase-dependent fash9）

ion . Full-length Bid can not induce apoptosis, while tBid induces apoptosis very rapidly and efficiently. tBid-induced
cell death is inhibited completely by BclXL.
Apoptosis is also accompanied by the internucleosomal degradation of chromosomal DNA. Recently, a caspase12）
activated DNase (CAD) and its inhibitor (ICAD) have been identified in the cytoplasmic fraction . Activation of

CAD downstream of the caspase cascade is responsible for internucleosomal DNA degradation during apoptosis andICAD works as an inhibitor of this process. In addition, of caspases, particularly caspase-3 is activated during apop13）
tosis and cleaves inhibitor (ICAD) of CAD and inactivates its CAD-inhibitory effect , suggesting that ICAD could

be inactivated by an apoptotic signal.
In this study, the mechanism on organotin-induced cell death in T-lymphocytes was investigated from the angle ofapoptosis as compared with dibutyltin and tributyltin.

Materials and methods
1. Culture of T-lymphocytes and treatment
Thymus of wistar-derived male rats (3 week ages) was gently pressed through a stainless mesh. T-lymphocytes
6were washed PBS and suspended RPMI 1640 medium (GIBCO) at 8.0 × 10 cells/mL. Cells were incubated with1 µM DBT or TBT in a 5.0 % CO2 humidified incubator at 37℃. T-lymphocytes were pretreated with caspase-9inhibitor (Z-LEHD-FMK) (Trevigen, Inc.) for 1 h.
2. Preparation of cytosolic fractions
T-lymphocytes were collected, washed PBS. Cells were resuspended in cell extraction buffer (10 mM Tris-HCl, pH
7.4, 10 mM KCl, 2 mM MgCl2, 5 mM 2-mercaptoethanol, 1 % Triton X-100, 10 % glycerol) and 2 % Protease Inhibitor
Cocktail (NACALAI TESQUE, INC.). Cell suspensions were homogenized by using a glass dounce homogenizer andsubjected to centrifugation at 10,000 rpm for 5 min. The resulting supernatants are cytosolic frantions. Cytosolic
fractions without mitochondria were extracted using Mitochondria/Cytosol Fraction Kit (BioVision, Inc.). Protein― 91 ―

content was determined with BCATM Protein Assay Kit (PIERCE).
3. Detection of caspase-8, -9, and -3 activity
Activity of caspase-8, -9, and -3 were evaluated by using Caspase-8 Apoptosis Detection Kit , Caspase-3 Apoptosis
Detection Kit (Santa Cruz Biotechnology, Inc.), and Caspase-9 Fluorometric Assay Kit (BioVision, Inc.). Cytosolic
fractions (5.0 µg) were placed in a 96-well plate with 50 µL reaction buffer and caspase substrate using fluorometriccaspase-8 substrate (IETD-AFC), caspase-9 substrate (LEHD-AFC), caspase-3 substrate (DEVD-AFC) was added
each well. Plates were incubated at 37℃ for 2 h and caspase activity was measured level of free AFC using a plate
reader (Flex Station96, Molecular Devices) with a 400 nm excitation filter and a 505 nm emission filter.
4. Western Blot analysis
Cytosolic fractions (5.0 µg) were separated by various appropriate concentrations of SDS-PAGE; 15 % forcytochrome c, Bid; 12 % for CAD, ICAD, β-actin, and blotted to PVDF membrane. Antibodies used were cytochrome c,Bid (Santa Cruz Biotechnology, Inc.), CAD (MERCK), ICAD (stressgen), β-actin (SIGMA).
5. WST-8 assay
WST-8 Assay was perfomed by Cell Counting Kit-8 (DOJINDO). Cell suspensions were cultured with DBT orTBT in a 5.0 % CO2 humidified incubator at 37 ℃ for 10, 60, 120 and 180 min. WST-8 solution was added and incu5bated for 2 h at 37 ℃. Cell suspensions were seeded at a density of 5.0 × 10 cells/well in 96-well plate. Theabsorbance at wavelength of 450 nm was measured by plate reader (SPECTRA MAX 190, Molecular Devices).
6. Detection of DNA fragmentation
14）
DNA was prepared for gel electrophoresis as previously described . Electrophoresis was performed in a 2.0 %agarose gel. DNA fragmentation was visualized by ethidium bromide staining.

Results and Discussion
1. Activation of caspases
Caspases are synthesized as precursor forms, and an apoptotic signal converts the precursors to mature enzymes,
which subsequently cleave other caspases that are downstream in the cascade. Caspases activated by apoptotic signals cleave various cellular substrates which may be responsible for the morphological changes that occur in thecells.
To evaluate the activation of caspases leading to apoptosis, time-dependent activities of caspase-8, -9, and -3 werefirst determined by using fluorometric assay. The weak activities of caspase-8, -9, and -3 were observed in DBTexposed T-lymphocytes. In contrast, the strong activities of caspase-8, -9, and -3 were observed in TBT-exposed
T-lymphocytes (Fig. 1). These results revealed that TBT-induced cell death was largely dependent on caspase activation, whereas DBT-induced cell death was independent on caspase cascade.
2. Release of cytochrome c into the cytosol
The release of cytochrome c into the cytosol triggers the interaction of Apaf-1 and caspase-9, which in turn resultsin the activation of caspase-9. Activated caspase-9 then cleaves and activates procaspase-3, an event that leads to the11）
cleavage of other death substrates, cellular and nuclear morphological changes, and ultimately, cell death .

Therefore, time-dependent release of cytochrome c into the cytosol was examined using western blot analysis. Asshown in Fig. 2, the strong expression was observed by TBT-treatment for 10 min. In other words, TBT-treatedT-lymphocytes caused release of cytochorome c from mitochondria into the cytosol.

― 92 ―

Fig. 1 Activity of caspase-8, -9, and -3 in T-lymphocytes treated with 1 µM DBT or TBT.
(A) activity of caspase-8, (B) activity ofcaspase-9, (C) activity of caspase-3 determined fluorometric assay. Cytosolic fractions were incubated at 37℃ for 2 h inreaction buffer containing the substrates
IETD-AFC, LEHD-AFC, DEVD-AFC for
caspase-8, -9, and -3, respectively.

Fig. 2 Expression of cytochrome c into the cytosol in T-lymphocytes treated with 1 µM DBT orTBT. Cytosolic fractions without mitochondria were analyzed by 15 % SDS-PAGE andtransferred to PVDF membrane and immunoblotted rabbit anti-cytochrome c, mouseanti- β-actin antibodies. β-actin was used as internal standard.

3. Cleavage of Bid
Bid, a BH3 domain-containing proapoptotic Bcl-2 family member, is a specific proximal substrate of caspase-8 during Fas-induced apoptosis that functions upstream of BclXL12）. The cleavage of Bid by caspase-8 mediates the mito13）
chondrial damage in the Fas pathway of apoptosis . Bid cleavage by activation of caspase-8 produces tBid which

induces first the clustering of mitochondria around the nuclei and translocates from the cytosol to mitochondria andreleases cytochrome c. Therefore, expression of Bid was investigated by using western blot analysis. As shown inFig. 3, expression of Bid was strongly decreased by TBT-treatment for 30 min. Bid cleavage was observed significantly in TBT-treated T-lymphocytes. Bid cleavage in DBT-treated T-lymphocytes was weak. These results concluded that TBT-induced cell death involved activation of caspase-8, Bid cleavage, and release of cytochrome c frommitochondria.

― 93 ―

Fig. 3 Detection of Bid cleavage. (A) Expression ofBid in T-lymphocytes treated with 1 µM DBTor TBT. Cytosolic fractions were analyzed by
15 % SDS-PAGE and transferred to PVDF
membrane and immunoblotted rabbit anti-Bid,
mouse anti-β-actin antibodies. (B) The relative
percentage (% of control) of Bid proteins normalized for β-actin. Results are means ± SD
(n = 4) and significance as ＊compared withControls (＊p < 0.05, ＊＊p < 0.01), significanceas #compared with TBT-treated T-lymphocytes (##p < 0.01).

4. Mitochondrial damage
Organotin compounds induce gross mitochondrial swelling. In addition, these compounds inhibit mitochondrial
ATP production. Therefore, mitochondrial damage was investigated using WST-8 assay evaluating the activity of
mitochondrial dehydrogenase. As shown in Fig. 4, mitochondrial dehydrogenase activity showed a down-regulationin T-lymphocytes treated with TBT. Mitochondrial function was lost in TBT-treated T-lymphocytes.
5. Link of caspase-9 and caspase-3
To confirm the link of caspase-9 and caspase-3, the activity of caspase-3 in T-lymphocytes preincubated with caspase-9 inhibitor (Z-LEHD-FMK) or treated with TBT were determined. As shown in Fig. 5, the activation of caspase3 with TBT was suppressed by the pretreatment of caspase-9 inhibitor. This means that TBT induced the activation of caspase-3 through caspase-9.

Fig. 4 Detection of mitochondrial damage evaluated by mitochondrial dehydrogenase activity using WST-8 assay. Cell suspensions were cultured
with 1 µM DBT or TBT in a 5.0 % CO2 humidified incubator at 37℃.

― 94 ―

Fig. 5 Activity of caspase-3 in T-lymphocytes treated with 1 µM TBT for 1h.
Caspase-9 inhibitor (Z-LEHD-FMK) was preincubated for 1h at a concentration of 50 µM. Results are means ± SD (n = 5) and significance as＊compared with controls (＊＊＊p < 0.001), significance as #compared withTBT-treated T-lymphocytes (###p < 0.001).

6. Cleavage of CAD inhibitor in CAD activation and DNA degradation
Activation of CAD downstream of the caspase cascade is responsible for internucleosomal DNA degradation dur12）
ing apoptosis and ICAD works as an inhibitor of this process . In addition, caspase-3 cleaves inhibitor (ICAD) of13）
CAD and inactivates its CAD-inhibitory effect , suggesting that ICAD could be inactivated by an apoptotic signal.

Therefore, to confirm the cleavage of CAD inhibitor in CAD activation and DNA degradation, expression of CAD/ICAD were analyzed by using western blot analysis. As shown in Fig. 6, expression of ICAD was not seen in TBTtreated T-lymphocytes for 1h. Reversely, strong expression of CAD was seen in TBT-treated T-lymphocytes., inother words, CAD was activated in TBT-treated T-lymphocytes. Therefore, DNA fragmentation was further confirmed in T-lymphocytes treated with TBT. As shown in Fig. 7, a ladder pattern of DNA fragmentation wasobserved in TBT-treated T-lymphocytes. These results concluded that CAD activated by caspase-3 induced DNAfragmentation in T-lymphocytes exposed with TBT.

Fig. 6 Expression of CAD/ICAD in T-lymphocytes treated with 1 µM TBT.
Cytosolic fractions were analyzed by
12 % SDS-PAGE and transferred to
PVDF membrane and immunoblotted rabbit anti-CAD, anti-ICAD,
mouse anti- β-actin antibodies. β-actin
was used as internal standard.

― 95 ―Fig. 7 Detection of DNA fragmentation. DNA wasanalyzed by 2.0 % agarose gel electrophoresis.
DNA extracted from (1) T-lymphocytes treatedwith 1 % ethanol, (2) T-lymphocytes treatedwith 1 µM TBT, (3) T-lymphocytes.

Conclusion
We could discuss the mechanism on apoptosis of T-lymphocytes exposed with organotin by the established invitro cell culture method. The results revealed that DBT induced caspase-independent cell death (necrosis), whereas
TBT induced caspase-dependent cell death (apoptosis). Furthermore, we demonstrated the mechanism on TBTinduced apoptosis. Bid cleavage by activation of caspase-8 produced tBid and released cytochrome c from mitochondria. Release of cytochrome c induced the activation of caspase-3 through caspase-9. CAD activated by caspase-3
induced DNA fragmentation. That is to say, it was elucidated that TBT induced caspase-dependent and mitochondria-mediated cell death.

References
1）Arakawa Y（1998）Chapter 10 Recent studies on the mode of biological action of the di- and tri-alkyltin compounds. In Chemistry of Tin（P. J. Smith ed.）. Blackie Academic & Professional, Chapman & Hall, Glasgow, U.K.
388−428.
2）Arakawa Y（1993）Chapter 4 Biological properties of alkyltin compounds. In Metal Ions in Biological Systems,Vol.29 Biological Properties of Metal Alkyl Derivatives（H. Sigel & A. Sigel ed.）. Marcel Dekker Inc., New York,USA. 101−136.
3）Arakawa Y（1996）Cellular and biochemical aspects of antitumor activity of organotin compounds. In Main
Group Elements and Their Compounds（Kumar Das VG ed.）. Narosa Publishing House, New Dehli, India. 422−445.
4）Arakawa Y（1992）Chapter 23 Antitumor activity of organotin compounds and inhibition of membrane signaltransduction. In Chemistry and Technology of Silicon and Tin（Kumar Das VG, Gielen M. ed.）. Oxford UniversityPress, Oxford. 319−333.
5）Arakawa Y（ 1988）Chapter 9 Suppression of ell proliferation by certain organotin compounds. In Tin andMalignant Cell Growth（J. J. Zuckerman ed.）CRC Press, Boca Raton, Florida, USA. 83−106.
6 2）:1−34.
6）Arakawa Y（1995）Tin and Immunity -Review- . Biomed Res Trace Elements （― 96 ―7）Arakawa Y（2000）Invasion of biofunctions by organotins -Immune system, brain nervous system and endocrine
system- Biomed Res Trace Elements 11: 259−286.
8）Arakawa Y（2003）Morbid aging of thymus immunity by metals. Biomed Res Trace Elements 14: 249−258.
9）Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X（1997）Cytochrome c anddATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91: 479−489.
10）Li H（1998）Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis.
Cell 94: 491−501.
11）Luo X（1998）Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response toactivation of cell surface death receptors. Cell 94: 481−490.
12）Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A, Nagata S（1998）A caspase-activated DNase that
degraded DNA during apoptosis, and its inhibitor ICAD. Nature 391: 43−50.
13）Sakahira H, Enari M, Nagata S（1998）Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 391: 96−99.
14）Murayama N, Suzuki H, Arakawa Y（2001）Organotin induced thymus atrophy and apoptosis. Trace NutrientsRes 18: 53−56.

― 97 ―