Markers of Acute Toxicity in Pancreatic Beta-Cells Exposed to Lethal Doses of the Organochlorine Pollutant DDT Determined by a Proteomic Approach

Many compounds have the potential to harm pancreatic beta-cells; organochlorine pollutants belong to those compounds. In this work, we aimed to find markers of acute toxicity of p,p‘-DDT among proteins expressed in human pancreatic beta-cells NES2Y and visible at 2-D electrophoresis. We exposed NES2Y cells to a lethal dose of p,p‘-DDT (150 μM) for 24 hours and 30 hours and determined changes in protein expression using 2-D electrophoresis. We also stained the cells exposed to p,p‘-DDT to visualize the altered phenotype of the exposed cells. Among proteins with changed expression, we identified proteins involved in ER stress (GRP78, and endoplasmin), mitochondrial proteins (GRP75, ECHM, IDH3A, NDUS1, and NDUS3), proteins with potential to change the cell morphology (EFHD2, TCPA, NDRG1, and ezrin), and some other proteins (HNRPF, HNRH1, K2C8, vimentin, PBDC1, EF2, PCNA, biliverdin reductase, G3BP1, FRIL, and HSP27). These proteins can be used as markers of acute DDT toxicity.


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Many compounds have the potential to harm pancreatic beta-cells and disrupt glucose 32 homeostasis in the human organism (1). Such compounds include pharmaceuticals like pentamidine (2), 33 or fluoxetine (SSRI antidepressant) (3) or saturated fatty acids palmitate (4), or stearate (5); and 34 potentially also organochlorine pollutants, such as the now-banned pesticide DDT (6, 7). Even decades 35 after most of the countries banned its use, DDT and its metabolites persist in the environment (8,9) and 36 represent a threat to living organisms (10,11). Epidemiologic studies (12-15) showed a correlation 37 between the presence of DDT in the human organism and the incidence of diabetes mellitus.

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Nevertheless, they did not specify if DDT affected insulin production by pancreatic beta-cells or insulin 39 signaling in target tissues (7,16,17).

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In our previous study, we used 2-D electrophoresis coupled to mass spectrometry to find 41 proteins possibly involved in mechanisms mediating prolonged (1 month) effect of non-lethal doses of 42 organochlorine pollutant p,p'-DDT in pancreatic beta-cells (6,18). In our present study, we aimed to find 43 markers of acute toxicity of lethal doses of p,p'-DDT in human pancreatic beta-cells NES2Y, also 44 employing 2-D electrophoresis. To achieve that, we exposed human pancreatic beta cells NES2Y to lethal 45 doses of p,p'-DDT for 24 and 30 hours and analyzed proteins with changed expression using a proteomic 46 approach (2-D electrophoresis coupled to MALDI-TOF mass spectrometry). Moreover, we stained the cells exposed to p,p'-DDT to visualize the altered shape of those cells. Obtained data could generally help 48 us to better understand the meaning and significance of changes in the expression of respective proteins 49 found after prolonged exposure to p,p'-DDT in NES2Y cells.

Viability of cells 62
The NES2Y human pancreatic β-cell line was kindly provided by Dr. Roger F. James (Department 63 of Infection, Immunity and Inflammation, University of Leicester). We seeded the cells in 24-well plate in 64 concentration 100 000 cells / 250 μl / well. After 24 hours, we exposed the cells to various p,p'-DDT

Gel image and analysis
118 After staining, we scanned gels using a calibrated UMAX PowerLook 1120 scanner running 119 LabScan software (both GE Healthcare, Uppsala, Sweden). We used Image Master TM 2D Platinum 6.0 120 software (GE Healthcare, Uppsala, Sweden) to analyze gels. We analyzed differences between 121 corresponding spots in each set of gels (NES2Y exposed to DMSO and p,p'-DDT 150 μM). We selected 122 spots with an approximately twofold (or bigger) difference in expression between the cell lysate exposed 123 to DMSO and the cell lysate exposed to 150 μM DDT as spots with a different expression. We  139 We seeded the NES2Y cells at a density of 60 000 cells / 0.5 ml of the medium onto coverslips.

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After 24 hours, we replaced the medium with a fresh one containing 150 μM p,p'-DDT. We used cells 141 cultivated in medium without pollutants as control cells.

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After 24 hours, we discarded the medium and washed the cells three times with PBS (5-10 min).

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We analyzed statistical significance of results of 2-D electrophoresis and western blot using the 156 Student´s t-test. We analyzed statistical significance of results of flow cytometry using a one-way ANOVA 159 3. RESULTS

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For our study, we wanted to establish a concentration of p,p'-DDT that would kill approximately 162 20 % of cells after 24 hours of exposure, so the remaining 80 % of cells would provide us with enough 163 proteins for 2-D electrophoresis. Tested concentrations were chosen based on our previous results (6).

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Almost all proteins that changed their expression after 24-hour exposure changed expression 238 also after 30-hour exposure (Fig. 4   (BIEA) and elongation factor 2 (EF2), (Fig. 4, Tab. 2). The position of the BIEA/EF2 spot did not correlate 285 with predicted size for EF2, which means that the spot contained an EF2 fragment. 286

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To visualize changes in the morphology of cells exposed to p,p'-DDT (150 μM), we employed 288 immunofluorescence of cytoskeletal proteins actin and tubulin. We found no cells undergoing mitosis or 289 cytokinesis among cells exposed to p,p'-DDT, but such cells occurred among cells exposed to solvent 290 control (DMSO), (Fig. 5). Many cells exposed to p,p'-DDT had a more elongated shape than control cells.
291 Some cells exposed to p,p'-DDT were even divided into two parts connected by a long thin "neck" and 292 with a nucleus located in one of those parts.

Proteins involved in the stress of endoplasmic reticulum
307 Strong upregulation of 78 kDa glucose-regulated protein (GRP78, also known as BiP) indicated 308 the presence of the stress of endoplasmic reticulum (ER stress) in cells exposed to p,p'-DDT (23, 24).

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Another protein, CHOP mediates the ER stress-induced apoptosis (25), and its upregulation in cells 310 exposed to p,p'-DDT supports the idea that ER stress played a role in cell death induced by p,p'-DDT in 311 pancreatic beta-cells.

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The exposure to p,p'-DDT also increased the expression of endoplasmin (or heat shock protein 313 90 kDa beta member 1) in pancreatic beta-cells (see Tab 349 We also tried to identify proteins that could play a role in the altered shape of exposed cells.

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Some of the cells exposed to p,p'-DDT achieved a singular shape: a prolonged one, with a long thin 351 middle section (see Fig. 5). We have identified several proteins with a changed expression that could play 352 a role in this phenomenon.

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We found a downregulated expression of a fragment of ezrin after exposure to p,p'-DDT. Ezrin 354 binds actin filaments to the plasma membrane (34) The exposure to p,p'-DDT reduced the level of heat shock protein 27 in pancreatic beta-cells.

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HSP27 inhibits apoptosis: it prevents the activation of caspase-9 by blocking the formation of 391 apoptosome (52, 53). The downregulation of HSP27 could be the reason why HSP27 failed to prevent the 392 activation of caspases; activated caspase-9 was detected in cells exposed to DDT by western blot 393 analysis. 394

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In this study, we aimed to find markers of acute toxicity of lethal doses of p,p'-DDT in human 396 pancreatic beta-cells NES2Y employing 2D electrophoresis. Moreover, we stained the cells exposed to 397 p,p'-DDT to visualize the altered shape of these cells.

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We have found 22 proteins that can be used as markers of acute cell toxicity of p,p'-DDT in 399 pancreatic beta-cells NES2Y. Those included proteins involved in ER stress (GRP78, and endoplasmin),