A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

Summary

Here we present a protocol to isolate mouse pancreatic islet cells for screening the ROS inductions by the xenobiotics in order to identify the potential diabetogenic xenobiotic chemicals.

Abstract

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic β cells which will lead to the development of type 2 diabetes mellitus (T2DM). Although the mechanisms for the chemical-induced β cell damage were unclear and likely to be complex, one recurring finding is that these chemicals induce oxidative stress leading to the generation of excessive reactive oxygen species (ROS) which induce damage to the β cell. To identify potential diabetogenic environmental chemicals, we isolated pancreatic islet cells from C57BL/6 mice and cultured islet cells in 96-well cell culture plates; then, the islet cells were dosed with chemicals and the ROS generation was detected by 2′,7′-dichlorofluorescein (DCFH-DA) fluorescent dye. Using this method, we found that bisphenol A (BPA), Benzo[a]pyrene (BaP), and polychlorinated biphenyls (PCBs), could induce high levels of ROS, suggesting that they may potentially induce damage in islet cells. This method should be useful for screening diabetogenic xenobiotics. In addition, the cultured islet cells may also be adapted for in vitro analysis of chemical-induced toxicity in pancreatic cells.

Introduction

Increases in the prevalence of T2DM have become a global health crisis in recent years posing a serious threat to public health¹. Many factors have been found to be causally linked to the development of T2DM, among which, recurring findings suggest that one common convergent point for these factors is the induction of oxidative stress which leads to the generation of excessive ROS^2,3.

A wide spectrum of environmental chemicals including PCBs, dioxins, and BaP has been found to induce oxidative stress, which may impair the function of pancreatic β cells and lead to insulin resistance and T2DM⁴. Although the physiological level of ROS plays an important role in cellular functions, exposure to ROS that exceeds the capacity of the antioxidant system results in the damage to cells/tissues and leads to diseases⁵. Pancreatic β cells express a low level of antioxidant enzyme, and thus are a sensitive target for the oxidative stress-mediated damage^6,7. Chronic exposure to high levels of ROS has been shown to cause stress-induced pancreatic cell dysfunction⁵ as well as insulin resistance in the liver and adipose tissue⁸.

The overall goal of this project is to develop a cell-based assay to screen chemicals for their diabetogenic potentials based on their induction of ROS in pancreatic cells. The pancreas lacks metabolic detoxification and is a sensitive target for xenobiotic-induced damage^6,7. Therefore, by directly measuring the ROS generated in the pancreatic cells, this assay should provide a direct approximation of the chemical-induced injury in the pancreas. To develop this method, we isolated mouse pancreatic islets, cultured the isolated islet under cell culture condition with chemicals, and utilized the chemical-induced ROS generation as the readout. This procedure is simple and effective in identifying ROS-inducing chemicals in the isolated islet; it can be further developed for investigation of the mechanisms of toxicity that are specific to the pancreas in vitro.

Protocol

All animal experiments were executed in compliance with all relevant guidelines, regulations and regulatory agencies. The protocol being demonstrated was performed under the guidance and approval of the Institutional Animal Care and Use Committee (IACUC) of the Texas A&M Institute for Genomic Medicine. 1. Solution Preparation Dilute 10x Hank's balanced salt solution to 1x with double distilled H2O, and store at 4 °C. Prepare the isolation solution …

Representative Results

A micrograph of the healthy Isolated islet is shown in Figure 2, in which islets have a round or oval shape with relatively uniform size (although size uniformity can vary from strain to strain). We next investigated the pancreatic islet functions in an in vitro assay by isolating the islet and stimulating the insulin secretion in the culture islets. Figure 3 shows our typical analysis of the GSIS assay from C57BL/6…

Discussion

Accumulating evidence suggests that exposure to environmental chemicals plays an important role in the development of T2DM. Xenobiotics-induced ROS has been recognized as a potential etiological factor contributing to the development of T2DM. Humans are exposed to a wide range of xenobiotic chemicals and there is a great need for novel research techniques to effectively identify the pancreatic toxicants and to investigate the mechanism of toxicity specific to the pancreatic cells.

In this stud…

Materials

10×Hank’s balanced salt solution	GIBCO	14185-052
Collagenase Type 4	Worthington Biochemical Corporation	CLS-4
polysucrose/sodium diatrizoate solution	Sigma	10771
2’,7’-dichlorofluorescein (DCFH-DA)	Sigma	D6883-50MG
fluorescence microplate reader	Biotek
L-glutamine	Sigma	G8540-25G
streptomycin	GIBCO	15140148
FBS	GIBCO	26140079
RPMI 1640	GIBCO	11875-085
avertin	Sigma	T48402-25G
Rat/Mouse Insulin ELISA Kit	Millipore	EZRMI-13K
Centrifuge	Sorval	Sorval RT7	for 96-well plate centrifuge
Microplate reader	Biotek	Epoch 2	for fluorescence reading