Journal of Immunotoxicology

ISSN: 1547-691X (Print) 1547-6901 (Online) Journal homepage: http://www.tandfonline.com/loi/iimt20

Exposure to DDT metabolite p,p′-DDE increases autoimmune type 1 diabetes incidence in NOD mouse model Marina Cetkovic-Cvrlje, Marin Olson, Broc Schindler & Hwee Kiat Gong To cite this article: Marina Cetkovic-Cvrlje, Marin Olson, Broc Schindler & Hwee Kiat Gong (2015): Exposure to DDT metabolite p,p′-DDE increases autoimmune type 1 diabetes incidence in NOD mouse model, Journal of Immunotoxicology To link to this article: http://dx.doi.org/10.3109/1547691X.2015.1017060

Published online: 27 Feb 2015.

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Date: 09 September 2015, At: 08:34

http://informahealthcare.com/imt ISSN: 1547-691X (print), 1547-6901 (electronic) J Immunotoxicol, Early Online: 1–11 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/1547691X.2015.1017060

RESEARCH ARTICLE

Exposure to DDT metabolite p,p0 -DDE increases autoimmune type 1 diabetes incidence in NOD mouse model Marina Cetkovic-Cvrlje1,2, Marin Olson1,2, Broc Schindler1, and Hwee Kiat Gong1 Department of Biological Sciences and 2Laboratory for Immunology, St. Cloud State University, St. Cloud, MN, USA

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Abstract

Keywords

The incidence of autoimmune Type 1 diabetes (T1D) has been steadily rising in developed countries. Although the exact cause of T1D remains elusive, it is known that both genetics and environmental factors play a role in its immunopathogenesis. Whereas a positive association between p,p0 -DDE, a DDT metabolite, and Type 2 diabetes (T2D) has been well established, its role in T1D development in an experimental animal model has never been elucidated. This study seeks to investigate the effects of DDE exposure on the development of T1D in a NOD mouse model. As T1D is a T-cell-mediated disease, the underlying mechanism of DDE action on T-cells was studied in vitro and, in the context of acute and chronic DDE exposure, in vivo by investigating lymphocytes’ viability, proliferation, their subsets and cytokine profiles. Chronic high-dose DDE treatment, initiated in pre-diabetic 8-week-old NOD females administered twice weekly intraperitoneally with 50 mg/kg DDE, significantly increased diabetes incidence and augmented disease severity in treated animals. Whereas T-cell proliferation and cell viability in the spleens of treated mice were not affected, diabetogenic action of chronic DDE exposure was associated with a decrease in regulatory T-cells and a suppression of secretion of protective cytokines, such as IL-4 and IL-10. Interestingly, an acute high-dose in vivo treatment of 8-week-old NOD males with 100 mg DDE/kg, administered intraperitoneally every other day over a period of 10 days, increased T-cell proliferation and potentiated pro-inflammatory and TH1-type cytokine secretion, without affecting the splenocytes viability and the T-cell subpopulations. These results confirm that high-dose DDE treatments affect the immune system, in particularly T-cell function. In conclusion, this study shows for the first time that high-dose chronic DDE exposure exhibits a diabetogenic potential, with an underlying immunomodulatory mechanism of action, in the development of T1D in an experimental mouse NOD model.

DDE, NOD mice, p,p0 -DDE, persistent organic pollutants, T-cells, type 1 diabetes

Introduction p,p0 -Dichlorodiphenyldichloroethylene [p,p0 -DDE (DDE)] is the main metabolite of one of the most used pesticides in the world, the organochlorine compound dichlorodiphenyltrichloroethane (DDT). As DDT and its metabolites exhibit a long half-life, they belong to a group of persistent organic pollutants (POP). p,p0 DDE is a highly lipophilic compound that possesses properties of bioaccumulation and biomagnification (Gray, 2002; Suedel et al., 1994). Usage of DDT was prohibited in the US in 1972 and banned by the Stockholm Convention on Persistent Organic Pollutants in 2004 (UNEP, 2011). However, DDT can be used these days in malaria-endemic areas for mosquito control. Besides, there are other possibilities for DDT exposure, such as residing in the areas where it was relatively recently used, as well as exposure to DDT in the areas where it was never used, due to its property of a long-range transport (Spencer & Cliath, 1990). Exposure to DDT and its metabolites is considered as a human health risk factor, as confirmed by the numerous studies that have

Address for correspondence: Dr Marina Cetkovic-Cvrlje, Laboratory for Immunology, Department of Biological Sciences, St. Cloud State University, 720 Fourth Avenue South, St. Cloud, MN 56301, USA. Tel: 320-308-3490. E-mail: [email protected]

History Received 29 October 2014 Revised 8 January 2015 Accepted 5 February 2015 Published online 27 February 2015

investigated the association of p,p0 -DDE exposure with neurotoxicity (Rocha-Amador et al., 2009), endocrine disruption (Li et al., 2008) and reproductive system changes (de Jager et al., 2006). Type 1 diabetes (T1D) is an autoimmune disease characterized by the immune cells-orchestrated attack on pancreatic insulinproducing cells, which results in a complete lack of insulin and consequent development of hyperglycemia. The incidence of T1D has been steadily rising in developed countries from the 1950s. However, it is even more alarming that the doubling of the incidence in children below 5 years-of-age is predicted by 2020 (Patterson et al., 2009). Although many hypotheses have been proposed, the reason for such an increase in T1D is still unknown. Both genetics and environmental factors, such as chemicals, hygiene, diet and infections, are known to play a role in the etiopathogenesis of disease (Bach, 2002; Gale, 2002). Thus, substantial research efforts have aimed to find environmental triggers for the disease development (Thayer et al., 2012). Several studies described an association between the environmental POP exposure and development of autoimmunity. Thus, trichloroethylene (TCE) has been shown to accelerate systemic lupus erythematosus and systemic sclerosis (Griffin et al., 2000; Seo et al., 2011), whereas bisphenol A (BPA) increased development of insulitis, a typical histopathological lesion of T1D, in NOD mice (Bodin et al., 2013).

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Several subsets of T-cells, characterized by different immunophenotypes and cytokine profiles, have been implied as major players in immunopathogenesis of T1D (Cetkovic-Cvrlje et al., 2003, 2012; Shoda et al., 2005). For years, besides cytotoxic T-cells, the T-helper (TH)1/TH2 paradigm, while over-simplified, has provided a conceptual scaffold for understanding immunopathogenesis of T1D: TH1 response, with its cytokines interleukin (IL)-2 and interferon (IFN)- , has been considered pathogenic, in contrast to protective TH2 response, mediated by IL-4 and IL-10 (Muller et al., 2002; Rabinovitch, 1998). More recently, the regulatory IL-10 and transforming growth factor (TGF)- -secreting T-regulatory (Treg) cells have been recognized by their regulatory influence and considered protective (Cetkovic-Cvrlje et al., 2012; Riley et al., 2009), whereas TH17 cells, characterized by secretion of IL-17, have been recognized as harmful in development of T1D (Bettelli et al., 2007). The NOD mouse model, with its spontaneous development of T-cell-mediated and T-cell-dependent T1D, has been accepted as the best experimental model for studying immunopathogenesis of this disease and the effects of different compounds in the context of T1D protection/aggravation (Shoda et al., 2005). The effects of DDT/DDE exposure on the immune system of humans and experimental animals have been poorly investigated. Whereas overall evidence suggests that DDT/DDE can affect immune responses, existing studies in humans have provided conflicting results, showing that exposure to these POP can be immunostimulative (Shiplov et al., 1972), ineffective (Svensson et al., 1994) or immunosuppressive (Vine et al., 2001). Inconclusive results were also obtained by studying humoral immune responses in mice treated by DDT, where both immunosuppression (Banerjee, 1987; Rehana & Rao, 1992) and immunoproliferation (Rehana & Rao, 1992) were observed. Interestingly, a study following the longest chronic exposure to high doses of DDE, although not measuring any functional parameters, has not found alterations in size and morphology of the thymus, spleen and lymph nodes in exposed mice (NCI, 1978). While a large number of studies about the association between DDE and the prevalence of T2D exist (Eden et al., 2014; Tang et al., 2014; Taylor et al., 2013), there is only one epidemiological study that explores DDE exposure and T1D development (Rignell-Hydbom et al. 2010). Moreover, there has been no study designed to directly answer that question in an experimental model of T1D. In the study here, we asked whether DDE affects development of autoimmune T1D in a NOD mouse model. As T1D is a T-cell-mediated disease, the actions of DDE on T-cells were studied in vitro and, in the context of acute and chronic DDE exposure, in vivo by investigating lymphocyte viability, proliferation, their subsets and cytokine profiles.

Materials and methods Animals NOD breeding pairs were initially purchased from The Jackson Laboratories (Bar Harbor, ME) and housed and bred in the Animal Housing Facilities at Saint Cloud State University. Mice were housed in OptimiceÕ caging system (Animal Care Systems, Centennial, CO) in temperature- (22.2  C) and relative humidity (40–60%)-controlled conditions with a 12-h light cycle. All mice were allowed food (Harlan Teklad 18% Global Protein Diet 2018) and water ad libitum. Pre-diabetic 8-week-old male and female mice were randomly assigned to control or treated groups. All protocols and procedures were approved by Saint Cloud State University Institutional Animal Care and Use Committee prior to the start of experimentation (Protocol title: The effects of the environmental contaminant

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p,p0 -dichlorodiphenyldichloroethylene (p,p0 -DDE) on the development of Type 1 diabetes (T1D) in NOD mouse model; PI: Cetkovic-Cvrlje). DDE preparation DDE (99% pure) and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO). A stock 80 mg DDE/ml solution was prepared by suspending 1 g DDE in 12.5 ml DMSO and sonicating in a waterbath for 10 min. DDE for intraperitoneal (IP) injections was prepared by suspending stock DDE in corn oil to reach the desired concentrations. DDE/corn oil injection volume was adjusted to 6.7-ml/g BW as described in Ter Veld et al. (2008). In vivo DDE treatment For acute studies, pre-diabetic 8-week-old male NOD mice were randomly assigned to low-dose- (1 mg DDE/kg), high-dosetreated (100 mg DDE/kg) or control groups. Mice were injected every other day IP for 10 days, for a total of five injections. Mice were euthanized by CO2 asphyxiation on Day 11, their spleens were removed and single cell suspensions prepared for analyses of cell viability, T-cell proliferation, immunophenotyping and cytokine profiles. For chronic studies, pre-diabetic female NOD mice were randomly placed into groups exposed to 25 mg DDE/kg, 50 mg DDE/kg or vehicle. Mice were injected IP bi-weekly for up to 16 weeks and glycemia levels and body weights recorded. Additional experiments were performed where mice treated with 50 mg DDE/kg and controls were sacrificed at particular timepoints, e.g. 2-, 10- or 16-weeks post-beginning of treatment. At necropsy, each mouse spleen was removed and single cell suspensions prepared for analyses of T-cell proliferation, immunophenotyping and cytokine production. Blood glucose determination and body weight analysis Testing of blood glucose levels was performed on a weekly basis starting at the first week of treatment and until the end of experimentation for chronic studies (Cetkovic-Cvrlje et al., 2003). A lateral tail vein puncture was performed and 0.6 ml blood placed onto an Accu-Check Aviva blood glucose meter strip (Roche Diagnostics, Indianapolis, IN) to determine blood glucose levels. A mouse was considered diabetic after two consecutive readings of 220 mg glucose/dl. Body weight was determined on a weekly basis starting at the first week of exposure and continuing throughout the entire experimental period. Single cell suspension preparation Single cell suspensions of the isolated spleens were done as described in Cetkovic-Cvrlje et al. (1997). In brief, a spleen was forced through a 70-mm nylon mesh strainer (BD Falcon, San Jose, CA); the resulting suspension was then treated with ACK Lysis Buffer (NH4CL 8.29 g/L, KHCO3 1.0 g/L, EDTA Na2 2H2O 0.0375 g/L; Lonza BioWhittaker, Walkersville, MD) to remove erythrocytes and washed three times using phosphate-buffered saline (PBS, pH 7.5). Trypan blue (Lonza BioWhittaker) exclusion was utilized to count cells in a hemocytometer and to determine cell viability. T-cell proliferation T-cell proliferation assays were performed as described previously (Cetkovic-Cvrlje et al., 2001, Cetkovic-Cvrlj & Uckun, 2005). In brief, splenocytes from the mice were suspended in complete media (at 4  106 cells/ml, in complete RPMI-1640 medium [containing 1 U penicillin/ml, 100 mg streptomycin/ml, and 10% fetal calf serum (FCS) (each Sigma)]). Dedicated wells received

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DOI: 10.3109/1547691X.2015.1017060

p,p0 -DDE increases autoimmune T1D in a NOD mouse model

the T-cell mitogen Concanavalin A (ConA; Sigma) at a final level of 3 mg/ml. Cells were placed in a 37  C 5% CO2 NuAire IR Autoflow incubator (Plymouth, MN) and cultured for 72 h. Proliferation was then quantified using an Alamar Blue colorimetric assay (Invitrogen, Grand Island, NY) wherein Alamar Blue was added to each well at 10% of total culture volume and the cells incubated at 37  C for a further 5–7 h. Thereafter, optical density in each well was measured using an ELISA plate reader (GeneMate, Kaysville, UT) at 570 nm.

Statistical analysis

Immunophenotyping

Effect of DDE exposure on T1D incidence and glycemia levels in NOD females

Imunophenotyping was performed as described previously (Cetkovic-Cvrlje et al., 2002, Cetkovic-Cvrlje & Ucken, 2004). Specifically, splenocytes (106 cells) obtained from each mouse were suspended in FACS buffer (0.1% NaN3, 1% FCS in PBS), stained with appropriate antibodies, incubated in the dark at 4  C for 30 min, washed three times with FACS buffer and then underwent analyses in a FACSCalibur flow cytometer (BD Biosciences, San Diego, CA). Antibodies (BD Biosciences) used to quantify immune cell populations were: peridinin chlorophyll-a protein (PerCP)-conjugated anti-CD4 (clone RM45), fluorescein isothiocyanate (FITC)-conjugated anti-CD8 (clone 53-6.7), allophycocyanin (APC)-conjugated anti-CD25 (clone 3C7), phycoerythrin (PE)-conjugated anti-CD3 (clone 1452C11), allophycocyanin (APC)-conjugated anti-CD45RB220 (clone RA3-6B2), FITC-conjugated anti-CD161 (clone PK136) and PerCP-conjugated anti-CDllb (clone M1/70). A minimum of 10 000 events was acquired for each analysis. Immunophenotype analyses were performed using CellQuest Pro software (BD Biosciences). As percentages of CD4+Foxp3+ splenocytes analyzed in our pilot experiments were similar and comparable to the percentages of CD4+CD25+ cells (Cetkovic-Cvrlje et al. 2012; and data not shown), CD4+CD25+ double staining was used as an indicator of Treg cell sub-population size. Cytokine analysis Levels of IL-2, IL-4, IL-10, IL-17, IFN and tumor necrosis factor (TNF)- in the supernatants obtained from 48-h cultures of the ConA-stimulated splenocytes were quantified using a BD Biosciences cytometric bead assay (CBA) mouse TH1/TH2/TH17 kit. Cytokine concentrations were analyzed by FCAP Array software (SoftFlow, New Brighton, MN). The level of sensitivity for IL-2, IL-4, IL-10, IL-17, IFN and TNF was, respectively, 0.1, 0.03, 16.8, 0.8, 0.5 and 0.9 pg/ml. In vitro DDE treatment Splenocytes obtained from non-treated NOD mice were cultured and stimulated by ConA (as described previously) in the presence of DDE. Serial dilutions of DDE in complete media were prepared starting at 100 mg/ml (0.314 mM) down to 0.39 mg/ml (0.001 mM). Cells were cultured (in triplicate) for 72 h and T-cell proliferation then quantified using Alamar Blue. Apoptosis detection Apoptosis among the in vitro-treated cells was detected using flow cytometry and a FITC Annexin V Apoptosis kit (BD Pharmingen, San Diego, CA). In brief, the splenocytes were cultured for 24 h at 4  106 cells/ml in the presence of 5, 25 or 100 mg DDE/ml. Thereafter, the cells were collected, washed with PBS and aliquots (i.e. 4  105 cells) stained with FITC-conjugated Annexin V and propidium iodide, according to manufacturer instructions, and then underwent analyses in the FACSCalibur flow cytometer.

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A life-table analysis, using the program SPSS (IBM, Armonk, NY), was used for diabetes incidence analysis (p50.05 considered statistically significant). All other statistical analyses were performed using Excel (Microsoft, Redmond, WA) and a twotailed unpaired Student’s t-test (p50.05 considered statistically significant).

Results

NOD females were exposed to DDE at a dose of 50 and 25 mg/kg (twice a week) during a chronic period of 16 weeks or until they became diabetic, whichever came first. The exposure started at 8 weeks when all experimental (n ¼ 32) and control mice (n ¼ 31) were normoglycemic. There were no mortality and no relevant clinical signs of toxicity observed in either of the drug-treated groups during the entire experimental period of 16 weeks. Along these lines, body weights of DDE-treated mice were not prominently different from the control mice (data not shown). As illustrated in Figure 1(a), exposure of mice to 50 mg DDE/kg significantly accelerated diabetes development and increased diabetes incidence compared to among controls (p50.05, lifetable analysis). Whereas 50% of DDE-treated mice became diabetic by 17 weeks-of-age, the same percentage of control mice was diabetic 7 weeks later. Furthermore, 82% of DDE-treated and 53% of control females became diabetic by the experimental period end, at 24 weeks-of-age. Chronic exposure of NOD females to a dose of 25 mg DDE/kg showed a trend of increase in diabetes incidence; however, statistical significance (versus controls) was not reached. Figure 1(b) shows the average glycemia levels were highest in mice treated with 50 mg DDE/kg compared to controls, exhibiting differences from 5 weeks post-beginning of DDE exposure until the study end. Thus, the data indicate that high-dose DDE treatment can affect T1D development, by increasing the incidence of disease, as well as potentiating the severity of disease in exposed mice. DDE effects on T-cell function in vitro Considering DDE-induced aggravation and a T-cell-dependent nature of T1D development, one should ask whether DDE could directly act on T-cells. To address this question, a T-cell proliferation assay was performed with the addition of DDE. NOD splenocytes were stimulated with a T-cell mitogen ConA and exposed to different concentrations of DDE, ranging from 100 mg/ml (0.3 mM) to 0.001 mg/ml (Figure 2a and data not shown). Positive control samples were stimulated by the mitogen in the same way, but instead of DDE, DMSO was added at the dose present in the highest drug dilution in order to exclude immunosuppressive effects of DMSO on T-cells (Ca´rdenasGonza´lez et al., 2013). Negative controls contained a mitogen and a medium only. Figure 2(a) shows that addition of 25, 50 and 100 mg DDE/ml significantly inhibited T-cell proliferation, whereas lower concentrations were ineffective. To determine a possible mechanism for the DDE-induced suppression of T-cell proliferation, flow cytometric detection of apoptotic cell death was performed in cultured splenocytes exposed to a high (100 mg/ml) and a low (5 mg/ml) concentration of DDE, as these two doses were previously shown (initial in vitro cell proliferation experiments) to, respectively, suppress and not affect the proliferative capability of T-cells. As illustrated in Figure 2(b), the addition of T-cell-suppressive DDE concentration (100 mg/ml) to the T-cell cultures induced a significantly higher level of apoptotic cell death (94.5 [±4.6]% of cells), compared to

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Figure 1. Diabetes incidence and average glycemia levels in NOD females treated with DDE up to 24 weeks-of-age. Mice received DDE at 50 or 25 mg/kg IP biweekly, starting at 8 weeks-of-age; control mice received vehicle. (a) Diabetes incidence presented as fraction of survival, where 1.0 represents 100% diabetes-free; *p50.05 (Mantel-Cox log-rank test). (b) Glycemia determined on a weekly basis. Data shown are mean ± SEM; n ¼ 15–30/regimen.

30.6 [±7.3] and 34.0 [±3.8]%, when an ineffective DDE concentration (5 mg/ml) and DMSO (control), respectively, were tested. Based on these finding, induction of apoptotic cell death is a likely mechanism for the inhibition of T-cell proliferation seen in cell cultures exposed to high concentrations of DDE. Effects of acute in vivo DDE treatment on T-cell sub-populations and cytokine secretion Conflicting data, showing both immunosuppressive and immunoproliferative properties of DDT and DDE in different animal models have been published (Banerjee, 1987; Rehana & Rao, 1992). To confirm DDE could influence T-cell sub-population sizes and/or their function in vivo, an acute high (100 mg/kg) and low (1 mg/kg) DDE treatment of 8-week-old NOD males was initiated over a period of 10 days (five injections). NOD males were chosen as the focus here was solely on studying immune parameters. As NOD males develop diabetes at a low frequency and later in their lifetime than females (Anderson & Bluestone, 2005), the influence of spontaneous disease development associated with changes in immune system parameters, present in females at that age, was thus eliminated. Figure 3 shows that the viability of splenocytes, obtained after either acute low(Figure 3a and b) or high- (Figure 3c and d) dose DDE treatment, when presented either as percentages or total cell numbers, was not affected. The flow cytometric analysis of splenic immune cell

populations (Figure 4), including T-cells (CD3+ cells and subpopulations including CD4+, CD8+ and CD4+CD25+ cells), Bcells, macrophages and NK cells, did not reveal a significant difference among any of the studied cell types in the high- (Figure 4b) or low-dose (Figure 4a) DDE-treated mice when compared to values associated with control mice. When the function of T-cells was analyzed following acute high-dose DDE treatment, prominent differences were observed (Figure 5b). For example, mitogen-induced T-cell proliferation was significantly increased compared to that by control mouse cells. In comparison, T-cells from spleens of NOD males after the low-dose DDE exhibited proliferative capacities similar to that of control mouse cells (Figure 5a). In agreement with differences observed in proliferation of T-cells between the high- and low-dose DDE exposures, significantly higher levels of IL-6, TNF and IFN were detected in the cultures of cells isolated from the mice treated acutely with high doses of DDE (Figure 6). Effects of chronic in vivo DDE treatment on T-cell sub-populations and cytokine secretion A set of experiments was also undertaken to study possible mechanism(s) of diabetogenic DDE action in NOD females. Mice were exposed to the same doses and drug regimens, as previously described for a chronic treatment of NOD females in which the glycemia and T1D incidence were studied. T-cell

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p,p0 -DDE increases autoimmune T1D in a NOD mouse model

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Figure 2. Proliferation and apoptosis among T-lymphocytes following in vitro exposure to DDE. (a) Proliferation was determined by culturing naı¨ve NOD splenocytes (in triplicate) for 72 h in the absence (non-stimulated) or presence (stimulated) of ConA (3 mg/ml) and with addition of 0.39–100 mg DDE/ml. Positive controls consisted of DMSO at the same concentration as in the highest DDE regimen; negative control consisted of complete media only. Data presented as mean ± SEM; *p50.05 versus stimulated positive control (Student’s t-test). Figure is representative of four experiments. (b) Apoptosis assessed in splenocytes cultured for 24 h with an addition of 5 or 100 mg DDE/ml; control cultures received DMSO at a concentration found in the 100 mg DDE/ml regimen. Apoptotic events were quantified by flow cytometry; apoptotic and live cells were detected as Annexin V FITC+/PI+ (dead) and Annexin V FITC/PI (alive), respectively. Data shown are mean ± SEM of three experiments; *p50.05 versus appropriate dead and live groups of cells exposed to 100 mg DDE/ml. Figure 3. Splenocyte viability after acute treatment of NOD males with DDE. Mice were treated with (a, b) 1 or (c, d) 100 mg DDE/kg (or vehicle) IP every other day for a total of five injections (over 10 days), then euthanized on Day 11. Cell viability was expressed as (a, c) a percentage or (b, d) absolute cell number (106). Viability was measured using Trypan blue. Data shown are mean ± SEM; n ¼ 9–10/regimen.

viability, proliferation, immune cell subset sizes, as well as levels of pathogenic and protective cytokines, were analyzed at 2-, 10and 16-weeks following beginning the 50 mg DDE/kg treatment. The results indicated that the DDE treatment did not affect the viability of splenocytes: comparable viability between the treated and control mice were obtained at all timepoints (data not shown). Figure 7 illustrates the proliferative capacity of T-cells obtained from DDE-treated mice did not differ from the control mice cells at 2- (Figure 7a), 10- (Figure 7b) or 16-week (Figure 7c)

timepoints. There were also no differences in T-cell population sizes or among their helper and cytotoxic subsets, nor among B-cells, macrophages and NK cells between control and DDEexposed mice at each studied timepoint (Figure 8a–c). However, T-regulatory (Treg) cell levels were significantly reduced in mice treated with DDE compared to control values (3.2 [±0.3] versus 4.3 [±0.2]%), at a 10-week timepoint. A significant reduction in IL-2 and TNF levels was observed at the same timepoint (Figure 9). Interestingly, the trend of decrease in the levels

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Figure 4. Immunophenotyping of splenocytes isolated from NOD males that underwent acute treatment with DDE. Mice were treated with (a, b) 1 or (c, d) 100 mg DDE/kg (or vehicle) IP every other day for a total of five injections (over 10 days), then euthanized on Day 11. Spleens were removed and single cell suspensions prepared; the cells were then stained with antibodies against the markers CD4 (TH), CD8 (TC), CD3 (T), CD45RB220 (B-cell), CD11b (Mac), NK1.1 (NK) and CD4/CD25 (TReg) and analyzed in a flow cytometer. Data shown are mean ± SEM; n ¼ 6–10/regimen.

of protective cytokines IL-4 and IL-10 was observed at all timepoints in mice exposed to DDE.

Discussion This study showed that a principal DDT metabolite, p,p0 -DDE, could affect development of T1D, as disease incidence as well as severity was aggravated in NOD females that underwent chronic treatment with the metabolite. To our knowledge, this is the first time a positive association between DDE exposure and disease development has been shown in the experimental NOD mouse model of T1D. A single epidemiological study (Rignell-Hydbom et al., 2010) has been published that investigated effects of DDE in the context of T1D, i.e. by studying whether in utero exposure to DDE triggered development of T1D in children. That study did not confirm a positive correlation between DDE and T1D, but implied a potential protective (although not statistically significant) effect of DDE exposure. However, the authors noted a caveat regarding interpretation of their results; all the subjects (pregnant mothers) exhibited, in addition to elevated levels of DDE, increased levels of PCB-153, another persistent organic pollutants (POP). Therefore, there is a possibility that effects of DDE exposure in a system where both POP are present might be skewed by PCB-153. Interestingly, our

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Figure 5. Proliferation by splenocytes (T-lymphocytes) isolated from NOD male mice that underwent acute treatment with DDE. Mice were treated with (a) 1 or (b) 100 mg DDE/kg (or vehicle) as outlined in the legends to Figures 3 and 4. Proliferation was then determined by culturing splenocytes in the absence (non-stimulated) or presence (stimulated) of ConA (3 mg/ml) for 72 h. Data shown are mean ± SEM; n ¼ 5–10/regimen. *p50.05 versus controls (Student’s t-test).

recent experiments in the NOD mouse model have provided evidence about immunosuppressive and anti-diabetogenic properties of PCB-153 exposure (manuscript in preparation). Numerous epidemiological studies indicate a link between DDE and induction of T2D (Eden et al., 2014; Tang et al., 2014; Taylor et al., 2013). Retrospective epidemiological analysis showed that increased levels of DDE in the human sera were positively correlated with increased insulin resistance (Lee et al., 2007) and prevalence of T2D (Lee et al., 2006). In addition, studies of a Swedish cohort (Rignell-Hydbom et al., 2007), the Great Lake fish consumers (Turyk et al., 2009), as well as recent analysis of 72 epidemiological studies (Taylor et al., 2013) and meta-analysis of 23 epidemiological studies (Tang et al., 2014), found a strong association between DDE exposure and increased incidence of T2D. However, it should be noted that these data were insufficient to establish causality. In contrast to underlying metabolic changes in T2D, T1D is an autoimmune disease, which occurs as a consequence of T-cell-mediated attack on pancreatic insulin-producing cells. Therefore, to elucidate whether DDE can directly affect T-cell function, its effects were studied in vitro on ConA-induced T-cell proliferation. The addition of high concentrations of DDE (25, 50 and 100 mg/ml) significantly reduced proliferation of T-cells in a dose-trend fashion. In addition, it was seen in this experimental system that induction of apoptosis was a mechanism of DDE-

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Figure 6. Quantification of cytokine levels after acute treatment of NOD males with DDE. Cytokines acquired from supernatants of splenocytes obtained from mice treated with 1 or 100 mg DDE/kg as outlined in the legends to Figures 3 and 4. After 48 h of culture in the presence of ConA (3 mg/ml), supernatants were analyzed using a CBA mouse TH1/TH2/TH17 kit that permits quantification of IL-6, TNF, IFN , IL-2, IL-4, IL-10 and IL-17. Data shown are mean ± SEM; n ¼ 3–6/regimen. *p50.05 versus corresponding control (Student’s t-test).

induced suppression of T-cell function. These data are in line with a previously published study showing apoptotic cell death as a mechanism of DDE-induced reduction of viability among human peripheral mononuclear blood cells (Alegrı´a-Torres et al., 2009). Moreover, that finding of effective (80 mg/ml) versus ineffective (10 mg/ml) in vitro doses of DDE on human T-cells correlated with the efficacy of DDE observed in the present model that used mouse cells (i.e. effective DDE dose 25 mg/ml versus ineffective 525 mg/ml). The first challenge faced in planning an in vivo study was choosing a dose of DDE to test its effects on the immune system and ultimately T1D development. Knowing that Banerjee et al. (1996) have described adverse effects of DDE exposure on humoral immune responses in rats treated with 22.2 mg DDE/kg daily for 6 weeks and that no treatment-related adverse effects on the thymus, spleen or lymph nodes were seen in mice chronically exposed to 49 mg DDE/kg daily over a period of 78 weeks (NCI, 1978), we chose doses of 25 and 50 mg DDE/kg to be administered twice a week for a chronic treatment and 1 and 100 mg/kg every second day over the period of 10 days for an acute exposure. The doses of p,p0 -DDE used in our experiments were certainly high compared to the ordinary environmental exposure levels. Whereas DDE levels in the sera of treated animals were not measured in our study, Makita and Omura (2006) reported that p,p0 -DDE concentrations in sera of rats treated with similar doses of DDE daily over the period of 42 days were comparable to those detected in human sera or tissues (Longnecker et al., 2001). There were no mortality and no clinical signs of toxicity observed in both treatment groups of mice during the entire period of 16 weeks of DDE exposure in the current study. The

body weights of DDE-treated mice were not prominently different from the control mice, confirming previously obtained data in male mice treated with 49 mg DDE/kg daily over a much longer period of 78 weeks (NCI, 1978). While people are expected to be exposed to DDE primarily through the oral route, it has been shown that tissue DDE levels obtained in experimental animal models post-oral/-intraperitoneal delivery were comparable (Ter Veld et al., 2008). Therefore, the intraperitoneal route of exposure was used in the current study to better control DDE dosage for each mouse. To shed light on a diabetogenic mechanism of DDE action in T1D in vivo, immune system parameters, such as viability of splenocytes, T-cell proliferation, T-cell and other immune cell populations and sub-population levels, as well as cytokine profiles, were investigated in NOD males treated acutely with high (100 mg/kg) and low (1 mg/kg) doses of DDE every second day over a period of 10 days. NOD males were chosen for use as they develop diabetes later and with prominently lower frequency than female counterparts (Anderson & Bluestone, 2005; Shoda et al., 2005). Thus, effects of DDE on the immune parameters in young pre-diabetic NOD males were studied without the influence of natural, spontaneous disease progression, observed in the females of the same age. Acute exposure to DDE in high and low doses did not affect immune cell viability, as both the percentages and total numbers of viable cells obtained from spleens following DDE treatments did not differ from control mouse values. Considering the observed suppression of T-cell proliferation induced by the high doses of DDE in vitro, we were surprised to find an increase in T-cell proliferation following acute high-dose exposure to DDE in vivo. Understandably, in vitro exposure exclusively determines

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Figure 7. Proliferation by splenocytes (T-lymphocytes) isolated from NOD females that underwent chronic treatment with DDE. Mice were treated for (a) 2, (b) 10 or (c) 16 weeks with 50 mg DDE/kg or vehicle. T-cell proliferation was determined by culturing isolated splenocytes in the absence (non-stimulated) or presence (stimulated) of ConA (3 mg/ml) for 72 h. Data shown are mean ± SEM; n ¼ 7–14/regimen.

the effect of DDE on T-cells; however, in vivo exposure needs to take into account a number of factors including other immune cells as well as metabolic processes. Interestingly, a study performed using sera of DDE-exposed loggerhead sea turtles found a significant positive correlation between induced proliferative responses of B-cells and serum DDE levels (Keller et al., 2006). In addition, exposure of mice to Aroclor 1254, which contains polychlorinated biphenyls (biologically similar to DDE), significantly increased ConA-induced T-cell proliferation (Segre et al., 2002). These studies indicated that DDE and similar POP exhibited immunoproliferative properties in vivo, thus supporting our T-cell proliferation data obtained by the acute high-dose DDE exposure. In general, only limited and somewhat conflicting data are available regarding the influence of DDE on the immune system. Several studies explored DDT/DDE exposure and immune system parameters in humans, finding: increased serum agglutinins after vaccination with additional subacute exposure to DDT (Shiplov et al., 1972); no changes in white cell counts, lymphocyte and subset levels or serum immunoglobulin levels post-chronic exposure to DDT via fish consumption (Svensson et al., 1994); and decreased ConA-induced lymphoproliferation, with slightly increased CD3+ and CD4+ T-cell levels and IgA in people who resided near a waste site and who exhibited elevated levels of serum DDE (Vine et al., 2001). Similarly conflicting data have been published regarding the DDT/DDE effects on the immune system of mice. Whereas exposure of mice to DDT over the period of 3–12 weeks induced immunosuppression, particularly of humoral immune response measured by plaque-forming cell assay (Banerjee, 1987; Banerjee et al., 1986), another study showed DDT-induced immunoproliferation if the mice were treated with DDT over 16 weeks, but immunosuppression of humoral responses if they were exposed to the same doses of DDT over the period of 24 weeks (Rehana & Rao, 1992). Interestingly, a study that described the longest

treatment with high-dose DDE in mice, i.e. B6C3F1 mice chronically exposed over 78 weeks to 49 mg DDE/kg/day orally found no treatment-related changes in the thymus, spleen and lymph nodes (NCI, 1978). Unfortunately, as T- or B-cell functions were not investigated, host immunocompetence was unevaluated. Overall, while those studies suggest DDT/DDE could affect humoral immune responses in mice, evidence has been lacking regarding the DDE influence on cellular immunity. Thus, our study provided needed evidence confirming that DDE could indeed affect T-cell responses. Furthermore, the current study showed acute DDE treatment with high or low doses of DDE did affect neither T-cell sub-populations (such as helper, cytotoxic or regulatory), nor other studied immune cells, such as B-cells, macrophages and NK cells. However, with regard to cytokine production, a significant increase in innate pro-inflammatory TNF and IL-6 and in the TH1/cytotoxic T-cell hallmark cytokine IFN was observed in the mice acutely exposed to high-doses of DDE. Taken together, these data showed that DDE, used in high doses over an acute period, exhibits immunomodulatory properties, reflected in promotion of T-cell proliferation, as well as in skewing the cytokine profile towards pro-inflammatory-/TH1-/ cytotoxic T-cell-type immune responses. Given the well-known role of pro-inflammatory cytokines as diabetogenic in the immunopathogenesis of T1D in the NOD mouse model (Padgett et al., 2013), these results suggested to us that DDE exposure might set the stage for promotion of T1D development in NOD mice. However, other possible mechanisms of DDE action in the promotion of T1D (not intended to be studied here), such as DDE effects on innate immunity (including related dendritic cells, macrophages, etc.) or direct effects on pancreatic -islet cells, should be considered as well. The last question asked here was what is the mechanism of diabetogenic DDE action observed in NOD females exposed chronically to high-doses of DDE, considering the results gained about DDE influence on the immune system of pre-diabetic

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Figure 8. Immunophenotyping of splenocytes recovered from NOD females that underwent chronic treatment with DDE. Mice were treated for (a) 2, (b) 10 or (c) 16 weeks with 50 mg DDE/kg or vehicle. Spleens were removed after each period was complete and single cell suspensions prepared. The isolated cells were stained with antibodies against the markers CD4 (TH), CD8 (TC), CD3 (T), CD45RB220 (B-cell), CD11b (Mac), NK1.1 (NK) and CD4/CD25 (TReg) and then analyzed in a flow cytometer. Data shown are mean ± SEM; n ¼ 9–17/regimen. *p50.05 versus controls (Student’s t-test).

NOD males during the acute exposure. Mice were sacrificed at 2-, 10- and 16-weeks following treatment with 50 mg DDE/kg twice a week and T-cell function, populations and sub-populations of different immune cells, as well as cytokine profiles were analyzed. Interestingly, the proliferative capacity of T-cells obtained from mice exposed to DDE did not differ from the controls at any analyzed timepoint. Although the majority of cell populations were unaffected by DDE exposure at all timepoints, a significant decrease in population of regulatory T-cells was observed at 10 weeks after beginning treatment. Cytokine analysis revealed an interesting trend (borderline statistical significance) of consistently decreased levels of IL-4 and IL-10 in DDE-treated females at all timepoints studied. In addition, decreased levels of TNF and IL-2 were observed at the 10-week timepoint only. These results indicate that the diabetogenic action of DDE during a chronic treatment is associated with decreased population of protective Treg cells, as well as decreased levels of protective cytokines such as IL-4 and IL-10. Additional studies involving measures of TGF should be performed to further characterize DDE effects on Treg cells. The protective role of Treg cells in the pathogenesis of T1D has been well documented (Cetkovic-Cvrlje et al., 2012; Riley et al., 2009). In addition, decreased levels of protective anti-diabetogenic cytokines have been linked with acceleration of diabetes development and increased incidence. Interestingly, the most prominent changes here were observed at the 10-week timepoint, which corresponds

with 18 weeks-of-age and a peak of diabetes occurrence in a majority of these animals. The puzzling difference regarding the effects of DDE on the immune system observed in the current studies of acute versus chronic treatments could be attributed to two factors, e.g. one being acute versus chronic exposure to the compound per se and the other the use of male versus female mice. In support of the first possibility and in agreement with our data, Rehana and Rao (1992) described immunoproliferation versus unaffected immune responses in mice exposed to the same DDE doses in acute versus chronic exposure regimens. The occurrence of clinical symptoms of diabetes (or very close to that stage), in addition to development of more aggressive autoimmune attack in NOD females compared to males of the same age, could contribute to measureable differences in DDE action. Shoda et al. (2005) clearly noted that NOD females exhibit altered sensitivity to treatment with the same compound at different stages of the disease.

Conclusions This study shows for the first time, using the animal experimental model, that DDE can affect T1D development by increasing disease incidence and potentiating the severity of hyperglycemia. The underlying mechanism of action is related to immunomodulation as DDE exposure exhibited an ability to skew immune

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Figure 9. Quantification of cytokine levels released by cultured splenocytes obtained from NOD females that underwent chronic treatment with DDE. Mice were treated for (a) 2, (b) 10 or (c) 16 weeks with 50 mg DDE/kg or vehicle. Spleens were removed after each period was complete and single cell suspensions prepared. After 48 h of culture in the presence of ConA (3 mg/ml), supernatants were analyzed using a CBA mouse TH1/TH2/TH17 kit that permits quantification of IL-6, TNF, IFN , IL-2, IL-4, IL-10 and IL-17. Data shown are mean ± SEM; n ¼ 3–8/regimen. *p50.05 versus corresponding control (Student’s t-test).

responses, either by increasing pathogenic (during acute treatment) or decreasing protective immune (over the chronic exposure) responses. Although high doses of DDE were used in this study and data observed in the mouse experimental model might not be directly extrapolatable to humans, this study indicates the need for greater awareness about the potential effects of DDE on human health. In addition, studies like this one emphasize that a cautious approach should be exercised regarding the potential reintroduction of DDT for prevention/control of diseases, such as West Nile virus disease. Taylor et al. (2013) emphasized several data gaps/areas that need to be researched regarding the association of POP and diabetes (diabetes here is used as umbrella term for T1D and T2D), one of them being the ‘‘relationships between POP and T1D’’, as to date only one prospective study (Rignell-Hydbom et al., 2010) had been done. In the context of this data gap, we believe our study adds to the knowledge about the relation between DDE and T1D.

Acknowledgments This study was supported by NSF grant 0821235 to MCC and SCSU OSP Student Research Grants to MO, BS and KG. We thank the undergraduate research students in the SCSU Immunology Laboratory, especially Allan Lea, Huong Phung, Sinduja Thinamany, Vivek Lamsal, Andrew Scott, Alex Scott, Christopher Prigge, Melissa Floren, Michelle Moran, Gayani Gamage, Katie Owen, Alesha McPhail, Tessa Hirdler, Mirza Zec and Jennifer Steen for their excellent technical assistance.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

References Alegrı´a-Torres, J. A., Dı´az-Barriga, F., Gandolfi, A. J., and Pe´rezMaldonado, I. N. 2009. Mechanisms of p,p0 -DDE-induced apoptosis in human peripheral blood mononuclear cells. Toxicol. In Vitro 23: 1000–1006. Anderson, M. S., and Bluestone, J. A. 2002. The NOD mouse: Model of immune dysregulation. Annu. Rev. Immunol. 23:447–485. Bach, J. F. 2002. Effect of infections on susceptibility to autoimmune and allergic diseases. New Engl. J. Med. 347:911–920. Banerjee, B. D. 1987. Sub-chronic effect of DDT on humoral immune response to a thymus-independent antigen (bacterial lipopolysaccharide) in mice. Bull. Environ. Contam. Toxicol. 39:822–826. Banerjee, B. D., Ramachandran, M., and Hussain, Q. Z. 1986. Subchronic effect of DDT on humoral immune response in mice. Bull. Environ. Contam. Toxicol. 37:433–440. Banerjee, B. D., Ray, A., and Pasha, S. T. 1996. A comparative evaluation of immunotoxicity of DDT and its metabolites in rats. Indian J. Exp. Biol. 34:517–522. Bettelli, E., Oukka, M., and Kuchroo, V. K. 2007. TH17 cells in the circle of immunity and auto-immunity. Nat. Immunol. 8:345–350. Bodin, J., Bølling, A. K., Samuelsen, M., et al. 2013. Long-term bisphenol A exposure accelerates insulitis development in diabetesprone NOD mice. Immunopharmacol. Immunotoxicol. 35:349–358.

Downloaded by [Texas A & M International University] at 08:34 09 September 2015

DOI: 10.3109/1547691X.2015.1017060

Ca´rdenas-Gonza´lez, M., Gaspar-Ramı´rez, O., Pe´rez-Va´zquez, F. J., et al. 2013. p,p0 -DDE, DDT metabolite, induces pro-inflammatory molecules in human peripheral blood mononuclear cells ‘in vitro’. Exp. Toxicol. Pathol. 65:661–665. Cetkovic-Cvrlje, M., and Uckun, F. M. 2004. Dual targeting of Bruton’s tyrosine kinase and Janus kinase 3 with rationally designed inhibitors prevents graft-versus-host disease (GVHD) in a murine allogeneic bone marrow transplantation model. Br. J. Haematol. 126:821–827. Cetkovic-Cvrlje, M., and Uckun, F. M. 2005. Effect of targeted disruption of signal transducer and activator of transcription (Stat)4 and Stat6 genes on the autoimmune diabetes development induced by multiple low doses of streptozotocin. Clin. Immunol. 114:299–306. Cetkovic-Cvrlje, M., Dragt, A. L., Vassilev, A., et al. 2003. Targeting JAK3 with JANEX-1 for prevention of autoimmune Type 1 diabetes in NOD mice. Clin. Immunol. 106:213–225. Cetkovic-Cvrlje, M., Gerling, I. C., Muir, A., et al. 1997. Retardation or acceleration of diabetes in NOD/Lt mice mediated by intra-thymic administration of candidate b cell antigens. Diabetes 46:1975–1982. Cetkovic-Cvrlje, M., Olson, M., and Ghate, K. 2012. Targeting Janus tyrosine kinase (JAK)3 with inhibitor induces secretion of TGFb by CD4+ T-cells. Cell. Mol. Immunol. 9:350–360. Cetkovic-Cvrlje, M., Roers, B. A., Schonhoff, D., et al. 2002. Treatment of post-bone marrow transplantation acute graft-vs.-host disease (GVHD) with a rationally designed JAK3 inhibitor. Leukemia Lymphoma 43:1447–1453. Cetkovic-Cvrlje, M., Roers, B. A., Waurzyniak, B., et al. 2001. Targeting Janus kinase 3 to attenuate the severity of acute graft-versus-host disease across the major histocompatibility barrier in mice. Blood 98: 1607–1613. de Jager, C., Farias, P., Barraza-Villarreal, A., et al. 2006. Reduced seminal parameters associated with environmental DDT exposure and p,p0 -DDE concentrations in men in Chiapas, Mexico: A cross-sectional study. J. Androl. 27:16–27. Eden P. R., Meek E. C., Wills R. W., et al. 2014. Association of Type 2 diabetes mellitus with plasma organochlorine compound concentrations. J. Exposure Sci. Environ. Epidemiol. [Epub ahead of print]. doi: 10.1038/jes.2014.69. Gale, E. A. 2002. A missing link in the hygiene hypothesis? Diabetologia 45:588–594. Gray, J. S. 2002. Biomagnification in marine systems: Perspectives of an ecologist. Mar. Pollut. Bull. 45:46–52. Griffin, J. M., Blossom, S. J., Jackson, S. K., et al. 2000. Trichloroethylene accelerates an auto-immune response by TH1 T-cell activation in MRL+/+ mice. Immunopharmacology 46:123–137. Keller, J. M., McClellan-Green, P. D., Kucklick, J. R., et al. 2006. Effects of organochlorine contaminants on loggerhead sea turtle immunity: Comparison of a correlative field study and in vitro exposure experiments. Environ. Health Perspect. 114:70–76. Lee, D. H., Lee, I. K., Jin, S. H., et al. 2007. Association between serum concentrations of persistent organic pollutants and insulin resistance among non-diabetic adults: Results from National Health and Nutrition Examination Survey 1999-2002. Diabetes Care 30:622–628. Lee, D. H., Lee, I. K., Song, K., et al. 2006. A strong dose-response relation between serum concentrations of persistent organic pollutants and diabetes: Results from the National Health and Examination Survey 1999-2002. Diabetes Care 29:1638–1644. Li, J., Li, N., Ma, M., et al. 2008. In vitro profiling of the endocrine disrupting potency of organochlorine pesticides. Toxicol. Lett. 183: 65–71. Longnecker, M. P., Klebanoff, M. A., Zhou, H., and Brock, J. W. 2001. Association between maternal serum concentration of the DDT metabolite DDE and preterm and small-for-gestational-age babies at birth. Lancet 358:110–114. Makita, Y., and Omura, M. 2006. Effects of perinatal combined exposure to 1,1-dichloro-2,2-bis-(p-chlorophenyl) ethylene and tributyltin on male reproductive system. Basic Clin. Pharmacol. Toxicol. 99: 128–132. Muller, A., Schott-Ohly, P., Dohle, C., and Gleichmann, H. 2002. Differential regulation of TH1- and TH2-type cytokine profiles in pancreatic islets of C57BL/6 and BALB/c mice by multiple low doses of streptozotocin. Immunobiology 205:35–50. National Cancer Institute (NCI). 1978. National Toxicology Program. Bioassays of DDT, TDE, and p,p0 -DDE for Possible Carcinogenicity. NCI Carcinogen Tech. Rep. Ser. 131:1–251.

p,p0 -DDE increases autoimmune T1D in a NOD mouse model

11

Padgett, L. E., Broniowska, K. A., Hansen, P. A., et al. 2013. The role of reactive oxygen species and pro-inflammatory cytokines in Type 1 diabetes pathogenesis. Ann. N.Y. Acad. Sci. 1281:16–35. Patterson, C. C., Dahlquist, G. G., Gyu¨ru¨s, E., et al, and the EURODIAB Study Group. 2009. Incidence trends for childhood Type 1 diabetes in Europe during 1989–2003 and predicted new cases 2005–20: A multicenter prospective registration study. Lancet 373:2027–2033. Rabinovitch, A. 1998. An update on cytokines in the pathogenesis of insulin-dependent diabetes mellitus. Diabetes Metab. Rev. 14: 129–151. Rehana, T., and Rao, P. R. 1992. Effect of DDT on the immune system in Swiss albino mice during adult and perinatal exposure: Humoral responses. Bull. Environ. Contam. Toxicol. 48:535–540. Rignell-Hydbom A., Elfving M., Ivarsson S. A., et al. 2010. A nested case-control study of intrauterine exposure to persistent organochlorine pollutants in relation to risk of Type 1 diabetes. PLoS One 5:e11281. Rignell-Hydbom, A., Rylander, L., and Hagmar, L. 2007. Exposure to persistent organochlorine pollutants and Type 2 diabetes mellitus. Human Exp. Toxicol. 26:447–452. Riley, J. L., June, C. H., and Blazar, B. R. 2009. Human T-regulatory cell therapy: Take a billion or so and call me in the morning. Immunity 30: 656–665. Rocha-Amador, D., Navarro, M., Trejo-Acevedo, A., et al. 2009. Use of the Rey-Osterrieth complex figure test for neuro-toxicity evaluation of mixtures in children. Neurotoxicity 30:1149–1154. Segre, M., Arena, S. M., Greeley, E. H., et al. 2002. Immunological and physiological effects of chronic exposure of Peromyscus leucopus to Aroclor 1254 at a concentration similar to that found at contaminated sites. Toxicology 174:163–172. Seo, M., Kobayashi, R., and Nagase, H. 2011. Immunotoxic effects of trichloroethylene and tetrachloroethylene. J. Health Sci. 57: 488–496. Shiplov, J., Graber, C. D., Keil, J. E., and Sandifer, S. H. 1972. Effect of DDT on antibody response to typhoid vaccine in rabbits and man. Immunol. Commun. 1:385–394. Shoda, L. K., Young, D. L., Ramanujan, S., et al. 2005. A comprehensive review of interventions in the NOD mouse and implications for translation. Immunity 23:115–126. Spencer, W. F., and Cliath, M. M. (Eds.). 1990. Movement of pesticides from soil to the atmosphere. In: Long Range Transport of Pesticides. New York: Lewis Publishers, pp. 1–4. Suedel, B. C., Boraczek, J. A., Peddicord, R. K., et al. 1994. Trophic transfer and biomagnification potential of contaminants in aquatic ecosystems. Rev. Environ. Contam. Toxicol. 136:21–89. Svensson, B. G., Hallberg, T., Nilsson, A., et al. 1994. Parameters of immunological competence in subjects with high consumption of fish contaminated with persistent organochlorine compounds. Int. Arch. Occup. Environ. Health 65:351–358. Tang, M., Chen, K., Yang, F., and Liu, W. 2014. Exposure to organochlorine pollutants and type 2 diabetes: A systematic review and meta-analysis. PLoS One 9:e85556. Taylor K. W., Novak R. F., Anderson H. A., et al. 2013. Evaluation of association between persistent organic pollutants (POP) and diabetes in epidemiological studies: A national toxicology program workshop review. Environ. Health Perspect. 121:774–783. Ter Veld, M. G., Zawadzka, E., van den Berg, J. H., et al. 2008. Foodassociated estrogenic compounds induce estrogen receptor-mediated luciferase gene expression in transgenic male mice. Chem. Biol. Interact. 174:126–133. Thayer, K. A., Heindel, J. J., Bucher, J. R., and Gallo, M. A. 2012. Role of environmental chemicals in diabetes and obesity: A National Toxicology Program workshop review. Environ. Health Perspect. 120:779–789. Turyk, M., Anderson, H., Knobeloch, L., et al. 2009. Organochlorine exposure and incidence of diabetes in a cohort of Great Lakes sport fish consumers. Environ. Health Perspect. 117:1076–1082. UNEP. 2011. Decoupling natural resource use and environmental impacts from economic growth. A Report of the Working Group on Decoupling to the International Resource Panel. Vine, M. F., Stein, L., Weigle, K., et al. 2001. Plasma 1,1-dichloro-2,2bis-(p-chlorophenyl)ethylene (DDE) levels and immune response. Am. J. Epidemiol. 153:53–63.