Article pubs.acs.org/est
Chronic Exposure to Tributyltin Chloride Induces Pancreatic Islet Cell Apoptosis and Disrupts Glucose Homeostasis in Male Mice Zhenghong Zuo,† Tian Wu,† Moudan Lin,† Shiqi Zhang,† Feihuan Yan,† Zhibin Yang,† Yuanchuan Wang,† and Chonggang Wang*,†,‡ †
State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, PR China State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen, PR China
‡
S Supporting Information *
ABSTRACT: It has been reported that organotin compounds such as triphenyltin or tributyltin (TBT) induce diabetes and insulin resistance. However, histopathological effects of organotin compounds on the Islets of Langerhans and exocrine pancreas are still unclear. In the present study, male KM mice were orally administered with TBT (0.5, 5, and 50 μg/kg) once every 3 days. The fasting plasma glucose levels significantly elevated, and the levels of serum insulin or glucagon decreased in the animals treated with TBT for 60 days. In animals treated for 45 days, the number of apoptotic cells in the islets and exocrine pancreas was elevated in a dosedependent manner. The percentage of proliferating (PCNApositive) cells was decreased in the islets, while it was increased in exocrine acinar cells. Immunohistochemistry analysis showed that estrogen receptor (ER) and androgen receptor (AR) were present in vascular endothelium, ductal cells, and islet cells, but absent from pancreatic exocrine cells. TBT exposure decreased the production of estradiol and triiodothyronine and elevated the concentration of testosterone, and resulted in a decrease of ERα expression and an elevation of AR in the pancreas measured by Western boltting. The results suggested that TBT inhibited the proliferation and induced the apoptosis of islet cells via multipathways, causing a decrease of relative islet area in the animals treated for 60 days, which could result in a disruption of glucose homeostasis. The different presence of ERs and AR between the islets and exocrine pancreas might be one of reasons causing different effects on cell proliferation.
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xenoestrogens.9 Widespread EDCs, such as dioxins, pesticides, and bisphenol A (BPA), cause insulin resistance and alter β-cell function in animal models.7 In 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD)-treated rats, insulin content is significantly decreased.10 The exposure of adult mice to BPA disrupts pancreatic β-cell function in vivo and induces insulin resistance.11 But mechanisms linking EDCs to insulin resistance and diabetes are largely unknown. Estrogens play an important role in blood glucose homeostasis. At physiological levels, 17β-estradiol (E2) is thought to be involved in maintaining normal insulin sensitivity and to be beneficial for β-cell function, while E2 levels above or below the physiological range may promote insulin resistance and type 2 diabetes.9 Estrogen receptor ERα is emerging as a key molecule involved in glucose and lipid metabolism.9 The use of ERα and ERβ agonists, as well as islets from ERα knockout (ERαKO) and ERβKO mice demonstrates that E2 action on insulin biosynthesis is mediated by ERα.12 Estradiol can protect β-cells
INTRODUCTION Organotin compounds such as tributyltin (TBT) have been used worldwide in agriculture and industry as biocides, heat stabilizers, and chemical catalysts. Wide distribution, high hydrophobicity, and persistence of organotin compounds have raised concern about their adverse effects on human health and the environment.1 Humans could be exposed to organotins through contaminated dietary sources (seafood and shellfish), drinking water, and so on. TBT levels in human blood collected in the USA are up to 8.18 ng/mL;2 concentrations of butyltin in human livers in Japan are in the range 59−96 ng/g wet wt;3 the levels of TBT in 41 of 74 molluscan products purchased from major supermarket outlets in seven cities across Canada are up to 233 ng/g;4 mean concentrations of TBT of 308.7 ng/ g wet wt are found in the muscle tissue of fish collected from Taiwanese harbors,5 and the average seafood consumption of 0.163 kg/day is estimated.6 Therefore, mammals, especially humans, are inevitably exposed to TBT. Endocrine disrupting chemicals (EDCs) in the environment play an important part in the incidence of metabolic diseases.7 Both epidemiological and experimental evidence indicate that some environmental pollutants are relative to diabetes mellitus.8 The pancreatic β-cell is a target of estrogens and © 2014 American Chemical Society
Received: Revised: Accepted: Published: 5179
March 24, 2013 March 31, 2014 April 2, 2014 April 2, 2014 dx.doi.org/10.1021/es404729p | Environ. Sci. Technol. 2014, 48, 5179−5186
Environmental Science & Technology
Article
from apoptosis though ERα.13 However, the existence of ERs in β-cells has not been extensively studied.9 There is little information available concerning the impact of EDC on the expression of ERs. As EDCs, organotin compounds such as TBT and triphenyltin (TPT) can disrupt the homeostasis of steroid hormones14,15 and thyroid hormones.16 Several studies indicate that TBT inhibits the activity of aromatase,17 which converts testosterone into estradiol, and leads to a decrease of 17βestradiol and an elevation of testosterone in male mammals.15 An oral administration of TPT can produce diabetes in rabbits18 and hamsters.19 Oral administration of a single dose of TPT induces diabetes with decreased insulin secretion in rabbits and hamsters after 2−3 days without any morphological changes in the pancreatic islets.20 The effect of TBT on adipocytes may be an environmental risk factor in the development of metabolic disorders such as obesity and type 2 diabetes, in which the adipocytes play a crucial role.21 Our previous study shows that chronic and repeated exposure to TBT (0.5, 5, 50 μg/kg) for 45 days results in obesity and hepatic steatosis, and elevates plasma insulin levels in mice.22 However, histopathological effects of TBT on the islets and its underlying mechanism are still unclear. In the present study, we investigated the effects of TBT on the islets and glucose homeostasis and looked into the toxicological mechanism involved.
were measured using a OneTouch Ultra glucometer (LifeScan, Milpitas, CA). Animals were then euthanized by injection intraperitoneally with sodium pentobarbital (60 mg/kg). Blood samples were collected from the eye socket and then stored at −80 °C for hormones analysis. The pancreas and thyroid gland were fixed in 10% neutral buffered formalin solution and then embedded in paraffin, cut into 4 μm sections, and stained with hematoxylin and eosin for histopathological study. Relative islet area was determined according to the reported method.23 For each individual, 16 areas from four different sections were randomly chosen for analysis under a light microscope at a magnification of ×100. The image analysis quantified total tissue area within this region, followed by islet area to generate the ratio of islet to total pancreas area. The pancreas and gonads were frozen in liquid N2 immediately after collection and stored at −80 °C prior to analysis. Hormone Measurement. Serum insulin or glucagon levels were detected using mouse insulin ELISA kit (Millipore, St. Charles, MO) or Mouse Glucagon ELISA Kit (ELAab Science Co., Wuhan, China). The testis was individually cut into small pieces of 0.1 g wet weight for analysis. Subsamples were homogenized in ethanol (5 mL) and frozen at −80 °C for at least 24 h. Homogenates were then extracted with diethyl ether (8 mL), followed with two further extractions with 4:1 diethyl ether/ethanol (2 × 10 mL) as previously described.24 The 17β-estradiol or testosterone of testis and serum was assayed using a commercial radioimmunoassay kit (Furui Biological Engineering Co., Beijing, China) and, for both, the intra- and interassay coefficients of variance were
