First Identification of the Toxicity of Microcystins on Pancreatic Islet

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First identification of the toxicity of microcystins on pancreatic islet function in humans and the involved potential biomarkers Yanyan Zhao, Qingju Xue, Xiaomei Su, Liqiang Xie, Yunjun Yan, Lixiao Wang, and Alan D. Steinman Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b03369 • Publication Date (Web): 09 Feb 2016 Downloaded from http://pubs.acs.org on February 16, 2016

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Environmental Science & Technology

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First identification of the toxicity of microcystins on pancreatic islet function in

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humans and the involved potential biomarkers

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Yanyan Zhaoa, Qingju Xuea, Xiaomei Sua, Liqiang Xiea, *, Yunjun Yanb,*, Lixiao

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Wangb, Alan D. Steinmanc

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a

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Geography and Limnology, Chinese Academy of Sciences, 73 East Beijing Road,

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Nanjing 210008, P.R. China

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b

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Life Science and Technology, Huazhong University of Science and Technology, 1037

State Key Laboratory of Lake Science and Environment, Nanjing Institute of

Key Laboratory of Molecular Biophysics of the Ministry of Education, College of

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Luoyu Road, Wuhan 430074, PR China

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c

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Drive, Muskegon MI 49441 USA

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*

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Liqiang Xie

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Institute of Geography and Limnology, Chinese Academy of Sciences, 73 East

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Beijing Road, Nanjing 210008, P.R. China

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Tel./fax: ＋86 25 86882199.

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E-mail: [email protected]

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Yunjun Yan

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Tel./Fax: ＋86 27 87792213

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E-mail: [email protected]

Annis Water Resources Institute, Grand Valley State University, 740 West Shoreline

Address correspondence to:

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ACS Paragon Plus Environment


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Abstract

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Microcystins (MCs) produced by cyanobacteria have been recognized as a major

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public health threat. However, the toxicity of MCs to humans is still largely unknown.

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In this study, we examined the changes in pancreatic islet function in fishers exposed

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to ambient levels of MCs at Lake Taihu, and using a mouse model, explored the

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molecular mechanisms involved in toxicity. MCs content in the serum of fishers

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tested positive, with a range from 0.10 to 0.64 µg /L. Both lower blood insulin levels

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(2.26 ± 0.96 uIU/mL) and impaired fasting glucose were found in participants from

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the Meiliang Bay area in Lake Taihu, where MC-LR levels were substantially greater

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than the MC threshold established by WHO for drinking water. Animal experiments

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showed that glucose level increased by 27.9% in mice exposed to 5 µg/kg bw and

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decreased by 41.5% in mice exposed to 20 µg/kg bw. Blood insulin levels declined by

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21.9% and 56.2% in mice exposed to 5 and 20 µg/kg bw MC-LR, respectively, which

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was consistent with the results observed in fishers. Furthermore, the diabetes gene

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pdx1 and several other proteins (such as Ppp3ca, Ide, Marcks, Pgk1, Suclg1, Ndufs4)

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involved in insulin secretion were identified for the first time in mice following

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MC-LR exposure; these biomarkers were considered responsible for MC-LR induced

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islet dysfunction. This study suggests that sub-chronic exposure to environmental

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levels of MCs may increase the risk of the occurrence of diabetes in humans.

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1. Introduction

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In the past few decades, the occurrence of toxic cyanobacterial blooms in

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eutrophic waters has become a worldwide problem. Some cyanobacterial genera are

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able to produce a series of natural toxins, including microcystins (MCs), which are the

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most common and heavily studied (1). To date, more than 100 structural analogues of

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MC have been identified (2). Among them, MC-LR is one of the most toxic and

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common congeners (3). Exposure to these widely distributed toxins imposes a health

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risk on animals and even humans (4, 5, 6, 7). Acute exposure to MCs results in

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cytoskeletal deformation, mitochondrial membrane rupture and involve damage to

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hepatic architecture with massive intrahepatic hemorrhaging (8, 9, 10). Sustained to

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sub-lethal level of MC-LR can lead to progressive liver tissue fibrosis and even

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tumorigenesis (11).

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People may be exposed to cyanotoxins in a chronic manner through several

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routes, such as ingestion of drinking water, consumption of contaminated food, and

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inhalation and dermal exposure during recreation (12). In humans, intake of

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cyanobacteria-contaminated water or food has been associated with vomiting,

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weakness, skin irritation, illnesses ranging from gastroenteritis to hepato-enteritis (4,

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12, 13, 14). Several reports indicated that chronic exposure to MCs may result in liver

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damage in adults or children (15, 16). The most severe case of human toxicity

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happened in 1996 in Brazil, when 100 dialysis patients developed acute liver failure

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due to MCs-contaminated water, resulting in the death of 70 patients (17).

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A large number of studies have focused on the hepatotoxicity of MCs on various 3



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kinds of animals (18, 19, 20, 21, 22), as MCs are predominantly absorbed, transported

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and accumulated into the liver (23). Organic anion transporting polypeptides (human

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OATP/rodent Oatp) are specifically required for active uptake of MCs into

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hepatocytes (24). More than 35 different OATPs/Oatps have been found in the human,

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mouse, and rat, but only OATP1B1, OATP1B3, and Oatp1b2 are capable of

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transporting MCs into hepatocytes (25, 26). In addition, OATP1B3, one of the MCs

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transporters, is also expressed in human pancreatic tissue, with the abundance of the

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transporter localized in the islets of Langerhans (27). Pancreatic islets can produce,

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store and release insulin, the only hormone in the body able to lower blood glucose

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levels. Pancreatic islet dysfunction plays an important role in the pathogenesis of

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diabetes (28). Therefore, it is possible that MCs are able to accumulate in the pancreas

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and exert toxic effects on islet function. However, this area has received very little

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attention to date.

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MCs are stable and widely distributed in freshwater systems. Although acute

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toxicity due to cyanotoxins is not very likely in humans, MCs are still of considerable

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health concern due to their potential long-term adverse effects even at low,

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environmentally relevant concentrations. In the present study, we targeted the effects

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of long term chronic exposure of MCs on pancreatic islet function in fishers, a group

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of individuals who are frequently exposed to toxic cyanobacterial blooms. We also

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tried to further validate the association between MCs contamination and pancreatic

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damage using a mouse model, and explored the related molecular biomarkers in

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response to MCs exposure using the iTRAQ (isobaric tags for relative and absolute 4


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quntification) technique. The aim of the study is to better understand the toxicity of

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widely distributed MCs in the pancreas and the potential molecular mechanism in the

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damage to the pancreas.

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2. Materials and Methods

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2.1 Study population and sampling site. Lake Taihu (119°54′-120°36′N,

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30°56′-31°33′E), the third largest freshwater lake in China, is located in the highly

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developed and densely populated Yangtze Delta. Its mean depth is 1.9 m with a

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surface area of 2428 km2. Due to rapid social development and the intensive use of

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water resources, the lake water is becoming more heavily polluted (29). The

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occurrence of severe cyanobacterial blooms during warm weather has increased in

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frequency and intensity during the past few decades, especially in an area called

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Meiliang Bay in the northern region of the lake (30). For this reason, Meiliang Bay

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was chosen as the study area (Figure 1A). From May 20 to May 30, 2014, all the

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fishers living on the lake in Meiliang Bay area for more than 10 years were invited to

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participate in the evaluation. Of the 40 invited fishers, 37 individuals agreed to

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participate in qualitative face-to-face interviews and health examination (including the

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detection of fasting plasma insulin and glucose level, and MCs content in serum).

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Their age, family and personal diseases (including diabetes mellitus, pancreatitis, and

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liver diseases), diet, smoking and alcohol use, and the source of daily food and

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drinking water were collected. We excluded participants if they were currently

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smoking, drinking or had family and personal diseases. This resulted in 30 individuals

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(75%) participating in the health examination. All individuals were examined in the 5



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morning after fasting overnight. Approximately 15 mL of blood was drawn per

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subject by a trained nurse at the local community health center. The biochemical

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analyses were examined using the Synchron clinical system CX3 (Beckman-Coulter

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Diagnosis, Fullerton, CA) in the local community health center. MCs content in

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serum were measured according to the method described by Chen et al. (15). All

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participants gave informed consent, and the study protocol was approved by the

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Ethics and Human Subject Committee of Huazhong University of Science and

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Technology. During the health examination period, water samples from seven

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sampling sites of Meiliang Bay area were also collected for the analysis of dissolved

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MCs.

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2.2 MCs analysis of water sample. MCs analysis was according to Park et al.

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(31). 1 L of water sample was filtered through a glass microfiber filter (Whatman

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GF/C). The filtered water was applied directly to an HLB cartridge (0.2 g, Oasis,

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Waters, Milford, Massachusetts, USA). The cartridge was rinsed with 5%

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methanol-water. The eluate from the cartridge was evaporated to dryness, and the

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residue was then dissolved in methanol. The methanol solution was analyzed by

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High-performance liquid chromatography (HPLC, Agilent 1200 series, Palo Alto, CA,

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USA) to determine the content of three MC congeners (-LR, -RR and -YR).

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2.3 Mouse model. 6-week-old male BALB/c mice were purchased from Wuhan

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Institute of Virology, CAS. Mice were assigned randomly to 3 groups (control and

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two treatments) and each group has 15 mice. They were housed in a laboratory animal

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center to acclimate to the laboratory environment for one week before treatment. 5 6


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mice were housed per cage. All cages were located in a climate-controlled room with

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temperatures ranging from 20 to 22 ℃ with a 12:12 h light:dark cycle, and mice

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were given free access to a standard rodent pellet diet and water. All procedures

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carried out on animals were approved by the Institutional Animal Care and Use

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Committee of Huazhong University of Science and Technology (permit NO.

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2018110).

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MC-LR (purity > 95%) standards purchased from Sigma Chemical (St. Louis,

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MO, U.S.A.) were dissolved in 0.9% saline solution at desired concentrations.

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Treatment groups received intraperitoneal injections (i.p.) of 5 or 20 µg MC-LR.kg-1

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body weight (bw) every 2 days for 16 weeks (low and high dose, respectively). The

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control group was treated with the same volume of 0.9% saline solution. At the end of

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the exposure period, mice were weighed, and anesthetized with 50 mg kg-1 bw sodium

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pentobarbital. Blood was obtained by cardiac puncture with a syringe containing

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EDTA. The pancreas was immediately removed, weighed, and transferred into 2-mL

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tubes and stored in liquid nitrogen during necropsy. We then transferred the frozen

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tissue samples into a -80 ℃ freezer until used for the following analyses.

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2.4 Blood insulin and glucose level in mice. Blood insulin was determined by

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enzyme-linked immunosorbent assay (ELISA) using a mouse insulin assay kit from

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Mercodia AB (Uppsala, Sweden). We determined fasting glucose in blood obtained

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from the tail vein using an Accu-check compact glucometer (Roche Diagnostic

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GmbH, Mannheim, Germany).

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2.5 Histological examination. Pancreas were fixed in freshly made 4% 7



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paraformaldehyde in PBS overnight at 4℃. The samples were embedded in paraffin

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after a stepwise dehydration in ethanol and xylene. 5 µm transverse sections were

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prepared and deparaffinized in xylene, and then, stained with hematoxylin and eosin.

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2.6 iTRAQ sample preparation. The pancreas samples were ground into

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powder in liquid nitrogen and extracted with Lysis buffer (7 M Urea, 2 M Thiourea,

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4℅ CHAPS, 40 mM Tris-HCl, pH 8.5). The suspension was sonicated at 200 w for 15

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min and then centrifuged at 30, 000 g for 15 min at 4 ℃. The extracted proteins were

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reduced, alkylated, digested, and labeled with iTRAQ-reagents as described in the

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iTRAQ protocol (Applied Biosystems, Foster City, CA). Additional details of iTRAQ

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sample preparation are provided in Supporting Information document.

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2.7 Peptide fractionation, LC-MS/MS analysis, and data processing and

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analysis. Labeled peptides were subjected to strong cation exchange (SCX)

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fractionation and then to C18 chromatography coupled directly to a Triple TOF 5600

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System (AB SCIEX, Concord, ON) fitted with a Nanospray source (AB SCIEX,

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Concord, ON) and a pulled quartz tip as the emitter (New Objectives, Woburn, MA).

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Database searches to identify the peptides were performed by using the Mascot search

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engine (Matrix Science, London, UK; version 2.3.02) against IPI mouse database

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(version, 3.87) containing 59534 sequences. Detailed procedures of the peptide

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fractionation, data acquisition, processing, and analysis are described in Methods in

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Supporting Information.

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2.8 Western blot analysis. The pancreas were lysed in 200 µL of RIPA buffer

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(50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 150 M NaCl, 0.25% deoxycholic acid, 1% 8


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NP-40, 1 mM PMSF, 10 µg/mL aprotinin, and 10 µg/mL leupeptin). Lysates were

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centrifuged at 15,000 rpm for 30 min at 4 ℃, and the protein concentrations were

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measured using the BCA protein assay (Pierce, Rockford, IL). Samples were

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separated by 12% (w/v) SDS-PAGE and transferred onto PVDF membranes using an

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electro blotting apparatus (Bio-Rad, America). The blots were probed with the

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following primary antibodies: protein phosphatase 3, catalytic subunit, alpha isozyme

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(Ppp3ca), pancreatic and duodenal homeobox 1 (Pdx1), phosphoglycerate kinase 1

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(Pgk1), succinate-CoA ligase, GDP-forming, alpha subunit (Suclg1) and NADH:

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ubiquinone oxidoreductase subunit S4 (Ndufs4) (Abcam Inc. Cambridge, MA), and

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glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam Inc. Cambridge, MA),

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followed by incubation in species-matched horseradish peroxidase (HRP)-conjugated

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secondary antibodies. The protein signal was developed using the NBT/BCIP system.

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The western blot results were quantified with Gene Snap software (Syngene,

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America). 2.9 Bioinformatics and statistical analysis. The classification and functions of

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the

proteins

identified

were

obtained

by

searching

Gene

Ontology

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(www.geneontology.org). Toxicity pathways were identified by using KEGG

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PATHWAY (http://www.genome.jp/kegg/pathway.html).

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A two-tailed Student’s t-test was used to determine the signiﬁcant differences

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between the control and treatment groups. Statistical analysis was performed using

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SPSS 13.0 software (SPSS, Chicago, IL), and p

First Identification of the Toxicity of Microcystins on Pancreatic Islet

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