Accepted Manuscript

Comparative toxicity of low dose tributyltin chloride on serum, liver, lung and

kidney following subchronic exposure

Sumonto Mitra, Ruchi Gera, Vikas Singh, Shashi Khandelwal

PII: S0278-6915(13)00793-X

DOI: http://dx.doi.org/10.1016/j.fct.2013.11.031

Reference: FCT 7704

To appear in: Food and Chemical Toxicology

Received Date: 14 September 2013

Accepted Date: 24 November 2013

Please cite this article as: Mitra, S., Gera, R., Singh, V., Khandelwal, S., Comparative toxicity of low dose tributyltin

chloride on serum, liver, lung and kidney following subchronic exposure, Food and Chemical Toxicology (2013),

doi: http://dx.doi.org/10.1016/j.fct.2013.11.031

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Comparative toxicity of low dose tributyltin chloride on serum, liver, lung and kidney

following subchronic exposure

Sumonto Mitra, Ruchi Gera, Vikas Singh, and Shashi Khandelwal*

Immunotoxicology Division, CSIR-Indian Institute of Toxicology Research (CSIR-IITR),

Lucknow-226001, India.

*Correspondence to-

Shashi Khandelwal,

Immunotoxicology Division,

CSIR-Indian Institute of Toxicology Research (CSIR-IITR), P.O.Box 80, Mahatma Gandhi

Marg, Lucknow – 226001, India,

Tel.: +91 522 2627586

Fax: +91 522 2628227

E-mail- skhandelwal_itrc@rediffmail.com

ABSTRACT

Tributyltin (TBT) pollution is rampant worldwide and is a growing threat due to its bio-

accumulative property. Isolated studies of TBT toxicity on different organs are available but

consolidated information is greatly lacking. We planned this study to delineate the effect of

subchronic (1 month) exposure to low dose TBT-chloride (TBTC) (1 and 5 mg/kg) in male

Wistar rats. Total tin concentration was found to be significantly increased in liver, kidney and

blood, and marginally in lungs. Organo-somatic indices were seen to be altered with little effect

on serum biochemical markers (liver and kidney function, and general parameters). Reactive

oxygen species but not lipid peroxidation content was observed to be significantly elevated both

in the tissues and serum. TBTC was found to act as a hyperlipidemic agent and it also affected

heme biosynthetic pathway. Hematological analysis showed that TBTC exposure resulted in

minor alterations in RBC parameters. Histological studies demonstrated marked tissue damage in

all the 3 organs. Calcium inhibitors (BAPTA-AM, EGTA) and antioxidants (NAC, C-PC)

significantly restored TBTC induced loss in cell viability, under ex-vivo conditions. Antioxidants

were evidently more efficient in comparison to the calcium inhibitors, implying major role of

oxidative stress pathways in TBTC toxicity.

Keywords: Tributyltin chloride, liver, kidney, lungs, subchronic exposure, multi-organ toxicity.

1. INTRODUCTION

Toxicity of tributyltin (TBT) compounds has gained prominence in recent times due to its

endocrine disruptive property. Industrial consumption of TBT and allied compounds has

increased enormously over the past few decades. An estimated amount of 5,000 tons of tin was

produced during 1955 which has increased to 35,000 tons in 1985 (Fent, 1996) and supposedly

included 9,000 tons of TBT alone (Laughlin et al., 1986). TBT compounds are primarily used as

a biocide in various industrial applications and humans are primarily exposed via food chain.

TBT was reported to bio-accumulate in fishes due to its lipophilic property (Batt, 2006).

Additional source come from corked bottles, carpets, sanitary products and canned foods (WWF

factsheet). Human sampling studies showed accumulation of TBT in liver (Kannan and

Falandysz, 1997; Nielsen and Strand, 2002; Takahashi et al., 1999) and blood (Kannan et al.,

1999; Whalen et al., 1999) primarily. A more detailed review about human exposure has been

given by Antizar-Ladislao (2008).

TBT compounds are moderately to highly toxic to laboratory mammals. Absorbed TBT-oxide

(TBTO) is rapidly distributed among tissues, mainly in liver and kidneys. TBTO can cross

blood-brain barrier and also the placenta (Chamorro-Garcia et al., 2013), showing its potential as

a develpmental toxicant. The major metabolites detected in blood within 3 h of parenteral

administration in animals appear to be dibutyl and monobutyl tin compounds (IPCS, 1999).

Following repeated oral administration, TBT compounds have been associated with various

outcomes (European Commision, 2011). In human beings, although TBT compounds are known

to cause irritation of the respiratory tract, eyes and skin, toxicological data are not available.

Most of the studies have investigated the mechanisms by which TBT compounds lead to toxic

manifestations in liver. Liu et al. (2006) suggested both oxidative and DNA damage in mammals

after 7 days repeated dosing of 10 mg/kg TBT in rats. It was also observed that subchronic low

dose (2 and 6 µg/kg) exposure of TBT in rats resulted in necrotic response in the liver without

any enzymatic alteration (Silva de Assis et al., 2005). In another study, marked mitochondrial

swelling appeared in the hepatocytes 4 h post intramuscular injection of 0.5 ml/kg TBT-oxide

(TBTO) (Yoshizuka et al., 1992). Tributyltin has also been attributed to induce apoptosis

involving the endoplasmic and mitochondrial signaling pathway (Grondin et al., 2007).

Interestingly, Cooke et al. (2004) has shown that TBT acts as a hepatotoxic agent even when

administered lower than the tolerable daily dose suggested for humans.

Regarding lung damage, a 4-h inhalation exposure caused nasal discharge, lung edema, and

congestion of the pulmonary circulation and enteritis in rats (Schweinfurth and Gunzel, 1987). In

guinea-pigs exposed to aerosols of TBTO in olive oil at 200 mg/m3 and above, death occurred

within 1 h of exposure. No animal deaths other than minor clinical signs (slight nasal discharge)

were noted in 10 male and 10 female rats exposed to almost saturated vapors of TBTO

(European Commision, 2011). Truhaut et al. (1979) noted a change in animal exploratory

behavior in mice exposed to either a single 1 h period or seven 1 h periods on successive days, to

TBTO concentrations in air ranging between 50 and 400 mg/m3.

TBT toxicity on kidney is rather less understood. A study by Silva de Assis et al (2005) reported

that there was no discernible change in the organ morphology after 30 or 60 days of exposure to

low level TBT. In another study, chronic toxicity of tributyltin oxide in rats resulted in decreased

kidney function and increased plasma enzyme activities (Wester et al., 1990).

Although, bioaccumulation of TBT has been reported in fat, liver and kidney in mammalian

species (Adeeko et al., 2003; Azumi et al., 2007; Harino et al., 2005; Strand and Jacobsen, 2005),

no correlation studies are available to highlight the time period and dosing of TBT exposure with

tissue organ effects and dysfunctions. This results in experimental discrepancies about the

dosage regimen and toxicity profile of TBT. The histological alterations by TBT have been

studied mainly in liver so far. Thus inter-relationship between biochemical responses,

histological structure and organ function following low level exposure of TBT (relevance to

human exposure levels mainly via food chain) needs exploration.

We therefore, planned this study to investigate the influence of TBT-chloride (TBTC) on serum

and blood parameters, oxidative damage, histological analysis (liver, kidney and lungs) and

organ function profiles after subchronically exposing male Wistar rats to low doses of TBTC (1

and 5 mg/kg). Cell densities along with metal distribution were also evaluated. This study is in

continuation of our ongoing research on the toxicity of TBTC in rat model. We have previously

reported its immune- (Gupta et al., 2011), neuro- (Mitra et al., 2013) and testicular toxicity

(Mitra et al., 2013). Oxidative stress appears to be one of the underlying mechanisms of TBTC

toxicity. In this in vivo study, we found that 1 mg/kg TBTC leads to significant loss in the

viability of hepatic cells followed by kidney and lastly in lungs. The involvement of calcium and

oxidative damage was further confirmed by various calcium inhibitors (BAPTA-AM, EGTA)

and antioxidants (NAC, C-PC). Since oxidative stress is generally encountered in metal

intoxication, the influence of TBTC on redox parameters such as reactive oxygen species (ROS),

lipid peroxidation (TBARS) and total antioxidant capacity (TAC) levels were analyzed. Our

study is the first attempt to delineate multi-organ toxicity of TBTC and will help to understand

the combinatorial effect of this toxicant upon exposure.

2. MATERIALS AND METHODS

2.1 Chemicals: Tributyltin chloride (TBTC) and tin metal standard solution was from Merck,

Germany; 3-(4,5-dimethyl-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), n-acetyl cysteine

(NAC), EGTA (ethylene glycol tetraacetic acid), BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-

N,N,N',N'-tetraacetic acid), heparin, δ-aminolevulinic acid, 2’,7’-dichlorofluorescein diacetate

(DCFH-DA), p-dimethylaminobenzaldehyde, Dulbecco’s phosphate buffered saline (PBS), were

purchased from Sigma Aldrich, USA; Dulbecco’s Modified Eagle’s medium (DMEM- high

glucose) was from Invitrogen, Life Technologies, USA; fetal bovine serum (FBS) was from

Gibco BRL, Life Technologies, USA. C-Phycocyanin (C-PC), Grade IV, was procured from

Delhi Nutraceuticals Pvt Ltd., New Delhi, India. All other chemicals used in the study were of

highest purity grade available in India.

2.2 Animals and Treatment: Male wistar rats (4 week old) were procured from IITR animal

breeding facility under standard conditions. They were maintained in IITR animal house facility

in plastic polypropylene cages (6 animals/cage) with a 12 h light/dark cycle and temperature of

25 ± 2ºC. They were fed with standard rodent pellet and water, ad libitum. Our animal house and

breeding facility are registered with Committee for the Purpose of Control and Supervision of

Experiments on Animals (CPCSEA), Government of India and CPCSEA guidelines were

followed (IAEC approval obtained).

18 animals were divided into 3 groups (6 rats / group) and designated as control, 1 mg/kg TBTC

and 5 mg/kg TBTC, respectively. TBTC treatment solutions were prepared in corn oil by

adjusting the dose concentrations to 1 ml/kg animal body weight, and were gavaged daily at

11:00 o’clock for 30 days. Control rats were gavaged with corn oil alone. Animal body weight

was recorded at regular intervals till the end of the experiment. At the time of sacrifice, blood

and tissues from each animal were collected and distributed randomly for various assessments.

After blood collection following cardiac puncture, all the animals were perfused with 50 ml

heparinised normal saline (ice cold) to remove blood from tissues and then the respective tissues

were isolated.

2.3 Estimation of tin accumulation: 100 mg tissue (liver, kidney and lungs) and 1 ml of blood

from each animal was used for metal estimation. Post acid digestion under constant heating in

HNO3 : HClO4 mixture (6 : 1), the volume of each sample was made up to 1 ml with 1 % HNO3.

Tin was estimated at 284 nm with Graphite furnace coupled Atomic Absorption Spectroscopy

(GF-AAS) with Zeeman background correction in a ZEEnit 700P instrument (Analytikjena)

attached with MPE60-autosampler module as already described elsewhere (Szoboszlai et al.,

2001). 20 µl sample was injected along with 5 µl palladium nitrate (0.1 %) matrix modifier and

argon was used as the source of neutral gas. Recovery of tin was in the range of 85-95 %.

2.4 Biochemical analysis: Serum was separated from blood by incubating it at room temperature

for ~30 min. Coagulated blood was centrifuged at 2000 x g for 30 min at 4 ºC, clear serum was

carefully pipetted out in a fresh tube kept on ice and utilized immediately. All the serum

parameters were carried out on the same day in a biochemical analyzer (Selectra Junior Spinlab

100, Vital Scientific, Dieren, Netherlands) with the help of the manufacturer’s kit provided.

Liver function was monitored by measuring aspartate aminotransferase (AST), alanine

aminotransferase (ALT), total bilirubin (TBIL) and albumin, whereas kidney function was

evaluated by measuring urea, uric acid, blood urea nitrogen (BUN) and creatinine. Lipid profile

(cholesterol and triglyceride levels) along with general parameters like ionic concentrations of

sodium, potassium, chloride and alkaline phosphatase and glucose levels were also analyzed.

The total antioxidant capacity (TAC) of serum was determined by using Cayman’s antioxidant

assay kit (Cat no. 709001) according to the manufacturer’s protocol.

To determine the effect of TBTC on heme biosynthetic pathway, enzymatic activity of δ-

aminolevulinic acid dehydratase (ALAD) and total porphyrin content were measured in

heparinized blood according to Granick et al. (1972). Since the far fetching effect of δ-ALAD

activity inhibition may result in alteration in hemoglobin production and on the activity of

RBC’s, we analyzed the hematological parameters on an identical set of 6 animals. Analysis was

carried out in HS9 hematology analyzer (Melet Schloesing Laboratories, France).

2.5 Determination of reactive oxygen species (ROS): Animal tissues were collected at the time

of sacrifice and 100 mg of liver, kidneys and lungs were immediately snap frozen in liquid

nitrogen and further stored at -80ºC freezer until analysis on the next day. Tissues were

homogenized in 1ml phosphate buffer (0.1 M) containing 5 mM EDTA and protease inhibitor

cocktail, using an Ultra Turrax Polytron (Janke & Kunkel, IKA-Labortechnik, Staufen,

Germany). The homogenate was centrifuged at 10,000 x g for 15 min at 4ºC and the clear

supernatant was used for the analysis. 20 µl of supernatant was immediately kept at -80ºC for

protein estimation for representation of the biochemical data. For ROS, 200 µl supernatant was

incubated with 100 µM 2’,7’-dichlorofluorescein diacetate (DCFH-DA) dye for 15 min in the

dark and fluorescence was measured in a plate reader (FLUOstar Omega) at excitation:485 nm

and emission:520 nm, respectively.

To measure ROS levels from serum samples, DCFH-DA dye was first treated with 0.1 M NaOH

for 30 min at room temperature (Possel et al., 1997) to cleave the -diacetate group and the DCFH

molecule was then reacted with the serum sample to yield fluorescent DCF which was measured

in a plate reader as mentioned above. Data is expressed as DCF fluorescence/mg protein for both

serum and tissue samples.

2.6 Estimation of lipid peroxidation: 10 % tissue homogenates as prepared in 0.1 M phosphate

buffer (as mentioned earlier) was also used for the estimation of lipid peroxidation. It is

measured as thiobarbituric acid reactive substances (TBARS) as described previously (Ohkawa

et al., 1979). Briefly, 50 µl homogenate or 50 µl serum was mixed with 100 µl SDS (8.1 %), 700

µl acetic acid (20 %, pH 3.5), 700 µl thiobarbituric acid (TBA) (0.8 %) and the volume was

made up to 2 ml with milli Q water. The samples were incubated in boiling water bath for 15 min

and then cooled to room temperature. After centrifugation at 1500 x g for 15 min, the supernatant

constituting TBARS was measured at 532 nm in a plate reader (FLUOstar Omega, BMG). Data

is expressed as µmole TBARS/mg protein.

2.7 Single cell suspension preparation: 100 mg tissue from each organ was taken and single

cells were isolated after enzymatic digestion with 2 mg/ml collagenase + 0.025 % trypsin.

Briefly, tissues were finely chopped and incubated with 5 ml enzyme mixture for 15 min at 37ºC.

Excess DMEM medium containing 10 % fetal bovine serum (FBS) was added and the total

suspension was filtered through 100 µm nylon mesh to get single cell suspension. After

centrifugation at 400 x g for 10 min, cell pellet was re-suspended in DMEM medium and the

cells counted using a hemocytometer under a light microscope.

2.8 Cell viability: For viability assay, 1x104 cells/well were plated in 96 well plates. 10 µl MTT

solution (5.4 mg/ml in PBS) was added to each well and the cells and the plate was incubated for

4 h to allow formation of purple formazan crystals (Mosmann, 1983). The plates were then

centrifuged at 1200 x g for 10 min and the supernatant was carefully removed so as not to disturb

the formazan crystals. 200 µl DMSO was added to solubilize the crystals and absorbance was

read at 530 nm with background correction at 660 nm in a plate reader (FLUOstar Omega,

BMG). Data was expressed as percent viability in respect to control values.

To delineate the role of Ca and ROS, cells plated as above were treated with calcium inhibitors

like 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM, 50µM), ethylene

glycol tetraacetic acid (EGTA, 5 mM ); and free radical scavengers like n-acetyl cysteine (NAC,

50 µM ) and c-phycocyanin (CPC, 1 µg/ml) for 1 h at 37ºC in a CO2 incubator. MTT was added

and further processed as mentioned above.

2.9 Histopathology: To understand the overall effect of TBTC exposure on the tissue

architecture, 100 mg tissue of the three organs from each animal was taken, washed with cold

0.1M PBS and post fixed in 4% paraformaldehyde fixative (in 0.1M PBS) for 48 h at 4ºC.

Paraffin embedded 5 µm sections were cut and stained with hematoxylin and eosin for light

microscopic examination. To semi-quantitatively score the histopathological alterations in the

tissue, sections from each rat were analyzed on a scale of 0 to 5 (0-no change, 1- mild, 2-

moderate, 3-high, 4-very high, and 5-severe).

2.10 Protein estimation: Protein was estimated according to the method of Bradford (1976)

using BSA as a standard.

2.11 Statistics analysis: Experimental data is expressed as mean ± S.E and p<0.05 is considered

significant. Data was analyzed by Prism3 software using Kruskal-Wallis statistics, followed by

Dunn's multiple comparison post-hoc test.

3. Results

3.1 TBTC and animal/organ weight

Tributyltin chloride exposure resulted in decreased weight gain in rats. Post 30 days, the control

rats gained ~150 g i.e. from 100 g to 250 g , whereas 1mg/kg TBTC exposed rats gained only

~135 g weight, i.e. 15 g less and the 5 mg/kg group, gained just ~80 g (p<0.01) which was ~70 g

less when compared to controls. The higher TBTC dose demonstrated greater loss in animal

weight gain (Fig 1a).

Effect of TBTC on liver, kidney and lung weights was also observed and all the three organs

were found to be affected. Liver and kidney weight decreased dose dependently (~6-8 % and ~30

% respectively) whereas lungs gained weight in the same order (~20 % and ~45 %) (data not

shown). A similar pattern was also observed in the organ weight / body weight ratios. A decrease

in the liver and kidney weights / body weight ratios of 5 mg/kg TBTC group indicates influence

of this compound on these organs. On the other hand, TBTC affects the lungs differently by

increasing the organ weight, as the relative ratio was enhanced (Fig 1b).

Although liver and kidney weight was decreased with the increasing TBTC dose, cell density

was inversely affected. With 5 mg/kg TBTC, the cell density increased to 1.71 folds in liver and

1.75 folds in kidneys respectively. While in lungs, with increasing lung weight the cell density

was reduced by 0.3 folds. The data indicates that TBTC either causes an increased cell number in

liver and kidneys or it could be a result of organ shrinkage (Fig 1c).

3.2 TBTC exposure resulted in Tin accumulation

Daily dosing of rats with TBTC for one month led to an increased tin (Sn) burden in blood, liver,

kidneys and lungs in a dose dependent fashion. Liver demonstrated highest Sn uptake leading to

3.2 ng Sn/g from the control value of 0.68 ng/g, increasing it by ~5 folds (p<0.01), followed by

kidneys ~3.5 folds (p<0.01)> blood ~1.8 folds (p<0.05) and the least was found in lungs (~1.5

folds) (Fig 2). The concentration of tin in corn oil and animal diet was found to be below

detectable range.

3.3 TBTC exposure leads to oxidative stress in tissue

TBTC exposure led to enhanced ROS production. Among the three organs, lung and liver

displayed a similar increased ROS profile, the effect being dose independent. ROS levels were

elevated to 1.7 and 1.3 folds in lungs and 1.4 and 1.2 folds in liver. As for kidney, 1.4 fold

increase in ROS levels was found in 1mg TBTC treated rats, while the higher TBTC group

exhibited no change. Similarly, change in lipid peroxidation levels were measured as TBARS

content and were found to be statistically insignificant (Table 1).

3.4 Influence of TBTC on cell survival

One month exposure to TBTC resulted in loss of cell viability in all the three organs to a varying

degree (Table 2). Almost 15 % cell death was observed in liver, followed by ~12 % in kidney

and ~10 % in lungs. Interestingly, the lower TBTC dose (1 mg/kg) exhibited higher cytotoxic

action as compared to 5 mg/kg TBTC dose. This is in accordance to the increase in ROS which

is also found to be more pronounced in the lower dose itself. The cell loss in these organs

signifies the influence of TBTC in these organs.

We also analyzed the influence of Ca2+ inhibitors (BAPTA-AM and EGTA) and antioxidants

(NAC and C-PC) on TBTC induced cell viability in the 1 and 5 mg/kg TBTC group. Data shows

that the antioxidants afforded higher protection than Ca2+ inhibitors and among the latter,

BAPTA-AM exhibited better efficacy. ROS and glutathione (GSH) therefore, play a vital role

along with intracellular Ca2+ in TBTC related cell loss. The tissue ROS data corroborates with

that of MTT indicating higher detrimental influence of 1 mg/kg TBTC dose.

3.5 TBTC inhibits heme biosynthetic pathway

We measured the activity of δ- ALAD and porphyrin levels in blood. δ-ALAD is a cytosolic

enzyme that catalyzes the second step of porphyrin formation, and it is expressed abundantly in

liver and erythroid tissues as well as in other tissues. We observed inhibition of δ-ALAD activity

to ~70 % of the control values in 1 and 5 mg/kg (p<0.05) TBTC treated rats (Fig 3a), leading to a

significant decrease in total porphyrin levels to 58 % of the control levels (p<0.05) in 5 mg

TBTC group (Fig 3b). Blood hematological analysis revealed minor reduction in hemoglobin

(~10%)(p=0.364) and hematocrit (~8.4%)(p=0.0734) values along with total RBC count (T-

RBC), mean corpuscular hemoglobin concentration (MCHC) and mean corpuscular volume

(MCV) respectively (Table 3). All the hematological changes remained mostly dose-dependent

but statistically insignificant.

3.6 Effect of TBT on tissue function and oxidative stress parameters (ROS, TAC &

TBARS) in serum

The two TBTC dosed groups displayed an almost comparable influence on serum TAC and ROS

levels (Fig 4a & 4b). Significant increment in ROS levels (~3.2 folds) (p<0.01) was seen,

irrespective of TBTC dosing, although the higher group received 5 times higher TBTC daily.

TAC levels were mildly reduced by ~0.2 folds in both the treated groups but significant only

with the 5 mg/kg TBTC dose (p<0.01). However, the TBARS levels were significantly raised

only in the 5 mg/kg TBTC group, increasing from 0.058 to 0.067 µmole TBARS/mg protein

(p<0.05) (Fig 4c).

3.7 Serum biochemical analysis

Functional status of liver and kidney was monitored in serum to determine the effect of TBTC on

these organs (Table 4). TBTC administered for one month had no influence on blood potassium

and chloride levels, whereas it significantly lowered sodium levels in 5 mg/kg group (p<0.01).

Liver function tests remained largely unaltered except mild increase in total bilirubin levels with

the higher dose. Similarly, kidney function tests also failed to show any discernible change after

TBTC exposure. Blood glucose showed a significant lowering trend (p<0.01) in 1 mg/kg TBTC

rats, while in the higher group the lowering was statistically insignificant. Alkaline phosphatase

was also found altered in both the dose groups but was statistically insignificant.

One of the parameters which were largely affected by TBTC exposure is the increase of

cholesterol and triglycerides in serum (Fig 5). Triglycerides were raised from 70.32 to 86.56

mg/dl in 1 mg/kg TBTC group which further increased to 124.7 mg/dl in the higher dose group

(p<0.01). Similarly cholesterol levels increased from 69.8 to 80.68 mg/dl and 91.32 mg/dl

(p<0.001) in the TBTC groups respectively. These results suggest that TBTC acts as a

hyperlipidemic compound resulting in high triglyceride and cholesterol levels particularly in the

higher dose group.

3.8 Structural alterations in liver, kidney and lung tissue was evident following TBTC

exposure

To understand whether TBTC influenced structural integrity of the tissues, tissue histopathology

was done (Fig 6). The liver morphology of both TBTC groups indicated mild cytoplasmic

vacuolation whereas in kidneys, the glomeruli appeared swollen with distinct capsular space. In

lungs, the bronchi appeared damaged. There was loss in mucosal epithelial lining with shrinkage

of submucosal and fibro cartilaginous shell. Semi-quantitative score of the tissue damage has

been shown in table 5.

4. Discussion

One month exposure to low dose of TBTC leads to tissue damage without any discernible

alteration in their function. It acts as a hyperlipidemic compound (Fig 5) and also severely affects

the heme biosynthetic pathway (Fig 3). The rise in ROS was more predominant in blood than in

the three organs. Depletion of blood antioxidant capacity could be attributed to the direct

interaction of organotin compounds with free -SH groups in proteins and also due to inhibition of

the activity of some enzymes, such as SOD (Milaeva et al., 2006). TBTC exposure led to modest

cytotoxicity and enhanced oxidant generation.

The increased burden of TBTC in blood and a recent report on the incorporation of tributyltin

into the hydrophobic region of the erythrocyte monolayer (Bonarska-Kujawa et al., 2012),

prompted us to evaluate its effect on porphyrin metabolism. Activity of δ-ALAD was previously

attributed to lead poisoning and was suggested to be utilized as a biomarker for the same

(Martinez et al., 2013). But in our study we found clear effect of TBTC on porphyrin

metabolism, indicating that TBTC also may ultimately lead to anemia like symptoms.

Previously, Chiba et al. (1980) showed profound inhibition of δ-ALAD activity by Sn in blood,

without altering its activity in liver although tin content in liver was considerably higher than that

of blood. A similar observation was made by us showing maximum Sn uptake in liver and

inhibition of δ-ALAD activity in blood. In another study, Chmielnicka et al. (1994) showed that

Sn caused hemolytic anemia depending on the abnormal iron utilization in rabbits. After i.p.

administration of tin, anemia was observed during the entire study period, whereas after oral

exposure, transient anemia was noticed. Effect of TBT on the hematocrit and hemoglobin levels

has already been demonstrated earlier (Silva de Assis et al., 2005). In our study, we observed

minor reduction in the total RBC count along with hemoglobin and hematocrit values suggesting

that even low doses of TBTC may disturb this delicate balance, paving the path for further

complications.

Although the exact mechanism of Sn(II)-induced inhibition of δ-ALAD has not yet been

thoroughly investigated, a study by Chiba and Kikuchi (1979) strongly suggests that Sn(II)

interacts in a more reversible way with the δ-ALAD than Pb(II), since the blood enzyme activity

returned to normal values more rapidly in Sn(II)-than in Pb(II)-intoxicated animals. In view of

the presence of 3 vicinal thiol groups in its active centre, δ-ALA-D can also be oxidized by

different soft electrophiles and by metals that compete with Zn(II) at its active centre (Rocha et

al., 2012). Inhibition of δ-ALAD activity by TBTC may be attributed to the strong binding

affinity of organotins with free –SH groups of proteins and GSH (Milaeva et al., 2006). We have

also demonstrated previously that TBTC exposure decreases the zinc content in brain cortical

tissue (Mitra et al., 2013).

Long duration exposure to TBTC also resulted in an increase in cholesterol and triglyceride

levels indicating hyperlipidemia. The hyperlipidemic action of TBTC could be due to decreased

lipoprotein lipase (LPL) activity as suggested by Matsui et al. (1982). The decreased LPL

activity seems to be related to the inhibition of insulin release from pancreatic islets, since fasting

blood glucose level was elevated. Subchronic TBTC exposure induced an increase in these

parameters which may serve as a pre-disposing factor for coronary arterial diseases.

In another study, Manabe and Wada (1981) showed that Triphenyltin fluoride (TPTF) inhibits

insulin release from rabbit islets, subsequently inducing diabetic lipemia due to insulin

deficiency. However, in our study we found lipemia (Fig 5) along with lowering in the random

blood glucose levels (Table 4) notably in the 1 mg TBTC group. Moreover, tributyltin (TBT) has

been suggested to be one of the environmental chemicals that lead to excessive accumulation of

adipose tissue which can result in obesity (Inadera and Shimomura, 2005). The obesogenic

property of TBT has also been shown to have trans-generationally inherited (Chamorro-Garcia et

al., 2013). This aspect of TBTC intoxication is of prime concern especially due to the gradual

increase of obesity which is now regarded as a social health problem worldwide, especially in the

advanced countries (WHO Report, 2000).

Apart from the little effect on the functional aspects of the various organs analyzed (Table 4),

TBTC affected oxidative stress indices (Table 1) along with the structural alterations (Fig 6) and

cell death (Table 2). Oxidative stress is involved in various pathologies involving liver (Zhu et

al., 2012), kidney (Palm and Nordquist, 2011) and lungs (Park et al., 2009). Prolonged oxidative

environment is also a leading cause of inflammatory responses and tumour development (Reuter

et al., 2010). Oxidative stress has been well linked to induce DNA damage and alterations

(Barzilai and Yamamoto, 2004). The protective efficacy shown by calcium inhibitors (BAPTA-

AM, EGTA) and antioxidants (NAC, C-PC) (Table 2) underlines the involvement of Ca2+ and

redox pathways in TBTC induced cell death and subsequent organ damage. Intracellular Ca2+ has

been well linked in inducing oxidative stress. In fact these two have been well reported to affect

each other through the involvement of mitochondria which primarily leads to cell death by

hampering oxidative phosphorylation (Brookes et al., 2004). On the other hand, altered lipid

peroxidation levels may hamper the cell signaling pathways as they are well linked to the

signaling pathway themselves serving as signal molecules (Higdon et al., 2012; Uchida et al.,

1999). Interestingly, we have observed a decrease in relative organ weight and simultaneous

increase in cell density. At the same time, a rise in cell death in these organs suggests TBTC

toxicity which may be the cause of tissue weight loss and tissue damage. Moreover, increased

cell density could be due to body’s natural response to replace the damaged cells. This

observation warrants further investigation to determine the underlying mechanisms.

So far, literature studies highlight oxidative damage by TBT compounds in liver (Liu et al.,

2006). In addition, we observed increased ROS production also in kidneys mainly with 1 mg

dose of TBTC. Oxidative stress in the kidney may affect the production of the hormone

erythropoietin which maintains RBC content in blood (Jelkmann, 2011). Oxidative damage may

also lead to the development of diabetic nephropathy and is also responsible for chronic renal

failure (Forbes et al., 2008; Martín-Mateo et al., 1999). Moreover, altered hematological

parameters will negatively affect the oxygen carrying capacity of the RBC’s destabilizing the

chain of body’s oxygen demand and supply. Simultaneously, liver damage may result in altered

lipid metabolism and thus may result in the built up of cholesterol content in serum.

Our study highlights the importance of subchronic daily exposure to low levels to TBTC which

is more of a practical scenario in today’s world. Since TBT compounds are making their way

into our daily lives from various sources (WWF factsheet), the exposure risk is very high.

Special focus on children should be given as TBTC is also a suspected developmental toxicant.

In our study, the total tin content in blood was found to be ~6 nM which is very low in respect to

the reported human blood accumulation of 50-400 nM (Whalen et al.,1999). Thus, we suspect

that the various alterations observed by us have direct implications on human health. Moreover,

the protection shown by calcium inhibitors and antioxidants may provide leads to a better

management of TBTC toxicity.

5. Conclusion

To sum up, our study highlights the toxicological impact of subchronic exposure of TBTC (1 and

5 mg/kg), relevant in context of human exposures encountered. Liver, being the main

detoxification organ is mainly affected, followed by kidney and lungs. The biochemical

responses responsible for cellular disorientation include redox imbalance and lipidemia. Various

modulators (Ca2+ inhibitors and antioxidants) showed protective efficacy and prevented cell loss

indicating their involvement in TBTC toxicity. Since total tin accumulation in our study is much

lower than those found in human tissue, the effects encountered may answer some of the

questions which relate to environmental toxicant exposure and increasing health ailments

reported globally.

6. Acknowledgement: We thank our Director, Dr. K.C. Gupta for his keen interest in the study,

Ms. Poonam Saxena for GF-AAS analysis and Mr. Pradeep Kumar for histopathology slide

preparation. The CSIR-IITR manuscript number is 3147.

7. Funding: This study was supported by CSIR sponsored supra-institutional project (SIP-08).

S.M and R.G are thankful to University Grants Commission (UGC) for providing Senior and

Junior Research Fellowship respectively; and V.S is thankful to CSIR for providing Junior

Research Fellowship.

8. Conflict of Interest: The Author(s) declare(s) that there is no conflict of interest.

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Figure Legends

Fig 1. Effect of TBTC on body weight and organo-somatic indices

(1a) TBTC significantly restricted the body weight gain in animals gavaged with 1 and 5 mg/kg

TBTC when compared to control rats. The body weight gain was measured at regular intervals

and expressed in g. The data represents mean ± S.E. of six rats. **p < 0.01 and *p < 0.05, when

compared to control using Kruskal-Wallis statistics.

(1b) The relative organ weight with respect to body weight was found to be altered in all the

three organs. After sacrifice, the total organ weight was measured and the data expressed as total

organ weight / body weight. Data represents mean ± S.E. of six rats.

(1c) The cell density was found to be increased in liver and kidney, while inversely affecting the

lungs following TBTC exposure. Data represents mean ± S.E. of total cell number / g tissue of

six rats.

Fig 2. TBTC exposure leads to tin accumulation

Maximum amount of tin accumulation was found in liver followed by kidney, blood and lungs.

After sacrifice, 100 mg tissue of three organs along with 1 ml blood of each rat was digested

with HNO3:HClO4 acid mixture (6:1) till complete oxidation.Total tin was estimated by GF-

AAS. Data represents mean ± S.E. (ng tin / g tissue) from six rats. **p < 0.01 and *p < 0.05,

when compared to control using Kruskal-Wallis statistics.

Fig 3. TBTC affects heme biosynthetic pathway

The effect of TBTC exposure on heme biosynthetic pathway was evaluated by measuring δ-

ALAD activity and total porphyrin content.

(3a) δ- ALAD activity in blood was found to be significantly affected by both the doses of TBTC

and was expressed as µ mole PBG ml-1 h-1. Data represents mean ± S.E. of six rats. *p < 0.05,

when compared to control using Kruskal-Wallis statistics.

(3b) Accordingly, the total porphyrin content in blood was significantly affected only by the

higher dose and only marginally by the lower dose of TBTC. Data represents mean ± S.E. of six

rats. *p < 0.05, when compared to control using Kruskal-Wallis statistics.

Fig 4. Oxidative stress induction in serum after TBTC exposure

(4a) Total antioxidant capacity (TAC) of serum is a measure of the antioxidant status of an

individual. Higher TBTC exposure resulted in significant lowering in TAC with nominal

alteration by the lower dose. Data represents mean ± S.E. of six rats. **p < 0.01, when compared

to control using Kruskal-Wallis statistics.

(4b) ROS levels were estimated by using deacetylated form of DCFH-DA dye by treating it with

0.1 M NaoH for 30 min. More than 3-fold increase in ROS levels was found in the treated

animals. Data represents mean ± S.E. of six rats. **p < 0.01, when compared to control using

Kruskal-Wallis statistics.

(4c) Lipid peroxidation was measured as thiobarbituric acid reactive substances (TBARS) and is

a sensitive indicator of oxidative damage to lipids. Marginal increment in TBARS content was

found after TBTC exposure, which was sigificantly altered only in the higer dose group. It is

expressed as µ mole TBARS/ mg protein. Data represents mean ± S.E. of six rats. *p < 0.05,

when compared to control using Kruskal-Wallis statistics.

Fig 5. TBTC acts as an obesogenic environmental toxicant and disturbs lipid profile

Serum was analysed in a biochemistry analyser for cholesterol and triglycerides contents

(5a) Cholesterol levels was found to be dose dependently and significantly increased upon TBTC

exposure. It was expressed as mg/dl serum. Data represents mean ± S.E. of six rats. ***p <

0.001, when compared to control using Kruskal-Wallis statistics.

(5b) Similarly, triglycerides were also found to be dose-dependently elevated which was

significant only with the higher dose of TBTC. It is expressed as mg/dl serum. Data represents

mean ± S.E. of six rats. **p < 0.01, when compared to control using Kruskal-Wallis statistics.

Fig 6. Changes in tissue morphology following TBTC exposure

After sacrifice, animals were perfused and organs were isolated. A portion of the tissue was post

fixed in 4% paraformaldehyde at 4ºC for 48 h, embedded in paraffin, sectioned at 10µm and

H&E staining was done. Pictures were obtained at 100X magnification. Arrows in the tissue

histology indicates marked cytoplasmic vacuolation in the liver tissue which was evident dose-

dependently. Kidney showed swollen glomeruli with increased capsular space indicating

damage. Lung tissue showed disruption of the mucosal epithelial lining surrounded by damaged

bronchi.

Fig 1. Effect of TBTC on body weight and organo-somatic indices

Fig 1b Fig 1a

Fig 1c

Fig 2. TBTC exposure leads to tin accumulation

Fig 3. TBTC affects heme biosynthetic pathway

Fig 3b Fig 3a

Fig 4. Oxidative stress induction in serum after TBTC exposure

Fig 4a Fig 4b

Fig 4c

Fig 5. TBTC acts as an obesogenic environmental toxicant and disturbs lipid profile

Fig 5b Fig 5a

Fig 6. Changes in tissue morphology following TBTC exposure

Table 1.TBTC causes oxidative stress in various organs

10% tissue homogenates in 0.1 M phosphate buffer were prepared. ROS was measured by incubating with DCFH-DA dye and measuring the fluorescence at 485/520 nm in a plate reader. Lipid peroxidation was measured as thiobarbituric acid reactive substances (TBARS) and absorbance was read in plate reader at 532 nm. ROS levels was found to be significantly elevated in all the tissues especially with the lower dose of TBTC. TBARS content on the other hand has marginal increment in all the samples assessed. Data represents mean ± S.E. of six rats. **p < 0.01 and *p<0.05, when compared to control, using Kruskal-Wallis statistics.

Table 2. Effect of TBTC on cell viability and the protective efficacy of various inhibitors

Single cell suspension (1x104cells/well) were incubated with 10 µl MTT solution (5.4 mg/ml in PBS) for 4 h. Likewise, calcium inhibitors (BAPTA-AM, 50µM & EGTA, 5 mM) and free radical scavengers (NAC, 50 µM & CPC, 1 µg/ml), were pre-incubated with the cells for 1 h at 37ºC in a CO2 incubator. Significant amount of cell death was evident in liver and kidney followed by lung . Interestingly, the various inhibitors used successfully restored the cell viability in the lower dose groups but not in the higher dose groups (except in the case of kidney). The free radical scavengers were found to be more efficient in restoring the cell viability than the calcium inhibitors. Data represents mean ± S.E. of six rats, where cell suspension of each rat was plated in triplicate. ap<0.001; bp<0.01 and cp<0.05, when compared to control using Kruskal-Wallis statistics; yp<0.01 and zp<0.05 when compared to TBTC alone using Kruskal-Wallis statistics.

44

T-RBC Hct MCV Hgb MCHC

(x 106

/ mm3

) (%) (micron3

) (g%) (g%)

Control 5.82 ± 0.21 36.2 ± 1.11 62.2 ± 2.63 11.05 ± 0.25 30.5 ± 1.86

1 mg/kg TBTC 5.79 ± 0.59 33.9 ± 2.59 59.0 ± 1.62 10.20 ± 0.80 30.0 ± 2.11

5 mg/kg TBTC 5.53 ± 0.38 33.45 ± 1.25 57.65 ± 2.11 9.55 ± 0.47 29.8 ± 1.27

Table 3. TBTC exposure results in hematological alterations

Heparinized blood was analyzed in a HS9 hematology analyzer. TBTC causes marginal reduction in the various blood indices. Data represents mean ± S.E. of six rats.

T-RBC: Total Red Blood Cell; Hct: Hematocrit; MCV: Mean Corpuscular Volume;

Hgb: Hemoglobin; MCHC: Mean Corpuscular Hemoglobin Concentration

45

Table 4. Effect of TBTC on various tissue function tests and general parameters

Serum was analyzed for various parameters in a biochemistry analyzer (Selectra Junior Spinlab 100). Among the liver function tests, only TBIL was found to be marginally altered whereas other parameters remained unaltered. Kidney function tests were also found unaltered along with the concentration of the electrolytes. Glucose levels were found to be highly repressed with the elevation of alkaline phosphatase activity, especially in the 1 mg/kg TBTC group. Data represents mean ± S.E. of six rats. **p < 0.01, when compared to control, using Kruskal-Wallis statistics.

46

TBTC

Control 1 mg/kg 5 mg/kg

Liver 0.33 2.50 b 3.16 a

kidney 0.16 2.33 b 3.00 a

Lungs 0.00 2.83 b 3.33 a

Table 5. Representation of the histological alterations in a semi-quantitative scale

The tissue morphological influence of TBTC was evaluated on a scale of ‘0’ to ‘5’ representing “no damage” to “severe damage”. Histological alterations by the higher dose (5 mg/kg) were found to be more severe than the lower dose (1 mg/kg). Scoring has been done for each animal

per group and data is represented as mean ± S.E. of six rats. ap < 0.001; bp <0.01 when compared

to control, using Kruskal-Wallis statistics.

47

Highlights

1. Subchronic exposure to low dose tributyltin results in multi-organ toxicity

2. Alterations in liver morphology but not serum parameters were observed

3. δ-ALAD activity in blood and lipid profile in serum were severely altered

4. Kidney and lungs were also affected along with changes in organo-somatic indices

5. Oxidative stress parameters were found to be involved in TBTC multi-organ toxicity