This work was supported by a pilot project grant from CREH center sponsored by NIEHS and by National Natural Science Foundation of China (No. 31572626).

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&#38;M Institute for Genomic Medicine.
1. Solution Preparation
<ol>
	<li>Dilute 10x Hank&#39;s balanced salt solution to 1x with double distilled H2O, and store at 4 &#176;C.</li>
	<li>Prepare the isolation solution ...

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCB126+induces+differential+changes+in+androgen,+cortisol,+and+aldosterone+biosynthesis+in+human+adrenocortical+H295R+cells.">PCB126 induces differential changes in androgen, cortisol, and aldosterone biosynthesis in human adrenocortical H295R cells.</a> Toxicological Sciences. 85 (1), 530-540 (2005).</li><li>Asahi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bisphenol+A+induces+endoplasmic+reticulum+stress-associated+apoptosis+in+mouse+non-parenchymal+hepatocytes.">Bisphenol A induces endoplasmic reticulum stress-associated apoptosis in mouse non-parenchymal hepatocytes.</a> Life sciences. 87 (13), 431-438 (2010).</li><li>Szot, G. L., Koudria, P., Bluestone, J. 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(7), e255 (2007).</li><li>Kirstetter, P., Lagneau, F., Lucas, O., Krupa, Y., Marty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+endothelium+in+the+modulation+of+isoflurane-induced+vasodilatation+in+rat+thoracic+aorta.">Role of endothelium in the modulation of isoflurane-induced vasodilatation in rat thoracic aorta.</a> British journal of anaesthesia. 79 (1), 84-87 (1997).</li><li>Brown, E., Umino, Y., Loi, T., Solessio, E., Barlow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+can+cause+sustained+hyperglycemia+in+C57/BL6J+mice.">Anesthesia can cause sustained hyperglycemia in C57/BL6J mice.</a> Visual neuroscience. 22 (5), 615-618 (2005).</li><li>Vaupel, D., McCoun, D., Cone, E. 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The authors declare that they have no competing financial interests.

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...

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 ROS2,3. 
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 &#946; 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 &#946; cells express a low level of antioxidant enzyme, and thus are a sensitive target for the oxidative stress-mediated damage6,7. 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 damage6,7. 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.

<table border="0" cellpadding="0" cellspacing="0" width="858">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">10&#215;Hank&#8217;s balanced salt solution&#160;</td>
			<td style="width:283px;">GIBCO</td>
			<td style="width:119px;">14185-052</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase Type 4</td>
			<td>Worthington Biochemical Corporation</td>
			<td>CLS-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polysucrose/sodium diatrizoate solution&#160;</td>
			<td>Sigma</td>
			<td>10771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2&#8217;,7&#8217;-dichlorofluorescein (DCFH-DA)</td>
			<td>Sigma</td>
			<td>D6883-50MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fluorescence microplate reader&#160;</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine</td>
			<td>Sigma</td>
			<td>G8540-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">streptomycin</td>
			<td>GIBCO</td>
			<td>15140148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>GIBCO</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td>GIBCO</td>
			<td>11875-085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">avertin</td>
			<td>Sigma</td>
			<td>T48402-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat/Mouse Insulin ELISA Kit</td>
			<td>Millipore</td>
			<td>EZRMI-13K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Sorval</td>
			<td>Sorval RT7</td>
			<td>for 96-well plate centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microplate reader</td>
			<td>Biotek</td>
			<td>Epoch 2</td>
			<td>for fluorescence reading</td>
		</tr>
	</tbody>
</table>

a murine pancreatic islet cell-based screening for diabetogenic environmental chemicals

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to the development of type 2 diabetes mellitus (T2DM). Although the mechanisms for the chemical-induced &#946; 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 &#946; 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.

A micrograph of the healthy Isolated islet is shown in Figure 2, in which islets have a round or oval&#160;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...

Watch this Scientific Journal Video about A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals at JoVE.com

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

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.

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to ...

Pancreas cells are sensitive target for ROS induced damages, which is associated with development of type-2 diabetes. A pancreas cell-based assays will help identify ROS inducing chemicals. The main advantage of this technique is to use isolated pancreas islet cells for simple and effective identification of the ROS inducing chemicals which may have the added diabetogenic potentials.This method can also be used for an in vitro analysis on the effects of chemicals on a physiological function of the pancreas such as a glucose-stimulated insulin secretion. Use a pair of curved forceps to clamp the duodenum close to the position of the sphincter of Oddi. With another pair of curved forceps, clamp the duodenum wall to block the bile pathway.Find the common bile duct and with a 30 gauge needle and a five milliliter syringe, slowly inject three milliliters of collagenase solution. Then, reposition the mouse with the head proximal and the feet distal. Ensure that the common bile duct and pancreas are inflated after the injection.Using the curved forceps and fine scissors, remove the distended pancreas to separate it from the descending colon, intestines, stomach and spleen. Then, place the organ in a 50 milliliter tube containing three milliliters of collagenase solution and keep it on ice. Place each 50 milliliter tube into a 37 degrees Celsius water bath and incubate them for 15 to 20 minutes.Vortex the tubes, then pool four to five digested organs in a large sterile sink strainer and use a scoop to thoroughly break apart the pancreas. With a 23 gauge needle and ten milliliter syringe containing wash solution, vigorously pipet the digested cells off the screen. Then, collect the wash solution in a 50 milliliter tube.Centrifuge the tubes at 97g and four degrees Celsius for one minute and decant the supernatant. Add 25 milliliters of wash solution to the tubes, and resuspend the tissue by gently vortexing. Then, spin down the tissue, and decant the supernatant.Repeat the wash three times, taking care not to dislodge the pelleted tissue. To purify the islets, add ten milliliters of purification solution to the washed tissue pellet and gently resuspend the contents. Gently overlay five milliliters of isolation solution onto the purification solution.Ensure that a sharp liquid interface is maintained between the two solutions. Centrifuge the tube at 560g at four degrees Celsius for 15 minutes, with slow acceleration and no break. Then, insert the pipet into the interface and collect the islet layer.Transfer the solution into new 50 milliliter tubes before washing the islet as just demonstrated. After removing the final wash, add ten milliliters of islet culture solution and gently and thoroughly mix to resuspend the islets. Then, using a multi-channel pipet transfer 100 microliters per well to the wells of a 96 well plate.Incubate the plate in a tissue culture incubator for 12 hours. Then, centrifuge the plate at 112g and remove the culture medium. Dilute the environmental chemicals in islet culture medium.Add 100 micro liters of each of the chemical diluents into separate wells of the plate. Resuspend the islets in each well and incubate the plate at 37 degrees Celsius for 12 hours. Following chemical treatment of the islets, use 1x PBS to wash the samples.Then, add five millimolar N-Acetyl Cysteine or NAC to the wells, as an antioxidant to quench ROS and incubate the plates at 37 degrees Celsius for 12 hours. Centrifuge the 96 well plate at 112g then, add five micromolar solution of fluorescent probe and incubate the plate at 37 degrees Celsius for 60 minutes. Use PBS to wash the treated islet cells three times and use a microplate reader at 480 nanometers to measure the fluorescence.Finally, perform the glucose-stimulated insulin secretion, or GSIS assay, on the ROS inducing xenobiotic treated and untreated islets, to measure the xenobiotic effects on the physiological function of the pancreatic islets. Shown here, are healthy pancreatic islets, isolated from C57 Black/6 mice, that are round or oval in shape and are relatively uniformed in size. In this GSIS assay, using isolated islets stimulated with glucose to secrete insulin, a dose-dependent response was observed.When isolated islets were treated with the xenobiotics listed here, several chemicals including Atrazine, BaP, BPA and PCB caused a significant induction of reactive oxygen species. However, the addition of NAC was able to quench the production of ROS by the chemical treatments. In addition, pancreatic islets pretreated with PCB126 and BPA showed a significant decrease in insulin secretion compared to the control group, confirming the important role of ROS in causing damage to the physiological function of pancreatic beta cells which lead to T2DM.Once mastered, the isolation of the pancreas islets can be done in two hours if it is performed properly. While attempting this procedure it's important to use gradient centrifugation to purify the islets away from the other pancreatic debris. Following this procedure, other methods like glucose-stimulated insulin secretion can be performed in order to answer additional questions like effects of the chemicals on the physiological functions of the pancreas islets.After its development, this technique paved the way for researchers in the field of toxicology to explore diabetogenic potentials of the environmental chemicals in isolated mouse pancreas islet cells. After watching this video, you should have a good understanding of how to isolate the mouse pancreas islets and use these islet cells for identification.

a-murine-pancreatic-islet-cell-based-screening-for-diabetogenic

Research

JoVE Journal

Immunology and Infection

7.2K Görüntüleme.  Hunan Agriculture University. Pancreas cells are sensitive target for ROS induced damages, which is associated with development of type-2 diabetes. A pancreas cell-based assays will help identify ROS inducing chemicals. The main advantage of this technique is to use isolated pancreas islet cells for simple and effective identification of the ROS inducing chemicals which may have the added diabetogenic potentials.This method can also be used for an in vitro analysis on the effects of chemicals on a physiological fun...