Markers of acute toxicity of DDT exposure in pancreatic beta-cells determined by a proteomic approach

Nela Pavlikova; Jan Sramek; Michael Jelinek; Petr Halada; Jan Kovar

doi:10.1371/journal.pone.0229430

. 2020 Oct 26;15(10):e0229430. doi: 10.1371/journal.pone.0229430

Markers of acute toxicity of DDT exposure in pancreatic beta-cells determined by a proteomic approach

Nela Pavlikova ^1,^*, Jan Sramek ^1,^#, Michael Jelinek ^1,^#, Petr Halada ^2,^#, Jan Kovar ¹

Editor: Ch Ratnasekhar³

PMCID: PMC7588079 PMID: 33104727

Abstract

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 exposure among proteins expressed in NES2Y human pancreatic beta-cells employing 2-D electrophoresis. We exposed NES2Y cells to a high concentration (150 μM, LC₉₆ after 72 hours) of p,p‘-DDT for 24 and 30 hours and determined proteins with changed expression using 2-D electrophoresis. We have found 22 proteins that changed their expression. They included proteins involved in ER stress (GRP78, and endoplasmin), mitochondrial proteins (GRP75, ECHM, IDH3A, NDUS1, and NDUS3), proteins involved in the maintenance of 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). The proteins we have identified may serve as indicators of p,p‘-DDT toxicity in beta-cells in future studies, including long-term exposure to environmentally relevant concentrations.

Introduction

Many compounds have the potential to harm pancreatic beta-cells and disrupt glucose homeostasis in the human organism [1]. Such compounds include pharmaceuticals like pentamidine [2], or fluoxetine (SSRI antidepressant) [3] or saturated fatty acids palmitate [4], or stearate [5], and potentially also organochlorine pollutants, such as the now-banned pesticide DDT [6, 7]. Even decades after most countries banned its use, DDT and its metabolites persist in the environment [8, 9] and represent a threat to living organisms [10, 11]. Nowadays, DDT in human serum/plasma/blood commonly range between 1–500 nM [12, 13] with maxima occasionally overcoming 1 μM [14]. Epidemiologic studies [15–18] showed a correlation between DDT in the human organism and the incidence of diabetes mellitus. Nevertheless, they did not specify if DDT affected insulin production by pancreatic beta-cells or insulin signaling in target tissues [7, 19, 20].

In our previous study, we used 2-D electrophoresis coupled to mass spectrometry to find proteins possibly involved in mechanisms mediating a prolonged (1 month) effect of non-lethal concentrations of organochlorine pollutant p,p‘-DDT in pancreatic beta-cells [6, 21]. In our present study, we aimed to find proteins that change expression in NES2Y human pancreatic beta-cells when exposed to a high concentration of p,p‘-DDT) and could be detected by 2-D electrophoresis. Such proteins would represent markers of acute toxicity of DDT exposure in NES2Y human pancreatic beta-cells. They could be used to evaluate the effects of lower, environmentally more relevant concentrations of p,p‘-DDT on pancreatic beta-cells. We also aimed to discuss the possible role of the changed expression of detected proteins in the damage caused to pancreatic beta-cells by exposure to a high concentration of p,p‘-DDT. To achieve that, we exposed NES2Y human pancreatic beta cells to 150 μM concentration of p,p‘-DDT for 24 and 30 hours and analyzed proteins with a changed expression using a proteomic approach (2-D electrophoresis coupled to MALDI-TOF mass spectrometry).

Material and methods

Material

We purchased p,p’-DDT (1,1,1-Trichloro-2,2-bis(4-chlorophenyl)ethane; product number 31041-100MG) from Sigma-Aldrich (www.sigmaaldrich.com), and propidium iodide from Abcam (www.abcam.cz: ab14085). For the western blot analysis, we used the following primary and secondary antibodies: anti-cleaved caspase-6 (#9761), anti-cleaved caspase-7 (#9491), anti-cleaved caspase-8 (#9496), anti-cleaved caspase-9 (#9505) anti-cleaved and total PARP (#9542), anti-GRP78 (#3177), and anti-CHOP (#2895) from Cell Signaling Technology (www.cellsignal.com). We purchased anti-actin (clone AC-40) primary antibody from Sigma-Aldrich (www.sigmaaldrich.com: A4700), and corresponding horseradish peroxidase-conjugated secondary antibodies from Proteintech (www.ptglab.com: SA00001-2, and SA00001-1).

Cell culture

The NES2Y human pancreatic β-cell line was kindly provided by Dr. Roger F. James (Department of Infection, Immunity and Inflammation, University of Leicester) [22]. We routinely cultured NES2Y cells in a medium based on RPMI 1640, which contained penicillin (100 U/ml), streptomycin (100 μg/ml), sodium pyruvate (110 μg/ml), extra L-glutamine (300 μg/ml), HEPES (15 mM), and phenol red. We also supplemented the medium with 10% fetal bovine serum (FBS). We regularly test the cells for mycoplasma when we thaw the frozen cells. We passaged the cells twice per week. We routinely maintained cells in a humidified atmosphere of 5% CO2, in the air, at 37°C 48. For the experiments, we used cells with a passage number between 15 and 20.

Viability of cells

We seeded the cells in a 24-well plate in a concentration of 100 000 cells / 250 μl / well. After 24 hours, we exposed the cells to a scale of p,p’-DDT concentrations, i.e., 100 μM, 125 μM, 150 μM, 175 μM, 200 μM, and to DMSO (solvent control). The concentration of DMSO in the final media was 0.5%. After 24 hours, we harvested the cells by centrifugation (2000 rpm, 9 min, 4°C). We resuspended cell pellets in a staining buffer containing propidium iodide (PI; dilution 1:100, ab14085, Abcam, Cambridge, UK) and incubated them for 10 minutes at room temperature in the dark. To detect the propidium iodide signal (emission = 585 nm) in dead cells, we used a signal detector FL2 of the FACS Calibur cytometer (https://www.bdbiosciences.com). We performed this experiment for three independent sets of samples.

Western blot analysis

We seeded the cells (approximately 1 000 000 cells into a 50 mm Petri dish). After 24 hours of cultivation, we replaced the culture medium with a medium containing p,p’-DDT at a concentration of 150 μM. After 24 or 30 hours of incubation, we harvested the cells, lysed them, and quantified the protein concentration in samples employing the BCA method. For western blot analysis, we mixed 7.5 μl of samples containing 20 μg of proteins with 7.5 μl of sample loading buffer (0.125 mM Tris-HCl, pH 6.8, 10% glycerol, 4% SDS, 250 mM DTT, 0.004% bromphenol blue), heated for 7 min at 95°C and then quickly cooled on ice. We separated proteins using a 10% polyacrylamide gel (with 4% polyacrylamide stacking gel) at 30 mA. We then blotted them onto a 0.2 μm nitrocellulose transfer membrane (Protran BA83, Schleicher-Schuell, Dassel, Germany) for 3 h at 0.25 A using a Mini-Protean 3 apparatus (www.bio-rad.com). We blocked the membrane for 20 min using 5% non-fat milk in TBS (100 mM Tris-HCl, 150 mM NaCl, pH 7.5) and then washed the membrane with 0.1% Tween-20/TBS three times. We incubated the washed membrane with respective primary antibodies in 0.1% Tween-20/TBS containing 1% non-fat milk overnight at 4°C [23]. We diluted all primary antibodies 1:1000. After the incubation with the primary antibody, we washed the membrane and incubated it with the corresponding horseradish peroxidase-conjugated secondary antibody for 2 h. After washing, we detected a secondary antibody signal by enhanced chemiluminescence employing a Carestream Gel Logic 4000 PRO Imaging System equipped with Carestream Molecular Imaging Software (www.carestream.com). We employed Image Master™ 2D Platinum 6.0 software (www.gehealthcare.com) to obtain data for densitometric analyses.

2-D electrophoresis and subsequent gel analysis

We run 2-D electrophoresis for at least three independent sets of samples.

Exposure to p,p‘-DDT

For the experiment, we seeded 5 000 000 NES2Y cells into a medium-sized flask. After 24 hours of cultivation, we replaced the medium with a medium containing 150 μM p,p’-DDT, or DMSO. After 24 or 30 hours, we harvested the cells. The concentration of DMSO (solvent control) in the medium was 0.5%.

Preparation of samples for 2-D electrophoresis

We trypsinized the cells, washed them 3x times with ice-cold PBS, and resuspended them in Protein Extraction Buffer-V ((GE Healthcare, www.gehealthcare.com)) (urea, thiourea, CHAPS) containing 2% of Protease Inhibitor Mix ((GE Healthcare, www.gehealthcare.com)). We purified all samples using a 2-D Clean-Up Kit (GE Healthcare, www.gehealthcare.com) following the manufacturer´s instructions. Consequently, we dissolved the proteins in Protein Extraction Buffer-V suitable for isoelectric focusing. We used the 2-D Quant Kit ((GE Healthcare, www.gehealthcare.com) weden) to determine protein concentrations.

2-D electrophoresis: Isoelectric focusing

We used an IPGphor focusing unit (GE Healthcare, Uppsala, Sweden) for the isoelectric focusing of samples loaded on 7 cm pH 4–7 Immobiline DryStrips (GE Healthcare, Uppsala, Sweden). We rehydrated each strip for 48 hours, as described previously [24]. After rehydration, pH 4–7 strips were focused with a limited current of 50 μA/strip at 20°C using the following conditions: gradient 0→150 V for 2 h, 150 V for 1 h, gradient 150→300 V for 1 h, 300 V for 2 h, gradient 300→1200 V for 3 h, 1200 V for 1 h, gradient 1200→3500 V for 5 h, and 3500 V for 5.5 h.

2-D electrophoresis: SDS-PAGE

Following isoelectric focusing, we equilibrated the strips for 20 min in an equilibration buffer [25] containing 2% DTT. Then, we changed the buffer for a new one containing 2.5% iodoacetamide instead of 2% DTT and equilibrated strips for another 20 min. After equilibration, we placed the strips on top of the gels, sealed them using 0.5% agarose containing bromphenol blue, and run SDS-PAGE. We employed a Mini-PROTEAN Tetra cell (Bio-Rad, www.bio-rad.com) device for the second dimension and used 10% polyacrylamide gels with 4% stacking gels for separation. We run gels at a constant voltage of 50 V until the blue line reached the bottom of the gels (approximately 3 h). After running the second dimension, we washed each gel 3 x 5 min in distilled water and stained in 50 ml of colloidal Coomassie brilliant blue (CBB) solution [26] overnight.

Gel image and analysis

After staining, we scanned gels using a calibrated UMAX PowerLook 1120 scanner running LabScan software (GE Healthcare, www.gehealthcare.com). We used Image Master^TM 2D Platinum 6.0 software (GE Healthcare, www.gehealthcare.com) to analyze the gels. We analyzed differences between corresponding spots in each set of gels (NES2Y exposed to DMSO and p,p‘-DDT 150 μM). We selected spots with an approximately twofold (or bigger) difference in expression between the cell lysate exposed to DMSO and the cell lysate exposed to 150 μM DDT as spots with a different expression. We determined the statistical significance of changes in protein expression using the student´s t-test. Spots with significantly different intensities were cut and sent for MS analysis.

Enzymatic digestion, MALDI-TOF mass spectrometry, and protein identification

After destaining CBB-protein spots, we incubated them overnight at 37 ᵒC in a buffer containing 25 mM 4-ethyl morpholine acetate, 5% acetonitrile, and trypsin (100 ng; Promega). We mixed the resulting peptides with a MALDI matrix consisting of an aqueous 50% acetonitrile/0.1% TFA solution of α-cyano-4-hydroxycinnamic acid (5 mg/ml; Sigma-Aldrich, www.sigmaaldrich.com). We measured mass spectra using an Ultraflex III MALDI-TOF (Bruker Daltonics, Bremen, Germany) in a mass range of 700–4000 Da calibrated externally using a mixture of PepMix II standard (Bruker Daltonics). We searched both MS and MS/MS data against the SwissProt 2017_03 database subset of human proteins using the in-house MASCOT software with the following settings: peptide tolerance of 30 ppm, missed cleavage site set to one, variable carbamidomethylation of cysteine, and oxidation of methionine. We considered proteins with MOWSE scores over the threshold of 54 (calculated for the settings used) as identified. We confirmed the identity of each protein candidate using MS/MS analysis.

Confocal microscopy

We seeded the NES2Y cells at a density of 60 000 cells / 0.5 ml of the medium onto coverslips. After 24 hours, we replaced the medium with a fresh one containing 150 μM p,p’-DDT. We used cells cultivated in a medium without pollutants as control cells.

After 24 hours, we discarded the medium and washed the cells three times with PBS (5‒10 min). Then, we fixed cells using 4% paraformaldehyde for 20 min, washed them by PBS, permeabilized them with 0.3% Triton X-100 for 10 min, and washed them again. After that, we blocked cells with 1% BSA for 60 minutes and stained them with the primary anti-tubulin antibody (ab6046 from Abcam, Cambridge, UK) diluted 1:300 in 1% BSA, at 4°C overnight. After washing the cells with PBS, we incubated the cells with a secondary goat anti-rabbit antibody (ab150077, from Abcam) diluted 1:300, in the dark at room temperature for 2 hours. To complete tubulin staining, we washed the cells again with PBS. Then, we stained actin in the cells by rhodamine-phalloidin (R415, from Invitrogen) diluted according to the manufacturer’s instruction for 20 min. The unbound staining solution was removed by PBS washing. Finally, we transferred the cells onto a droplet of Vectashield® Vibrance™ Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA) and sealed them. Samples were analyzed using a Leica TCS SP5 confocal microscope (Bannockburn, IL, USA).

Statistical analysis

We analyzed the statistical significance of the results of 2-D electrophoresis and western blot using a student´s t-test. We analyzed the statistical significance of the results of flow cytometry using a one-way ANOVA Dunnett´s test (SigmaPlot 14 Software). We considered the differences with p < 0.05 as statistically significant.

Results

Effect of various concentrations of p,p‘-DDT on the viability of NES2Y cells

For our study, we wanted to establish a concentration of p,p‘-DDT that would kill approximately 20% of cells after 24 hours of exposure, so the remaining 80% of cells would provide us with enough proteins for 2-D electrophoresis. The tested concentrations were chosen based on our previous results [6]. After 24 hours of exposure, the two lower concentrations of p,p‘-DDT (100 μM and 125 μM) caused only a non-significant decrease in cell viability when compared with the control (Fig 1). The three higher concentrations (150 μM, 175 μM, and 200 μM) caused a significant reduction of viability of cells (Fig 1), (p = 0.11 for 150 μM, p < 0.001 for 175 μM, p < 0.001 for 200 μM). For the planned experiments, we chose a concentration of 150 μM that reduced cell viability to 82% of the control after 24 hours of exposure. Further testing established a 150 μM concentration of p,p‘-DDT as LC₉₆ (lethal concentration) for NES2Y after 72 hours (see S1 Fig in S1 File).

Fig 1 — We determined the number of viable cells using flow cytometry after staining with propidium iodide. We used cells exposed to DMSO (final concentration of 0.5%) as solvent control. The graph represents the mean of results from 3 independent experiments. * means a statistically significant difference (p < 0.05) when compared to the solvent control (one-way ANOVA, Dunnett’s test).

Effect of p,p‘-DDT on apoptotic and ER stress markers

In order to confirm that p,p‘-DDT induced apoptotic cell death in NES2Y cells, we tested the presence of the main markers of apoptosis, i.e., activated caspase-8, -9, -6, -7 and cleaved PARP (poly (ADP-ribose) polymerase)—a substrate of caspase-7, (Fig 2, S4–S10 Figs in S1 File). We did not detect caspase-3 since we know that it is not functional in NES2Y cells [23]. The level of cleaved caspase-8 increased to 235% of the control (p = 0.039) after 24-hour exposure, and to 391% of the control (p = 0.015) after 30-hour exposure (Fig 3). The level of cleaved caspase-9 also increased after both 24-hour exposure (581% of the control, p = 0.018) and 30-hour exposure (423% of the control, p = 0.004), (Fig 3). The level of cleaved caspase-6 increased to 555% of the control (p = 0.042) after 24 hours of exposure, and to 635% of the control (p = 3.77 x 10⁻⁴) after 30-hour exposure (Fig 3). The level of cleaved caspase-7 represented the highest increase; it reached 1434% of the control (p = 0.003) after 24-hour exposure, and after 30-hour exposure, it reached 1820% (p = 0.008) of the control. The level of cleaved PARP reached 240% of the control after 24-hour exposure (p = 0.022), and after 30-hour exposure, it reached 226% of the control (p = 0.003), (Fig 3).

Fig 2 — The picture shows the expression of chosen proteins in NES2Y cells exposed to DMSO as a solvent control (con) and 150 μM p,*p‘-*DDT for 24 and 30 hours. We used actin as a loading control. CHOP means C/EBP homologous protein, GRP78 means 78kDa glucose-regulated protein, and PARP means poly (ADP-ribose) polymerase.

Fig 3 — Columns represent mean values ± SEM of protein levels from 3 independent sets of experiments. CHOP means C/EBP homologous *protein*, *GRP78 means 78kDa glucose-regulated protein*, and PARP means poly (ADP-ribose) polymerase. *, **, *** means a statistically significant difference with p<0.05, p<0.01, and p<0.001, respectively, when compared to the control at the relevant time (analyzed by the student’s t-test).

To confirm that p,p‘-DDT induces ER stress in NES2Y cells, we have tested the effect of p,p‘-DDT on the expression of proteins BiP and CHOP—the main markers of ER stress. The level of CHOP increased to 1599% of the control (p = 0.021) after 24-hour exposure, and to 893% of the control (p = 0.002) after 30-hour exposure. The expression of GRP78 was upregulated to 1213% of the control (p = 0.002) after 24-hour exposure, and to 1875% of the control (p = 0.034) after 30-hour exposure (Figs 2 and 3, S2 and S3 Figs in S1 File).

Proteins with changed expression after 24 hours of exposure to p,p‘-DDT

The 24-hour exposure to 150 μM p,p‘-DDT changed the expression of 10 spots; 4 spots were upregulated, and 6 spots were downregulated when compared with control (Fig 4 and S11 Fig in S1 File, Table 1). 78 kDa glucose-regulated protein (GRP78, also known as BiP) was represented by three upregulated spots (GRP78*, GRP78**, GRP78***); spot GRP78* was upregulated to 1610% of the control (p = 0.045), spot GRP78** to 240% of the control (p = 0.003), and spot GRP78*** to 285% of the control (p = 0.044). Another glucose-regulated protein, 75 kDa glucose-regulated protein (GRP75, also known as mortalin), was found upregulated too (spot GRP75, expression increased to 1083%, p = 3.38 x 10⁻⁴).

Fig 4 — Part A shows spots/proteins that changed their expression after both 24-hour and 30-hour exposure. *, **, *** mark different spots identified as the same protein. Part B shows spots/proteins that changed their expression only after 30-hour exposure. *, ** mark different spots identified as the same protein. BIEA/EF2, NDUS3/HSP27, and PBDC1/PCNA represent spots that contained two different proteins. For the full names of detected proteins, see Tables 1 and 2.

Table 1. Differentially expressed proteins after both 24-hour and 30-hour exposure to 150 μM p,p‘-DDT identified in 2-DE experiments using three independent sets of samples.

Fold change—24 h	Fold change—30 h	Protein name	DTB No.	No. peptides	Coverage [%]	MS/MS confirmation	MW protein	pI
0.44	0.39	ECHM ↓ (enoyl-CoA hydratase, mitochondrial)	ECHM_HUMAN	5	25	ASGANFEYIIAEKR AQFAQPEILIGTIPGAGGTQR	31	8.3
10.83	8.57	GRP75 ↑ (75 kDa glucose-regulated protein)	GRP75_HUMAN	10	18	VQQTVQDLFGR NAVITVPAYFNDSQR STNGDTFLGGEDFDQALLR	74	5.9
16.10	- - -	GRP78^* ↑ (78 kDa glucose-regulated protein)	GRP78_HUMAN	13	22	ITPSYVAFTPEGER VTHAVVTVPAYFNDAQR IEIESFYEGEDFSETLTR	72	5.1
2.40	2.42	GRP78^** ↑ (78 kDa glucose-regulated protein)	GRP78_HUMAN	15	27	ITPSYVAFTPEGER IEIESFYEGEDFSETLTR	72	5.1
2.85	1.97	GRP78^*** ↑ (78 kDa glucose-regulated protein)	GRP78_HUMAN	14	24	ITPSYVAFTPEGER VTHAVVTVPAYFNDAQR IEIESFYEGEDFSETLTR	72	5.1
0.48	0.50	HNRPF ↓ (heterogeneous nuclear ribonucleoprotein F)	HNRPF_HUMAN	6	17	HSGPNSADSANDGFVR ATENDIYNFFSPLNPVR	46	5.4
0.54	0.42	HSP27 ↓ (heat shock protein 27)	HSPB1_HUMAN	9	43	RVPFSLLR LFDQAFGLPR LATQSNEITIPVTFESR	23	6.0
0.41	0.43	IDH3A ↓ (isocitrate dehydrogenase [NAD] 3 subunit alpha, mitochondrial)	IDH3A_HUMAN	5	17	IAEFAFEYAR TIPIDGNFFTYTR	40	6.5
0.41	0.26	K2C8 ↓ (keratin, type II cytoskeletal 8)	K2C8_HUMAN	7	15	LEGLTDEINFLR ASLEAAIADAEQRGELAIK	54	5.5
0.51	0.36	VIME ↓ (vimentin)	VIME_HUMAN	16	43	ISLPLPNFSSLNLR EMEENFAVEAANYQDTIGR QVQSLTCEVDALKGTNESLER	54	5.1

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The table covers the proteins that changed expression after both 24 hours and 30 hours of exposure. The table includes fold change after 24 hours and 30 hours, protein name, SwissProt database number, the number of peptides matched to the identified protein, sequence coverage, peptide sequences confirmed by MS/MS, theoretical molecular weight, and pI of the protein.

*, **, *** mark different spots identified as the same protein.

The 24-hour exposure to 150 μM p,p‘-DDT downregulated the expression of enoyl-CoA hydratase mitochondrial to 44% of the control (spot ECHM, p = 0.006), the expression of vimentin to 51% of the control (spot VIME, p = 4.82 x 10⁻⁵), the expression of heat shock protein 27 to 54% of the control (spot HSP27, p = 0.026), the expression of isocitrate dehydrogenase [NAD] subunit alpha mitochondrial to 41% of the control (spot IDH3A, p = 3.84 x 10⁻⁵), the expression of keratin, type II cytoskeletal 8 to 41% of the control (spot K2C8, p = 3.09 x 10⁻⁸), and the expression of heterogeneous nuclear ribonucleoprotein F to 48% of the control (spot HNRPF, p = 0.021) (Fig 4, Table 1).

Proteins with changed expression after 30 hours of exposure to p,p‘-DDT

Almost all proteins that changed their expression after 24-hour exposure also changed expression after 30-hour exposure (Fig 4 and S12 Fig in S1 File, Table 1). The only exception was spot GRP78* (78 kDa glucose-regulated protein). The spot was smeared and blended with surrounding spots; therefore, it could not be analyzed. The 30-hour exposure to 150 μM p,p‘-DDT upregulated the expression of 78 kDa glucose-regulated protein to 242% of the control (spot GRP78**, p = 7.59 x 10⁻⁸, and to 197% of the control (spot GRP78***, p = 1.88 x 10⁻⁵), and the expression of 75 kDa glucose-regulated protein to 857% of the control (spot GRP75, p = 7.75 x 10⁻⁴).

The 30-hour exposure to 150 μM p,p‘-DDT downregulated the expression of more proteins than were upregulated. The expression of enoyl-CoA hydratase mitochondrial (spot ECHM, p = 3.91 x 10⁻⁵) decreased to 39% of the control, vimentin (spot VIME, p = 2.97 x 10⁻⁴) to 36% of the control, heat shock protein 27 (spot HSP27, p = 8.34 x 10⁻⁵) to 42% of the control, isocitrate dehydrogenase [NAD] 3 subunit alpha mitochondrial (spot IDH3A, p = 1.86 x 10⁻⁴) to 43% of the control, keratin type II cytoskeletal 8 (spot K2C8, p = 8.94 x 10⁻⁶) to 26% of the control, and heterogeneous nuclear ribonucleoprotein F (spot HNRPF, p = 1.11 x 10⁻⁴) to 50% of the control. The changes of expression remained quite similar after both exposure times in most of the proteins (see Table 1).

Besides 10 proteins with changed expression detected after both 24-hour exposure and 30-hour exposure, there were also 14 more spots with changed expression detected only after 30-hour exposure (Fig 4, Table 2). Those 14 spots represented 14 proteins; nevertheless, some spots contained two proteins, and some proteins occurred as two different spots. We found 3 proteins upregulated: N-myc downregulated gene 1 protein (spot NDRG1, p = 0.019) to 312% of the control, EF-hand domain-containing protein D2 (spot EFHD2, p = 3.01 x 10⁻³) to 274% of the control, and one of the two spots representing endoplasmin (spot ENPL*, p = 1.31 x 10⁻⁴) to 199% of the control (Fig 4, Table 2).

Table 2. Differentially expressed proteins identified in 2-DE experiments.

Fold change—30 h	Protein name	DTB No.	No. peptides	Coverage [%]	MS/MS confirmation	MW protein	pI
0.43	BIEA ↓ (biliverdin reductase A),	BIEA_HUMAN	4	17	FGFPAFSGISR GSLLFTAGPLEEER	33	6.6
0.43	and EF2 ↓ (elongation factor 2)	EF2_HUMAN	3	4	VNFTVDQIR ALLELQLEPEELYQTFQR	95	6.4
2.74	EFHD2 ↑ (EF-hand domain-containing protein D2)	EFHD2_HUMAN	6	27	FEEEIKAEQEER VFNPYTEFKEFSR	27	5.2
1.99	ENPL^* ↑ (endoplasmin)	ENPL_HUMAN	13	18	SILFVPTSAPR FQSSHHPTDITSLDQYVER	92	4.8
0.51	ENPL^** ↓ (endoplasmin)	ENPL_HUMAN	8	10	FAFQAEVNR SILFVPTSAPR	92	4.8
0.50	EZRI ↓ (ezrin)	EZRI_HUMAN	8	11	QLLTLSSELSQAR RKEDEVEEWQHR	69	5.9
0.49	FRIL ↓ (ferritin light chain)	FRIL_HUMAN	6	40	DDVALEGVSHFFR LNQALLDLHALGSAR LGGPEAGLGEYLFER KLNQALLDLHALGSAR	20	5.5
0.53	G3BP1 ↓ (Ras GTPase-activating protein-binding protein 1)	G3BP1_HUMAN	6	17	FYVHNDIFR	52	5.4
0.53	HNRH1^* ↓ (heterogeneous nuclear ribonucleoprotein H)	HNRH1_HUMAN	7	24	GLPWSCSADEVQR HTGPNSPDTANDGFVR ATENDIYNFFSPLNPVR EGRPSGEAFVELESEDEVK	49	5.9
0.33	HNRH1^** ↓ (heterogeneous nuclear ribonucleoprotein H)	HNRH1_HUMAN	4	13	ATENDIYNFFSPLNPVR	49	5.9
3.12	NDRG1 ↑ (N-myc downstream regulated 1)	NDRG1_HUMAN	3	14	SIIGMoxGTGAGAYILTR GNRPVILTYHDIGMoxNHK	43	5.5
0.53	NDUS3 ↓ (NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, mitochondrial),	NDUS3_HUMAN	9	31	VVAEPVELAQEFR	30	7.0
0.53	and HSP27 ↓ (heat shock protein 27)	HSPB1_HUMAN	6	31	LFDQAFGLPR LATQSNEITIPVTFESR	23	6.0
0.25	NDUS1 ↓ (NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial)	NDUS1_HUMAN	10	15	FEAPLFNAR	79	5.9
0.56	PBDC1 ↓ (polysaccharide biosynthesis domain containing 1),	PBDC1_HUMAN	5	25	IQFFAIEIAR FNGIVEDFNYGTLLR	26	4.7
0.56	and PCNA ↓ (proliferating cell nuclear antigen)	PCNA_HUMAN	6	26	SEGFDTYR CAGNEDIITLR	29	4.6
0.40	TCPA ↓ (T-complex protein 1 subunit alpha)	TCPA_HUMAN	6	12	EQLAIAEFAR	60	5.8

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The table covers proteins that changed expression ONLY after 30 hours of exposure. Some spots contained two different proteins. The table includes fold change after 30 hours, protein name, SwissProt database number, the number of peptides matched to the identified protein, sequence coverage, peptide sequences confirmed by MS/MS, theoretical molecular weight, and pI of the protein.

*, **, *** mark different spots identified as the same protein.

Downregulated proteins included Ras GTPase-activating protein-binding 1 (spot G3BP1, p = 1.57 x 10⁻⁴) with the expression downregulated to 53% of the control, NADH-ubiquinone oxidoreductase 75 kDa subunit mitochondrial (spot NDUS1, p = 1.71 x 10⁻⁸) with the expression downregulated to 25% of the control, and T-complex protein 1 subunit alpha (spot TCPA, p = 7.28 x 10⁻⁶) with the expression downregulated to 40% of the control. The expression of ferritin light chain (spot FRIL, p = 3.23 x 10⁻⁵) was downregulated to 49% of the control, and the expression of ezrin (spot EZRI, p = 1.11 x 10⁻⁴) to 50% of the control. The position of spot EZRI on the 2-D gel did not correlate with its predicted molecular; spot EZRI represents a fragment of ezrin. We identified two other spots as heterogeneous nuclear ribonucleoprotein H (HNRH1): expression of HNRH1** (a fragment of a protein) decreased to 33% of the control (p = 2.50 x 10⁻⁴), and expression of HNRH1* (a whole form of protein) to 53% of the control (p = 1.61 x 10⁻⁵). Expression of endoplasmin fragment (spot ENPL**, p = 3.69 x 10⁻⁵) decreased to 51% of the control (Fig 4, Table 2).

Several spots with downregulated expressions contained two proteins each. The spot with expression downregulated to 53% of the control (spot NDUS3/HSP27, p = 7.28 x 10⁻⁴) contained both NADH dehydrogenase [ubiquinone] iron-sulfur protein 3 mitochondrial and heat shock protein 27. The spot with the expression downregulated to 56% of the control (spot PBDC1/PCNA, p = 1.37 x 10⁻³) contained protein PBDC1 and proliferating cell nuclear antigen (PCNA). Furthermore, the spot with the expression downregulated to 43% of the control (spot BIEA/EF2, p = 3.11 x 10⁻⁴) contained both biliverdin reductase (BIEA) and elongation factor 2 (EF2) (Fig 4, Table 2). The position of the BIEA/EF2 spot did not correlate with the predicted size for EF2, which means that the spot contained an EF2 fragment.

Morphology of the cells exposed to p,p‘-DDT

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

Fig 5 — Cells incubated without p,*p‘-*DDT represented control cells. After 24 hours of incubation, we stained actin (red), tubulin (green), and nuclei (blue) of the cells. The figure shows representative pictures.

Discussion

In this study, we used 2-D electrophoresis coupled to mass spectrometry to find markers of acute toxicity of DDT exposure in NES2Y human pancreatic beta-cells. Together, 2-D electrophoresis revealed 22 proteins with altered expression. We have sorted these proteins into groups based on their function and tried to evaluate the meaning and importance of changes in the expression of individual proteins.

Proteins involved in the stress of endoplasmic reticulum

Strong upregulation of 78 kDa glucose-regulated protein (GRP78, also known as BiP) indicated the presence of the stress of endoplasmic reticulum (ER stress) in cells exposed to p,p‘-DDT [27, 28]. Another protein, CHOP, mediates the ER stress-induced apoptosis [29], and its upregulation in cells exposed to p,p‘-DDT supports the idea that ER stress played a role in cell death induced by p,p‘-DDT in pancreatic beta-cells.

The exposure to p,p‘-DDT also increased the expression of endoplasmin (or heat shock protein 90 kDa beta member 1) in pancreatic beta-cells (see Table 2). Endoplasmin plays a vital role in cell survival under ER stress [30–32]. Therefore, its upregulation supports the idea that exposure to p,p‘-DDT induces ER stress in beta-cells. Another spot identified as endoplasmin had a smaller size than predicted, and its expression was downregulated. Endoplasmin is a dimer [31], and theoretically, the downregulated spot could represent the endoplasmin monomer.

Mitochondrial proteins

We found four mitochondrial proteins downregulated in pancreatic beta-cells after exposure to p,p‘-DDT: ECHM, IDH3A, NDUS1, and NDUS3. Enoyl-CoA hydratase (ECHM) participates in β-oxidation, isocitrate dehydrogenase [NAD] 3 subunit alpha (IDH3A) in the citric acid cycle, NADH-ubiquinone oxidoreductase 75 kDa subunit (NDUS1), and NADH dehydrogenase [ubiquinone] iron-sulfur protein 3 (NDUS3) belong to the complex I of the respiratory chain. The 24-hour exposure to p,p‘-DDT reduced the expression of ECHM and IDH3; the 30-hour exposure followed that trend. The 30-hour exposure to p,p‘-DDT also reduced the expression of both NDUS1 and NDUS3. We hypothesize that DDT exposure affected first the metabolic pathways that precede the respiratory chain and later the respiratory chain itself.

Another protein with changed expression, 75 kDa glucose-regulated protein (GRP75), is a molecular chaperone localized preferentially (but not exclusively) in mitochondria. This chaperone interacts with many proteins, including NDUS3 mentioned above [33]. In mitochondria, GRP75 helps to maintain mitochondrial shape and function [34, 35]. The exposure to p,p‘-DDT strongly upregulated expression of GRP75 (see Tables 1 and 2), which may suggest that, after the exposure to DDT, mitochondria needed full support to maintain their functionality.

Heterogeneous nuclear ribonucleoproteins

We found two members of the heterogeneous nuclear ribonucleoprotein family downregulated after exposure to p,p‘-DDT: heterogeneous nuclear ribonucleoprotein F (HNRPF) and heterogeneous nuclear ribonucleoprotein H (HNRH1). HNRPF and HNRH1 bind to the p53 transcript and protect it against degradation [36]; therefore, their downregulation could play a role in apoptosis initiation.

Cytoskeletal proteins

In 2-D electrophoresis, researchers usually see the presence of fragments of cytoskeletal proteins as a sign of cell degradation [37]. Nevertheless, we found only two cytoskeletal proteins with altered expression, i.e., vimentin (VIME) and keratin type II cytoskeletal (K2C8). Both spots were at positions corresponding to their predicted size, which means that they were full-size proteins and not fragments. It is interesting to compare these data to our previous results, where we exposed NES2Y work to a non-lethal concentration of p,p‘-DDT for 1 month and found changed expression of several cytoskeletal proteins and their fragments [6]. We hypothesize that the presence of fragments of cytoskeletal proteins does not necessarily correlate with the presence of cell death.

Proteins involved in the maintenance of the cell morphology

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

We found a downregulated expression of a fragment of ezrin after exposure to p,p‘-DDT. Ezrin binds actin filaments to the plasma membrane [38], but it is difficult to evaluate the meaning of the downregulation of its fragment. EF-hand domain-containing protein D2 (EFHD2), upregulated after exposure to p,p‘-DDT, represents another protein with an altered expression that can bind actin [39]. EFHD2 participates in forming structures associated with actin, such as lamellipodia and membrane ruffles [40]. T-complex protein 1 subunit alpha (TCPA), downregulated after exposure to p,p‘-DDT, is a part of a chaperonin called TCC (chaperonin containing t-complex polypeptide 1) responsible for the proper folding of actin and tubulin [41, 42]. N-myc downstream-regulated gene 1 (NDRG1) expression can be induced by DNA damage [43], Fe deprivation [44], Ca²⁺ deprivation, or hypoxia [45]. Interestingly, NDRG1 overexpression led to an altered shape of prostate cancer cells DU145 [46]. Therefore, increased expression of NDRG1 could also play a role in the altered shape of NES2Y cells exposed to p,p‘-DDT. So could the changed protein expression of EFHD2 and TCPA.

Proteins involved in processes connected with DNA or RNA

The exposure to p,p‘-DDT downregulated the expression of three proteins connected with transcription, translation, or replication: PBDC1, EF2, and PCNA. Polysaccharide biosynthesis domain containing 1 protein (PBDC1, also known as CXorf26) is an unexplored protein with predicted function connected with RNA polymerase II and ribosomes [47]. Elongation factor 2 (EF2) regulates peptide chain elongation on the ribosome during translation [48], but we found only a fragment of this protein downregulated. Proliferating cell nuclear antigen (PCNA) recruits participant proteins to the replication fork [49]. Downregulation of PBDC1 due to p,p‘-DDT treatment could, therefore, negatively affect protein synthesis; downregulation of PCNA could negatively affect replication in cells. It is difficult to predict the effect of downregulation of only a fragment of EF2 on protein synthesis.

Proteins involved in oxidative stress

The exposure to p,p‘-DDT downregulated the expression of biliverdin reductase A (BIEA). The bilirubin/biliverdin system represents powerful protection against oxidative stress [50]. Recently, Lee and coworkers [51] described that the forced upregulation of BIEA protected INS1E rat pancreatic beta-cells from ER stress and oxidative stress. Pancreatic beta-cells have a low antioxidative defense [52]. BIEA, with better quenching ability than glutathione [51], can represent an effective defense mechanism against ROS and ER stress in pancreatic beta-cells.

The exposure to p,p‘-DDT reduced the expression of Ras GTPase-activating protein-binding protein 1 (G3BP1). G3BP1 controls an antioxidative ability of stress granules under stress conditions [53, 54]. The exposure to p,p‘-DDT also reduced the expression of ferritin light chain (FRIL). The downregulation of FRIL can indicate that p,p‘-DDT-treated cells are less able to quench ROS [55]. Together with the downregulation of biliverdin reductase A, these changes in protein expression suggest that after 30-hour exposure to p,p‘-DDT, beta-cells lose their defense against oxidative stress.

Heat shock protein 27

The exposure to p,p‘-DDT reduced the level of heat shock protein 27 in pancreatic beta-cells. HSP27 inhibits apoptosis: it prevents the activation of caspase-9 by blocking the formation of apoptosome [56, 57]. The downregulation of HSP27 could be the reason why HSP27 failed to prevent the activation of caspases; activated caspase-9 was detected in cells exposed to DDT by western blot analysis.

Conclusions

In this study, we aimed to find markers of acute toxicity of a high concentration of p,p‘-DDT in NES2Y human pancreatic beta-cells employing 2D electrophoresis.

We have found 22 proteins that can be used as markers of acute toxicity of p,p‘-DDT exposure in NES2Y pancreatic beta-cells. Those included proteins involved in ER stress (GRP78, and endoplasmin), mitochondrial proteins (GRP75, ECHM, IDH3A, NDUS1, and NDUS3), heterogeneous nuclear ribonucleoproteins (HNRPF, and HNRH1), cytoskeletal proteins (K2C8, and vimentin), proteins involved in the maintenance of the cell morphology (EFHD2, TCPA, NDRG1, and ezrin), proteins involved in processes connected with DNA or RNA (PBDC1, EF2, and PCNA), proteins involved in oxidative stress (BIEA, and G3BP1, and FRIL), and heat shock protein 27. The proteins we have identified may serve as indicators of p,p‘-DDT toxicity in beta-cells in future studies, including long-term exposure to environmentally relevant concentrations. Also, we have found that p,p‘-DDT-induced cell death is apoptotic cell death and that a high concentration of p,p‘-DDT induces ER stress in NES2Y cells.

Supporting information

S1 File

(PDF)

Click here for additional data file.^{(821KB, pdf)}

Data Availability

All relevant data are within the manuscript and its Supporting Information file.

Funding Statement

This work was supported by the research projects UNCE 204015 and PROGRES Q36 of Charles University in Prague, Czech Republic, and by the project BIOCEV CZ.1.05/1.1.00/02.0109 from the European Regional Development Fund. The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0229430.r001

Decision Letter 0

Ch Ratnasekhar

29 Jul 2020

PONE-D-20-03380

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

PLOS ONE

Dear Dr. Nela,

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Kind regards,

Ch Ratnasekhar, Ph.D.

Academic Editor

PLOS ONE

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[This work was supported by research projects UNCE 204015 and PROGRES Q36 of Charles University in Prague, Czech Republic, and by the project BIOCEV CZ.1.05/1.1.00/02.0109 from the European Regional Development Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.]

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Comments to the Author

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Reviewer #1: No

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

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Reviewer #1: The manuscript “Markers of Acute Toxicity in Pancreatic Beta-Cells Exposed to Lethal Doses of the Organochlorine Pollutant DDT Determined by a Proteomic Approach” by Pavlíková et al, employs various biochemical techniques to determine the acute markers of acute toxicity in vitro.

Few are the points that must be considered to improve the overall quality of the manuscript

The term ‘lethal dose’ is incorrect to use since the dose chosen for any toxicant is always sublethal.

The authors have concluded that the detected proteins serve as the markers for acute DDT toxicity. However, they are not the exclusive markers for DDT toxicity. Maybe the authors can modify the statement or direct their conclusion towards the pathways affected following DDT exposure.

It would be advisable to add few inhibitors experiments against the targets to ascertain if the changes are solely due to DDT exposure or it’s a general response.

Are the effects observed mediated through direct binding of DDT or through its metabolites?

A thorough Grammatical proofread is required to improve the flow for better understanding

Abstract must be rewritten to highlight the rationale, potential results and findings in a precise manner

The technique however is a valuable part of the manuscript to achieve the objectives. What is the importance of acute time points and how the data obtained in the present study will be utilized for translatable value?

Please mention the culture conditions of the NES2Y cell, medium used, passage number etc.

Line 68-69 “We detected the fluorescence of cells using a FACS Calibur cytometer (Becton Dickinson, San Jose, CA, USA) channel FL2”. What does this sentence imply?

Please try to be consistent while mentioning the cell number 100 000 cells vs 1×106 cells

Please restructure the material and method section to contain the essential information like

1. What concentration of DMSO was used as solvent control?

2. Why did the authors select the time points - 24h and 30 h? Will it be feasible to assess the cellular activity at initial time points of 12 and 18 h?

Western blotting details should be explained properly in terms of methodology, antibodies and concentration.

Statistical analysis, please mention significance level.

Is there any specific mechanism the authors were interested to explore? Mentioning the requirement to kill 20% cells for 2D purpose does not make sense rationally since this percentage of cell mortality is obvious at sublethal doses of any toxicant.

Study rationale needs to be improved as in what are authors interested in and why? Is it the cell viability or the mechanism or both? This point must be clearly stated.

Please avoid repetition of sentences or same information throughout the manuscript

Reviewer #2: The manuscript by Pavlikova et al. provides an interesting perspective that highlights the acute toxic effect of DDT on pancreatic beta cells NES2Y. In the context of environmental pollutants induced diabetes, this study seems relevant.

Major concerns

1-The author should mention the doses corresponded to the comparable levels that were toxic in humans in relation to diabetes and pancreatic beta cells. If the author provides some in-vivo references or data supporting their results to show the translatability of their in-vitro findings will definitely enhance the impact of this study.

2- In figure 2, the western blots for CHOP, GRP78, cleaved caspase-9, cleaved caspase 8, cleaved caspase -7 the band thickness does not match with the densitometric quantification in figure 3. In the control groups, the bands have completely vanished.

Minor Concerns

1-The author should mention the cell viability at 30 hours.

2- In result 3.1, the author has used neutral red assay for cell viability while in figure 1, it is mentioned as propidium iodide.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: Yes: Nagendra Kumar Rai

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Oct 26;15(10):e0229430. doi: 10.1371/journal.pone.0229430.r002

Author response to Decision Letter 0

25 Sep 2020

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

We have made the required changes in our manuscript.

*************************************************************************************

Please provide information about any quality control testing procedures (authentication, characterisation, and mycoplasma testing) performed on the cell line used in this work. For more information, please see http://journals.plos.org/plosone/s/submission-guidelines#loc-cell-lines.

We have added this information to the section Cell culture.

*************************************************************************************

We note that you have included the phrase "data not shown" in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

We have placed the mentioned data to the Supplementary Information file and put a reference into the main text.

*************************************************************************************

PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission's figures or Supporting Information files. This policy and the journal's other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

We have added this information to the Supporting information file. Nevertheless, the gel pictures are partially cropped by the software we use for taking the pictures. In the case of WB membranes, we cut them into strips and expose them to different primary antibodies. Therefore, the uncropped picture is a strip and not the whole membrane.

*************************************************************************************

In your cover letter, please note whether your blot/gel image data are in Supporting information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

We have added the required information to the cover letter.

*************************************************************************************

Thank you for stating the following in the Acknowledgments Section of your manuscript:

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

[The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.]

We have removed the funding information from the manuscript and put it into the Cover letter for revision.

*************************************************************************************

Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

We have included captions for Supporting Information file at the end of our manuscript and updated in-text citations.

************************************************************************************************

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: No

Reviewer #2: Yes

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: No

Reviewer #2: Yes

________________________________________

5. Review Comments to the Author

*************************************************************************************

Reviewer #1: The manuscript "Markers of Acute Toxicity in Pancreatic Beta-Cells Exposed to Lethal Doses of the Organochlorine Pollutant DDT Determined by a Proteomic Approach" by Pavlíková et al, employs various biochemical techniques to determine the acute markers of acute toxicity in vitro.

Few are the points that must be considered to improve the overall quality of the manuscript

1.1

The term 'lethal dose' is incorrect to use since the dose chosen for any toxicant is always sublethal.

We took, incorrectly, some deliberation in using the words "lethal dose". We have realized that according to toxicological standards, for a toxicant applied in the liquid (e.g., a cell medium), we should use the words "lethal concentration". We have established 150 μM concentration of p,p'-DDT as LC96 (it killed 96% of exposed cells) after 72 hours (see Figure S1 in Supplementary Information file). Nevertheless, because we used shorter exposure times than 72 hours in our experiments, we have replaced words “lethal dose” with words “a high concentration”.

*************************************************************************************

1.2

When we called detected protein "markers of acute toxicity," we did not mean that the changed expression of those proteins suggested that the samples had been exposed to DDT and not to anything else. We see the changed expression of those proteins as signs of damage caused to pancreatic beta-cells by DDT. We will search for the changed expression of those proteins in samples exposed to lower, environmentally relevant concentrations DDT for a long time (months, years) to see if the environmental exposure can reach the same damage.

To make it more clear, we have renamed our article to “Markers of acute toxicity of DDT exposure in pancreatic beta-cells determined by a proteomic approach”.

*************************************************************************************

1.3

It would be advisable to add few inhibitors experiments against the targets to ascertain if the changes are solely due to DDT exposure or it's a general response.

In this study, we did not aim to examine the exact mechanism of cell death induced by DDT. We focused on the damage caused by a high concentration of DDT and reflected by changed protein expression.

*************************************************************************************

1.4

Are the effects observed mediated through direct binding of DDT or through its metabolites?

Animals (humans included) metabolize DDT very slowly, so we do not believe that DDT metabolites can play a significant role in 24-hour or 30-hour experiments. Moreover, we think it is unlikely that pancreatic beta-cells have enzymes necessary for metabolic reactions that usually occur in the liver. In our previous studies (http://dx.doi.org/10.1016/j.envres.2015.06.046, https://doi.org/10.1038/s41598-019-54579-z), we exposed pancreatic beta-cells to lower concentrations of DDT and its metabolite DDE for 1 month. In both cases, we found only one protein that changed its expression after both DDT exposure and DDE exposure. For these reasons, we do not believe that DDT metabolites played a role in the present experiment.

*************************************************************************************

1.5

A thorough Grammatical proofread is required to improve the flow for better understanding.

We have had our manuscript proofread by a native speaker.

*************************************************************************************

1.6

Abstract must be rewritten to highlight the rationale, potential results and findings in a precise manner.

We have rewritten the abstract as requested.

*************************************************************************************

1.7

We exposed the cells for 24 hours because 24-hour exposure represents one of the standard lengths for acute toxicity tests. We exposed the cells for 30 hours because the results of 24-hour exposure failed to satisfy us (it changed the expression of only a few proteins visible at 2-DE). We chose 30 hours rather than, e.g., 36 hours as our longer exposure time for a technical reason (to have the number of survived cells high enough to provide us with enough proteins for 2-DE).

The obtained data represent a 2-DE fingerprint of severe damage caused by high DDT concentration in beta-cells. As we described above, we will search for the changed expression of those proteins in samples exposed to lower, environmentally more relevant DDT concentrations for a long time (months, years) to see if the environmental exposure can reach the same damage.

*************************************************************************************

1.8

Please mention the culture conditions of the NES2Y cell, medium used, passage number etc.

We have added this information to the new chapter in "Material and Methods" called "Cell culture." (line 81 in the manuscript with tracked changes)

*************************************************************************************

1.9

Line 68-69 "We detected the fluorescence of cells using a FACS Calibur cytometer (Becton Dickinson, San Jose, CA, USA) channel FL2". What does this sentence imply?

This sentence describes the detection of PI signal in the analyzed cells. Now the sentence is modified by the following: “For detection of PI signal (emission = 585 nm) in dead cells, we used signal detector FL2 of FACS Calibur cytometer (Becton Dickinson, San Jose, CA, USA)”. (line 99 in the manuscript with tracked changes)

*************************************************************************************

1.10

Please try to be consistent while mentioning the cell number 100 000 cells vs 1×106 cells

We have unified the description of cell numbers (lines 94, 105, and 129 in the manuscript with tracked changes).

*************************************************************************************

1.11

Please restructure the material and method section to contain the essential information like

1. What concentration of DMSO was used as solvent control?

2. Why did the authors select the time points - 24h and 30 h? Will it be feasible to assess the cellular activity at initial time points of 12 and 18 h?

We have added this information to the new chapter in "Material and Methods" called "Cell culture." (line 82 in the manuscript with tracked changes)

We chose 24 hours as the first exposure time. It resulted only in several changes visible at 2-DE. We prolonged the exposure to 30 hours and checked if it would lead to more protein expression changes at 2-DE and still get enough living cells to harvest proteins. We did not test shorter exposure times because, at 2-DE, it would probably fail to show any changes.

*************************************************************************************

1.12

Western blotting details should be explained properly in terms of methodology, antibodies and concentration. Statistical analysis, please mention significance level.

We have added this information to the Material and methods section (line 104, and 205 in the manuscript with tracked changes)

*************************************************************************************

1.13

We did not intend to explore any specific cell death induction mechanism, only to check if using 2-DE can reveal any proteins that could serve as navigation for potential exploring of the mechanism of cell death induction.

The requirement to kill 20% of cells for 2-DE purposes is a technical requirement. The chosen experiment setup provided us with enough proteins to run 2-DE. It helped us to determine a proteomic description of beta-cells exposed to a concentration of DDT so toxic that would eventually (after 72 hours) kill the exposed cells. As we describe above, we used the DDT concentration of 150 μM that killed 18% of cells after 24 hours and 96% of cells after 72 hours (S1 figure).

In our previous studies (http://dx.doi.org/10.1016/j.envres.2015.06.046, https://doi.org/10.1038/s41598-019-54579-z), we exposed beta-cells to DDT concentration of 10 μM for one month. That exposure failed to induce cell death in the exposed cells; at the end of exposure the cells showed no difference in appearance or cell growth when compared to control cells. Therefore, we do not see 20% mortality ratio as obvious at sublethal doses of any toxicant.

*************************************************************************************

1.14

Study rationale needs to be improved as in what are authors interested in and why? Is it the cell viability or the mechanism or both? This point must be clearly stated.

We have rewritten the introduction and conclusions to make our study rationale clearer.

*************************************************************************************

1.15

Please avoid repetition of sentences or same information throughout the manuscript

We have done our best to remove repetition throughout the manuscript.

*************************************************************************************************************************************************************************

Major concerns

2.1

The author should mention the doses corresponded to the comparable levels that were toxic in humans in relation to diabetes and pancreatic beta cells. If the author provides some in-vivo references or data supporting their results to show the translatability of their in-vitro findings will definitely enhance the impact of this study.

We have added information about typical DDT levels in human serum or plasma to the introduction (lines 48 and 49 in the manuscript with tracked changes).

The epidemiological studies usually divide people into two groups: those with diabetes and those without it, and detect the concentrations of organochlorine pollutants in their serum or plasma. Then they search for statistical differences between the concentrations of individual pollutants in people with diabetes compared to healthy people. The outcome is which pollutants can play a role in diabetes. The actual concentrations can differ among these studies. There is no clear line between what is not toxic yet and what already is.

In the present experiment, we have intentionally used the concentration much higher than the environmental levels to induce cell death. We hypothesize that long-term exposure to the environmental level of DDT could eventually lead to beta-cell death. The proteins we have found can be used as markers of the beta-cell damage in future experiments when we use lower (environmentally relevant) DDT concentrations for a long time.

*************************************************************************************

2.2

In figure 2, the western blots for CHOP, GRP78, cleaved caspase-9, cleaved caspase 8, cleaved caspase -7 the band thickness does not match with the densitometric quantification in figure 3. In the control groups, the bands have completely vanished.

The densitometry represents the mean of three experimental values; the picture represents a typical example of how the western blot of the samples looked. So there can be a difference. We have added all western blots used for densitometry into the Supplement information file.

*************************************************************************************

Minor Concerns

2.3

The author should mention the cell viability at 30 hours.

We have tested the viability of the cells exposed for 30 hours using flow cytometry, and the viability was similar to that after 24 hours. But we believe that the real viability was lower than that because some of the dead cells already disintegrated into small particles (debris), and the method failed to detect them as cells.

*************************************************************************************

2.4

In result 3.1, the author has used neutral red assay for cell viability while in figure 1, it is mentioned as propidium iodide.

We used neutral red assay to test if 72-hour exposure to 150 μM DDT would kill the exposed cells. We used a neutral red assay because it is cost- and time-effective. Nevertheless, the neutral red assay cannot distinguish if the cells die more or grow more slowly due to exposure. It only determines the total number of living cells. Naturally, when only cca 4% of cells survive the experiment, this difference does not matter. In contrast, using flow cytometry with propidium iodide staining allowed us to determine the number of dead or dying cells together with the total number of cells so we could determine the concentration that killed approximately 20% of cells.

**************************************************************************************************************************************************************************

________________________________________

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose "no", your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Nagendra Kumar Rai

Attachment

Submitted filename: response to reviewers.pdf

Click here for additional data file.^{(639KB, pdf)}

PLoS One. doi: 10.1371/journal.pone.0229430.r003

Decision Letter 1

Ch Ratnasekhar

12 Oct 2020

Markers of acute toxicity of DDT exposure in pancreatic beta-cells determined by a proteomic approach

PONE-D-20-03380R1

Dear Dr. Nela,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Ch Ratnasekhar, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

**********

6. Review Comments to the Author

Reviewer #1: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

PLoS One. doi: 10.1371/journal.pone.0229430.r004

Acceptance letter

Ch Ratnasekhar

16 Oct 2020

PONE-D-20-03380R1

Markers of acute toxicity of DDT exposure in pancreatic beta-cells determined by a proteomic approach

Dear Dr. Pavlikova:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Ch Ratnasekhar

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 File

(PDF)

Click here for additional data file.^{(821KB, pdf)}

Attachment

Submitted filename: response to reviewers.pdf

Click here for additional data file.^{(639KB, pdf)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information file.

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PERMALINK

Markers of acute toxicity of DDT exposure in pancreatic beta-cells determined by a proteomic approach

Nela Pavlikova

Jan Sramek

Michael Jelinek

Petr Halada

Jan Kovar

Roles

Abstract

Introduction

Material and methods

Material

Cell culture

Viability of cells

Western blot analysis

2-D electrophoresis and subsequent gel analysis

Exposure to p,p‘-DDT

Preparation of samples for 2-D electrophoresis

2-D electrophoresis: Isoelectric focusing

2-D electrophoresis: SDS-PAGE

Gel image and analysis

Enzymatic digestion, MALDI-TOF mass spectrometry, and protein identification

Confocal microscopy

Statistical analysis

Results

Effect of various concentrations of p,p‘-DDT on the viability of NES2Y cells

Fig 1. Viability of NES2Y cells exposed to various concentrations of p,p‘-DDT for 24 hours.

Effect of p,p‘-DDT on apoptotic and ER stress markers

Fig 2. Representative western blots of selected markers of ER stress (CHOP, GRP78) and apoptosis (cleaved caspase-6, -7, -8, -9, and PARP).

Fig 3. Densitometry of western blots of chosen markers of ER stress and apoptosis.

Proteins with changed expression after 24 hours of exposure to p,p‘-DDT

Fig 4. Representative 2-DE gels (pI range 4–7) of NES2Y cells exposed to 150 μM p,p‘-DDT in DMSO for 24 hours and 30 hours.

Table 1. Differentially expressed proteins after both 24-hour and 30-hour exposure to 150 μM p,p‘-DDT identified in 2-DE experiments using three independent sets of samples.

Proteins with changed expression after 30 hours of exposure to p,p‘-DDT

Table 2. Differentially expressed proteins identified in 2-DE experiments.

Morphology of the cells exposed to p,p‘-DDT

Fig 5. Effect of 150 μM p,p‘-DDT on the cell shape of NES2Y pancreatic beta-cells.

Discussion

Proteins involved in the stress of endoplasmic reticulum

Mitochondrial proteins

Heterogeneous nuclear ribonucleoproteins

Cytoskeletal proteins

Proteins involved in the maintenance of the cell morphology

Proteins involved in processes connected with DNA or RNA

Proteins involved in oxidative stress

Heat shock protein 27

Conclusions

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Ch Ratnasekhar

Roles

Author response to Decision Letter 0

Decision Letter 1

Ch Ratnasekhar

Roles

Acceptance letter

Ch Ratnasekhar

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

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