s2fieldsofstudy: Biology Chemistry

text:
Hydralazine Protects Nigrostriatal Dopaminergic Neurons From MPP+ and MPTP Induced Neurotoxicity: Roles of Nrf2-ARE Signaling Pathway

Although the pathogenic mechanisms of Parkinson's disease (PD) remain unclear, ample empirical evidence suggests that oxidative stress is involved in the pathogenesis of this disease. The nuclear factor E2-related factor 2 (Nrf2) is known to activate several antioxidant response element (ARE)-driven antioxidative genes that prevents oxidative stress in vitro and in vivo. Moreover, it was documented that hydralazine is a potent Nrf2 activator. In this study, we tested whether hydralazine can attenuate 1-Methyl-4-phenylpyridinium (MPP+) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- induced neurotoxicity in vitro and in vivo by activating Nrf2 and its downstream network of antioxidative genes. We found that treatment with hydralazine attenuated MPP+ or H2O2-induced loss of cell viability in human neuroblastoma cell line (SH-SY5Y). In addition, hydralazine significantly promoted the nuclear translocation of Nrf2, and upregulated the expression of its downstream antioxidative genes. Further, knockout of Nrf2 abolished the protection conferred by hydralazine on MPP+ -induced cell death. Similar findings were observed in vivo. Before, during, and after MPTP 30 mg/kg (i.p.) administration for 7 days, the mice were given hydralazine (Hyd) 51.7 mg/kg per day by oral gavage for 3 weeks. Oral administration of hydralazine ameliorated oxidative stress, MPTP-induced behavioral disorder, and loss of neurons of dopaminergic system in the substantia nigra (SN) and striatum, all of which were attributed to its ability to activate the Nrf2-ARE pathway. Hydralazine increased the migration of Nrf2 to the nucleus in dopaminergic neurons, enhanced the expression of its downstream antioxidative genes. Together, these datasets show that the Nrf2-ARE pathway mediates the protective effects of hydralazine on Parkinson's disease.

INTRODUCTION
Currently, the pathogenesis of Parkinson's disease (PD) is elusive. It has been postulated that prolonged increased in reactive oxygen species (ROS) plays important role in modulating the occurrence of the disease (1,2). This is because ROS compromises the mechanisms that balances oxidant and antioxidant systems (3). One of the mechanisms that prevent cell damage caused by oxidative stress is the antioxidant defense system. By increasing the level of antioxidant enzymes, phase II detoxifying enzymes, quinone oxidoreductase 1 (NQO1), NAD(P)H, heme oxygenase-1 (HMOX1), glutamate-cysteine ligase subunits (GCLC and GCLM), the nuclear factor E2related factor 2 (Nrf2) modulates the pathophysiological and physiological processes of various diseases (4). These enzymes are involved in glutathione (GSH) synthesis and maintains the form of GSH by suppressing its oxidized form GSSG (5,6). Several structural and molecular studies on Nrf2 revealed that Nrf2 is expressed in the cytoplasm in physiological conditions via a Kelch-like ECH-associated protein 1 (Keap1)-dependent ubiquitination-proteasomal degradation, and this process is enhanced by electrophiles and oxidants (7,8). It was revealed that the N terminal region and double glycine repeat domain (DGR) of Keap1 contains the bric-a-brac, tramtrack, broad-complex (BTB) domain, termed as Kelch repeats in the C terminal region. Keap1 keeps the Nrf2 within the cytoplasm in normal cellular conditions by binding to a scaffold protein of Nrf2 ubiquitin ligase (E3), Cul3 through its BTB domain, and binding to the substrate of Nrf2 through its DGR domain, which causes Nrf2 degradation (9) and ubiquitination (10). When stimulated by oxidants or electrophiles, Nrf2 undergoes modifications by some mechanisms which compromise Keap1/Nrf2 interactions, which leads to the dissociation of Nrf2 from Keap1 complex, become stabilized, and then moves into the nucleus. This is followed by binding to the antioxidant response elements (AREs), a consensus gene sequence located in the promoter region of several genes that encode antioxidant enzymes (11). Several studies have provided evidence that Nrf2-ARE signal transduction participates in PD (12,13). Downregulation of Nrf2 renders the dopaminergic neurons susceptible to oxidative stress damage (14)(15)(16), while activation of Nrf2 confers neuroprotection (17)(18)(19)(20)(21). Based on this background, the Nrf2-ARE interaction is thought to play important role in neurodegenerative conditions including PD.
Administration of pharmacological antioxidants seem to be the most direct approach to suppress oxidative damage. However, clinical trials on antioxidants e.g., N-acetylcysteine, vitamin E, glutathione, and vitamin C have revealed rather disappointing results for PD (22)(23)(24)(25). One possible reason is that the efficacy of these antioxidants is largely based on their ability to stoichiometrically scavenge for oxidants. An alternative promising therapeutic strategy of restoring redox homeostasis in PD by activating the transcription factor Nrf2 may have significant advantages over conventional strategies. Hydralazine (Hyd), an FDA approved treatment for hypertension, is a watersoluble carbonyl-scavenger due to its nucleophilic hydrazine group (26). Because of this, hydralazine has been found to be a powerful antioxidant properties (27)(28)(29), Moreover, it was reported that hydralazine could activate the Nrf2 signaling pathway in vitro [human neuroblastoma cell line (SH-SY5Y)] and in vivo (Caenorhabditis elegans) model system (30), and possesses anti-aging properties (31). As mentioned above, oxidative stress and the deregulation of Nrf2-ARE signaling pathways are both involved in PD. Thus, we hypothesized that hydralazine may provide strong neuroprotective effects in Parkinson's disease in both in vitro and in vivo settings. To test this possibility, we investigated the mechanisms that orchestrate the neuroprotective effects of hydralazine using an 1-Methyl-4-phenylpyridinium (MPP + )-induced cytotoxicity model and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mice model of PD. We found that hydralazine displayed promising therapeutic efficacy toward PD by activating the Nrf2 signaling pathway.

EXPERIMENTAL PROCEDURES
Preparation of Human Neuroblastoma SH-SY5Y Cells SHSY5Y cells were obtained from ATCC (ATCCCRL-2266) and grown in DMEM/F12 medium (hyclone) supplemented with 10% FBS (EVERY GREEN, Zhejiang Tianhang Biotechnology Co., Ltd, China), 100 µg/mL streptomycin, and 100 U/ml penicillin (Beijing solarbio science & technology co., Ltd) in high humidity condition with 5% CO 2 at 37 • C. After culturing the cells in 100 mm dishes to reach a ∼70% confluence, they were subjected to hydralazine, H 2 O 2 or MPP + treatment. The dose and duration of application of hydralazine, H 2 O 2 or MPP + are provided in the figures and text. SiRNA interference were performed by treating the cells with Nrf2 SiRNA (sc-37030) or control SiRNA (sc-37007) (Santa Cruz Biotechnology, Santa Cruz, CA) in 6-well plates for 24 h using the Lipofectamine 3000 reagent (Thermo Fisher Scientific Co., Carlsbad, CA, USA) as indicated in the instructions provided by the manufacturer. After transfection for approximately 24 h, SHSY5Y cells were exposed to hydralazine with or without MPP + . After these treatments, cells were used for biochemical analysis.

Cell Viability Evaluation by CCK-8 Assay
The cell counting kit-8 solution (CCK-8) assay was performed to determine the cell viability. Briefly, after seeding the SH-SY5Y cells in 96-well plates at a density of 1 × 10 4 cells/well, they were treated with reagents. This was followed by incubating with 10 µl CCK-8 buffer for 1 h at 37 C following the instructions provided by the kit company. A microplate reader (BioTek, Winooski, VT, USA) was used to measure the absorbance at 450 nm. All samples were assessed in triplicate.

Animals and Treatment
The mice used in this study were kept and handled according to the guidelines of the NIH Guide regarding the Use and Care of Laboratory Animals. All animals were given free water and food ad libitum, and the housing conditions were regulated to a 12 h dark-light cycle and temperature of (22 ± 2 • C). This study conformed to the guidelines of the Animal Care and Use Committees of Maximum efforts of Huazhong University of Science and Technology (HUST). Care was taken to use few animals and reduce discomfort. Male C57/BL6 mice, 8 weeks old, were used all of which were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. There are four groups (n = 8 or 9 for per group) in this experiment. The first group mice (MPTP group) only received injections of MPTP-HCl (30 mg/kg, i.p., Sigma) in saline for consecutive 7 days, an MPTP model of PD was generated as previously described (32). The second group mice (Hyd+MPTP group) were administered hydralazine (51.7 mg/kg per day in saline, Sigma) (33) by oral gavage for 3 weeks before, during, and after MPTP administration. The third group mice (Hyd group) were administered hydralazine (51.7 mg/kg per day in saline, Sigma) (33) by oral gavage for 3 weeks, and the fourth group (Control group) received vehicle only. Behavioral test of the animals were performed after the last oral gavage, after behavior test, mice were killed ( Figure 3A).

Rotarod Testing
The general motor deficits were evaluated using the rotarod test. Mice were put in a rod which was 7 cm in diameter, and then tested at a constant speed of 30 rpm. In each test, the mouse was subjected to the rod for 1 min prior to the test. Each animal was put on the roller for 5 min, and then the latency to fall off the rolling rod was calculated. Prior to assessing the behavior of the animals, they were given pre-trials for habituation to the test system for 4 days. An average of five trials were performed for each mouse.

Pole Testing
Each mouse was placed on the top of a vertical pole (50 cm long and 1 cm in diameter) wrapped with gauze to avoid slip and fall. After pre-trials for habituation to the test system for 4 days, time of the mouse head orient downward (named as Turn) and total time climbing down the pole as an indication of the locomotion activity (TLA) were recorded. Each mouse received five successive trials for average.

Tissue Preparation
One day after the behavior test, animals were killed by decapitation under anesthesia. Afterwards, brains were removed immediately was fixed in 4% buffered paraformaldehyde and embedded in paraffin for the next immunofluorescent staining. The ventral midbrain containing the substantia nigra and striatum of the other mice were dissected and frozen immediately in liquid nitrogen and stored at −80 • C for protein extraction.

Immunofluorescent Staining
After isolation, the substantia nigra and striatum tissues were embedded in paraffin and sectioned to 5 µm thickness. The sections were deparaffinized with xylene, followed by dehydration using ethanol. Finally, EDTA (PH 9.0) antigen retrieval buffer was used for antigen retrieval. These section were put on slices followed by treatment with 3% BSA at room temperature for 30 min, before they were incubated with the following primary antibodies at 4 • C overnight, mouse polyclonal anti-TH (1:200, Proteintech, China), rabbit polyclonal anti-Nrf2 (1:200, Genetex), rabbit monoclonal anti-IBA1(1:200, Abcam), rabbit monoclonal anti-GFAP (1:200, Abcam). Subsequently, secondary antibodies, Alexa Fluor 488-conjugated (1:500, Life Technologies) and/or Alexa Fluor 647-conjugated (1:500, Life Technologies) were added and incubated for 1 h at room temperature. SH-SY5Y cell were fixed with 4% paraformaldehyde for 30 min, washed in PBS, and permeabilized with 0.3% Triton X-100 in PBS for 10 min at room temperature. The cells were treated with rabbit polyclonal anti-Nrf2 (1:200, Genetex) antibody overnight at 4 • C after blocking with 10% normal goat serum for 1 h, and then incubated with Alexa Fluor 488conjugated (1:500, Life Technologies) secondary antibody for 1 h at room temperature. Images from the sections were examined on an OLYMPUS IX71 fluorescent microscope. Then quantitative analysis of the optical density and of number positive neurons was performed using Image pro plus.

Determination of Protein Expression
Nuclear and cytosolic fractions were extracted from the substantia nigra, striatum specimen and human neuroblastoma SH-SY5Y cells using cytoplasmic and nuclear protein extraction kit (Beyotime, Beijing, China) following the instructions given by the manufacturer. Briefly, cells were subjected to various treatments and then suspended in PBS. The next step involved the addition of the cytoplasmic protein isolation reagents A and B. The cytosolic fraction was obtained by centrifugation. The nuclei pellets were suspended in the isolation reagent for nuclear protein. Similarly, the nuclear fraction was obtained by centrifugation. All the isolated proteins from mouse striatum, ventral, and SH-SY5Y cells were treated on ice with RIPA lysis buffer comprising of phosphatase inhibitor A and B, protease inhibitor PMSF (PMSF:RIPA = 1:99), cocktail (Servicebio, Wuhan, China), followed by centrifugation at 12,000 g, for 15 min at 4 • C, to obtain the supernatants. BCA Protein Assay Kit was applied to measure the protein concentration. This was followed by denaturation of the protein specimen using the sodium dodecyl sulfate solution together with boiling for 5 min at 98 • C. About 30 µg sample was loaded onto SDS-PAGE after which electrophoresis was performed to transfer the proteins to a PVDF membrane. Thereafter, 5% milk was using to block the membranes at room temperature for 1 h before primary antibodies, rabbit monoclonal anti-β-actin (1:3000, Antgene, China), rabbit polyclonal anti-Histone H3 (1:1000, Servicebio, China), mouse monoclonal anti-GCLC (1:500, Santa Cruz), rabbit polyclonal anti-Nrf2 (1:1000, Genetex), rabbit monoclonal anti-NQO1 (1:10000, Abcam), rabbit monoclonal anti-HMOX1 (1:2000, Abcam), rabbit monoclonal anti-GCLM (1:5000, Abcam), and rabbit polyclonal tyrosine hydroxylase antibody (1:1000, Proteintech, China) were added and incubated overnight at 4 • C. Finally, HRP-conjugated secondary antimouse antibody (1:5000, Antgene, China) or HRP-conjugated secondary anti-rabbit (1:5000, Antgene, China) was added to the membranes after washing with TBST thrice (5 min/each time) for 1 h at room temperature. After washing, ECL was loaded onto the membrane to detect the immunoreaction using the Bio-Rad imaging system. The branes were analyzed with Imagine J software. Relative band intensities were measured with β-actin or Histone H3 serving as internal control.

Determination of the Oxidative Activity by Lipid Peroxidation Assay
The generation of malondialdehyde (MDA) was measured using the lipid peroxidation commercial assay kit following the instructions provided by the manufacturer. Briefly, and tissue were homogenized, sonicated, and centrifuged. The MDA concentration on in the supernatants was measured as well as the protein concentration. The protein concentration was determined using the assay kit (Bio-Rad Laboratories, Hercules, USA). This was followed by performance of the MDA assay following the instructions provided with the Lipid Peroxidation MDA assay kit (Beyotime). The multimode microplate reader was used to quantify the MDA levels in the samples at 532 nm.

Determination of Cellular Levels of GSSG and GSH Using HPLC
Substantia nigra samples were homogenized, sonicated and centrifuged. In brief, 30 µl supernatant was isocratically eluted through a 4.6 × 150 mm C18 column (ESA, Inc.) with a mobile phase containing 50 mM LiH 2 PO 4 , 1.0 mM 1-octanesulfonic acid, and 1.5% (v/v) methanol, and then detected by a 2-channel Coulochem III electrochemical detector (ESA, Inc), set with a guard cell potential 950 mV, Channel 1 potential for GSH detection and Channel 2 potential 880 mV for GSSG detection (34). The quantities of GSSG and GSH were presented as nmol per mg protein.

Data Analysis
All data are expressed as mean ± SEM and were performed using the SPSS20 software. Between-group comparisons were conducted by Student's t-test, while the one-way ANOVAs was used to compare three or more groups followed by Tukey's multiple comparison test. A P-value of < 0.05 was considered to be statistically significant.

RESULTS
Hydralazine Prevents the MPP + and H 2 O 2 -Induced Cell Death in SH-SY5Y Cells, and Activates Nrf2-ARE Signaling in Both Treated and Non-treated MPP + Cells in vitro Analysis of the influence of MPP + on SH-SY5Y viability revealed that the cell viability decreased to about 50% or below based on CCK-8 assays when exposed to 1000 µM (1 mM) MPP + for 24 or 36 h ( Figure 1A). Therefore, we treated SH-SY5Y cells with MPP + 1 mM for 24 h to establish a model of MPP +induced cytotoxicity. Next, the effects of hydralazine on the survival of cells following exposure to MPP + were tested. Four doses of hydralazine (2.5, 5, 10, or 20 µM) were tested against MPP + . Hydralazine alone at the micromolar range (2.5-20 µM) had no overt effects on these cells. But, hydralazine (10 and 20 µM) conferred significant protection against MPP + according to the CCK-8 assays ( Figure 1B). Moreover, we chose H 2 O 2 (100 µM) as another control of toxicity (30). Similarly, hydralazine protected the cells from H 2 O 2 -induced cytotoxicity ( Figure 1C). Moreover, we observed that hydralazine (10 and 20 µM) alone increased the viability of SH-SY5Y in the absence of MPP + and H 2 O 2 (Figures 1B,C). It is wellknown that during cellular metabolism, spontaneous oxidative damage to unsaturated lipids generates many electrophilic carbonyl compounds which are potential threats to cell survival. Since hydralazine is a well-known carbonyl scavenger (35), we speculated that it can improve SH-SY5Y viability by scavenging Carbonyl moieties.
Thereafter, we tested the ability of hydralazine to induce nuclear translocation of Nrf2. SH-SY5Y cells were treated with 10 µM hydralazine with or without MPP + , but the control groups were treated with saline following Nrf2 partition quantification and subcellular fractionation. Hydralazine treatment increased the nuclear translocation of Nrf2, which was accompanied by a corresponding decreased in the cytosolic fraction relative to the nuclear Nrf2 fraction (Figures 1D-F). The same phenomenon of nuclear translocation was observed based on immunofluorescence ( Figure 1G). As showed in Figures 1D-G, treatment with hydralazine alone triggered a remarkable increase in the nuclear translocation of Nrf2, but the reasons for this effect remain unclear. It is likely that Nrf2 was modified by hydralazine in a way that disrupted the Nrf2:Keap1 interaction, and then, translocated to the nucleus. Further studies are required to answer this question. Next, we determined whether hydralazine (10 µM) would increase the downstream protein of the Nrf2-ARE pathway, . Cell viability was determined by CCK-8 assay. *p < 0.05, **p < 0.01, n = 6, compared with the two indicated groups. SH-SY5Y cells were incubated with or without hydralazine (2.5, 5, 10, 20 µM) in the absence or presence of 1 mM MPP + for 24 h (B). The viability of cells under MPP + -mediated cytotoxicity was significantly improved with hydralazine (10 and 20 µM) treatment. *p < 0.05, **p < 0.01, ***p < 0.001, n = 6, significant difference between the two indicated groups. SH-SY5Y cells were incubated with or without hydralazine (2.5, 5, 10, 20 µM) in the absence or presence of H 2 O 2 (100 µM) for 24 h (C). The viability of cells under H 2 O 2 -induced cytotoxicity was significantly improved with hydralazine (10 and 20 µM) treatment. *p < 0.05, **p < 0.01, ***p < 0.001 n = 6, significant difference between the two indicated groups. The results of immunofluorescence and western blot analysis indicated Nrf2 translocated to the nucleus with hydralazine treatment when exposured to MPP + or not in SH-SY5Y (D-G). Scale bar =500 µm. Treated cells were subjected to cell fractionation and western blot analysis. β-actin or Histonen H3 served as a loading control. *p < 0.05, **p < 0.01, ***p < 0.001, n = 6, significant difference between the two indicated groups. Hydralazine induces expression of Nrf2-dependent antioxidant enzymes measured by western blot analysis (H,I). *p < 0.05, **p < 0.01, ***p < 0.001, n = 6, significant difference between the two indicated groups. Data were presented as mean ± SEM. GCLC, GCLM, HMOX1, and NQO1 by western blot analysis. All four protein levels of target genes were significantly increased (Figures 1H,I). Collectively, the in vitro findings indicate that hydralazine induce nuclear translocation of Nrf2 which activates the ARE genes, thereby conferring protection against MPP + .
Hydralazine Activate Nrf2-ARE Signaling and Attenuate MPP + -Mediated Cytotoxicity in an Nrf2-Dependent Pattern To confirm whether the Nrf2-dependent pathway mediated the neuroprotective effects of hydralazine, the inhibitory effect of hydralazine on MPP + neurotoxicity was assessed in SH-SY5Y cells after transfection with either control SiRNA or Nrf2 SiRNA. Results from western blotting and real-time PCR indicated that Nrf2 was successfully knocked-down in SH-SY5Y cells by Nrf2 SiRNA compared to control SiRNA (Figures 2A-C). Treatment with hydralazine alone triggered a remarkable increase in the nuclear translocation of Nrf2 (Figures 2D-F). However, Nrf2 interference abolished the increase in nuclear Nrf2 caused by hydralazine in SH-SY5Y cells. On the other hand, cytosolic Nrf2 displayed a corresponding decrease relative to the control group transfected with control SiRNA. Furthermore, the hydralazineinduced increase in expression of Nrf2-mediated ARE genes, such as GCLC, GCLM, HMOX1, and NQO1 were inhibited following Nrf2 SiRNA in SH-SY5Y cells based on western blot analysis (Figures 2G,H). According to these results, we speculate hydralazine activate Nrf2-ARE signaling in an Nrf2dependent manner.
Meanwhile, our results indicated that hydralazine treatment increased cell viability, which is opposite to the CCK8 assay results recorded in MPP + exposure alone group in SH-SY5Y cells transfected with control SiRNA (Figure 2I, p < 0.01). However, hydralazine failed to increase cell viability following Nrf2 knockdown and MPP + exposure ( Figure 2I). Nrf2 SiRNA transfection in SH-SY5Y cells increased their vulnerability to MPP + neurotoxicity as compared to the control SiRNA transfection group (Figure 2I, p < 0.05). These data reveal that the protective effect of hydralazine on SH-SY5Y cells exposed to MPP + neurotoxicity is dependent on the Nrf2 pathway.

Hydralazine Confers Protection in Dopaminergic Neurons in the MPTP Model of Parkinson's Disease
To explore the therapeutic effect of hydralazine in PD, the MPTP mice model was used. Mice were divided into four groups (n = 9 for per group) for this experiment. The first group mice (MPTP group) only received injections of MPTP-HCl (30 mg/kg, i.p., Sigma) in saline for consecutive 7 days, an MPTP model of PD was generated as previously described (32). The second group of mice (Hyd+MPTP group) were administered with hydralazine (51.7 mg/kg per day in saline, Sigma) (33) by oral gavage for 3 weeks before, during, and after MPTP administration. The third group mice (Hyd group) were administered with hydralazine (51.7 mg/kg per day in saline, Sigma) (33) by oral gavage for 3 weeks, and the fourth group mice (Control group) received vehicle only. The pretreatment paradigm in the MPTP-induced PD Model was utilized ( Figure 3A). We observed that the weight of MPTP-treated mice was much lower compared to normal group due to the neurotoxicity of MPTP. But administration of hydralazine alleviated the loss of weight ( Figure 3B). Behavior testing of the animals was performed after the last oral gavage (Figure 3A). The rotarod test was applied to evaluate motor and coordination abilities. In this test, the decrease in latent time on the rod in the MPTP mice compared to the vehicle treated control mice (p < 0.001) was reversed by hydralazine treatment (p < 0.01) (Figure 3C). Similar results were obtained in Pole test. The time to orient downward (Turn) and to descend (TLA) was increase in the MPTP mice compared to the control mice (p < 0.05), indicating a motor deficit, and this effect was reversed by hydralazine (p < 0.05) (Figure 3D). These results suggested that hydralazine conferred protection in locomotion function.
After the behavioral tests, we tested whether in vivo hydralazine can maintain the integrity of dopaminergic neurons in the SNpc and their terminal fibers in striatum. Thus, we performed immunofluorescence staining using dopaminergic neuronal specific marker, anti-TH antibody. MPTP administration caused remarkable loss of dopaminergic neurons in the SNpc (Figures 3E,F) and striatal DA terminal fibers (Figures 3I,J) when compared to control. Meanwhile, we found that reduction of TH-positive neurons was abolished in the similar anatomic level sections of SN in the MPTP mice treated with hydralazine than MPTP alone (Figure 3E). Quantification analysis using the cell counting kit in a double blind way indicated a statistically significant preservation of TH positive dopaminergic SN neurons in the MPTP-treated mice with hydralazine groups compared with MPTP mice (Figure 3F, p < 0.01). This observation was further confirmed by western blotting assay on SN lysates ( Figure 3G). The ratio of TH/βaction in the MPTP group was significantly lower than in control group (p < 0.001) and MPTP mice with hydralazine groups (Figure 3H, p < 0.05). Hydralazine-treated mice in the presence of MPTP showed significant reductions in the loss of the TH positive DA terminals in the striatal region when compared to MPTP alone mice (Figures 3I,J, p < 0.01). Consistent with TH-immunofluorescent in the striatal region, western blotting of the striatum lysates rescued the loss of TH positive DA terminals in the hydralazine-treated mice with MPTP compared to MPTP alone mice (Figures 3K,L, p < 0.001), Taken together, these findings provide evidence that hydralazine confers protection on nigrostriatal dopaminergic neurons in the subacute model of MPTP neurotoxicity.

Hydralazine Alleviate Oxidative Stress and Activates Nrf2-Triggered Gene Expression in vivo
Subsequently, we investigated whether hydralazine activates Nrf2-ARE pathway in vivo using mice. Mice were treated as described above to assess the level of MDA, a key product produced from membrane lipid oxidation. Mice treated FIGURE 2 | Hydralazine activate Nrf2-ARE signaling and attenuate MPP + -mediated cytotoxicity in an Nrf2-dependent pattern. SH-SY5Y cells were treated with either an Nrf2 SiRNA or a control SiRNA. The protein and mRNA levels of Nrf2 were measured by western blotting and real-time PCR (A-C). *p < 0.05, **p < 0.01, n = 6, significant difference between the two indicated groups. Hydralazine treatment alone induced a remarkable increase in the nuclear translocation of Nrf2, however, Nrf2 interference abolished the increase in nuclear Nrf2 induced by hydralazine in SH-SY5Y (D,E), in the meantime, cytosolic Nrf2 had corresponding decrease in contrast (Continued) FIGURE 2 | with control group transfected with control SiRNA (D,F) by western blot analysis. *p < 0.05, **p < 0.01, n=6, significant difference between the two indicated groups. Selective activation of Nrf2 mediated gene transcription by hydralazine in SH-SY5Y cells transfected by control SiRNA, but not in SH-SY5Y cells transfected by Nrf2 SiRNA measured by western blotting (G,H). *p < 0.05, n = 6, significant difference between the two indicated groups. Hydralazine significantly protected SH-SY5Y cells from MPP + induced death in an Nrf2-dependent manner measured by CCK-8 assay (I). *p < 0.05, **p < 0.01, n = 6, significant difference between the two indicated groups. Data were presented as mean ± SEM.
with MPTP displayed a remarkably increase in MDA levels which were suppressed by hydralazine treatment in the SNpc ( Figure 4A) and in striatum ( Figure 4B). Glutathione (GSH) is an antioxidant which has the capacity to prevent damage to important cellular components. Glutathione disulfide (GSSG) is its oxidized form and the ratio of oxidized glutathione to reduced glutathione is frequently used to evaluate oxidative stress. MPTP decreased GSH levels and increased GSSH levels and these effects were reversed by hydralazine in the SNpc, as supported by the increased ratio of GSH/GSSG (Figures 4C,D).
It can be inferred that Nrf2-ARE signaling suppressed cellular oxidative stress caused by several factors. These experimental results demonstrate that hydralazine suppresses oxidative stress triggered by MPTP. We further explored whether this effect was due to regulation of the Nrf2-ARE signaling pathway. Figures 4E-G, shows that hydralazine induced a dramatic increase in the nuclear translocation of Nrf2 in the SNpc of hydralazine-treated mice with MPTP, compared with the MPTP mice (p < 0.001), corresponding decrease in cytosolic Nrf2 fraction (p < 0.01). Moreover, hydralazine treatment alone enhanced the nuclear Nrf2 levels when compared to control (Figures 4E,F, p < 0.01). The cytosolic Nrf2 fraction decreased slightly compared to nuclear Nrf2 fraction (Figures 4E,G). This observation was further confirmed by double immunofluorescence (Figure 4H) in the SNpc. Consistent with SNpc, western blotting assay on the striatum lysates showed a statistically significant increase in the Nrf2 nuclear translocation, and a corresponding decrease in cytosolic Nrf2 fraction, in both hydralazine-treated mice with MPTP and hydralazine alone mice when compared to control (Figures 4I,J). Based on these results, it was observed that hydralazine activated Nrf2-ARE signaling. Moreover, we observed that Nrf2 colocalized with TH (dopaminergic neuron marker) in the SNpc, but not with ionized calcium binding adapter molecule 1 (IBA1, microglial cell marker) or glial fibrillary acidic protein (GFAP, astrocyte marker) as revealed by double immunofluorescence in the SNpc of hydralazine-treated mice with MPTP. Hydralazine increased the nuclear translocation of Nrf2 in the dopaminergic neuron (Figure 4L), which explained why hydralazine protected dopaminergic neurons from MPTP induced neurotoxicity to a certain extent.
In the hydralazine treatment group, western blot assay revealed that Nrf2-ARE targeted genes, NQO1, HMOX1, GCLM, and GCLC, were significantly increased in the SNpc (Figures 4M,N) and in striatum (Figures 4O,P) after compare with MPTP alone. Additionally, treatment with hydralazine alone increased the expression of above ARE genes compared to control group in both SNpc (Figures 4M,N) and striatum (Figures 4O,P). Based on the above discussion, the mechanism by which hydralazine activated Nrf2-ARE signaling even in the absence of toxicant was not clear. This should be explored in further studies. In conclusion, these data provide in vivo evidence that hydralazine can activate the Nrf2-mediated ARE gene transcription which provides antioxidant effects in vivo.

DISCUSSION
Current treatment for PD are primarily designed relief some of the clinical symptoms which hardly translated to improved quality of life in most patients. In addition, there is no known therapy that prevents the pathogenesis and progress of neurodegeneration. Although several studies have been performed to assess the etiology of PD, the associated mechanisms are still not fully understood. Mounting evidence suggests that oxidative stress appears to be involved in the pathogenesis of PD (36). Consequently, we have reasons to believe that increasing the capacity of dopaminergic neurons to confront oxidants could serve as an important strategy to prevent the onset and/or to delay the progression of PD. However, the prevailing knowledge from clinical trials on antioxidants such as ubiquinone, glutathione, N-acetylcysteine, vitamin C, and vitamin E reveals there are conflicting outcomes on their efficacy (22)(23)(24)(25). It is likely that oxidants may exert influence by activating death-related pathways rather than directly killing dopaminergic neurons (37), relying only on stoichiometric scavenging of oxidants has proved to be ineffective. The Nrf2 signaling cascade modulates transcriptional expression of oxidative stress to restore redox homeostasis and is a versatile pathway for neurotherapeutics. Several genetic studies have demonstrated that a functional haplotype in the Nrf2 gene promoter, which conferred high transcriptional activity had a protective effect (38), and the deficiency of Nrf2 increased hypersensitivity of the dopaminergic neurons to neurotoxicity caused by MPTP (14,39,40) and 6-OHDA (15) in animal models. Moreover, in vitro Nrf2 activation also provides neuroprotective effect against the neurotoxins paraquat (41), 6-OHDA (42,43), and MPP + (44). These datasets imply that the Nrf2 pathway may provide neuroprotection which can be exploited to develop drugs that treat PD.
Hydralazine, which was developed by Swiss researchers in the early 1950s (45), was approved drug by FDA for antihypertensive treatment. In this study, we demonstrate that hydralazine displays remarkable neuroprotective effects in a model of PD in vivo and in vitro. Oral administration of hydralazine decreased MDA levels, increased the ratio of GSH/GSSG, alleviated weight loss and the associated motor deficits, attenuated dopaminergic neurons loss and activated Nrf2 signaling pathways, all of which exerted profound neuroprotective activities in MPTP model of PD (Figure 3), and these effects were further confirmed in human  D). Performance on the rotarod and pole test was impaired in MPTP-treated group. However, impairment was ameliorated in MPTP model treated with hydralazine. *p < 0.05, **p < 0.01, ***p < 0.001, significant difference between the two indicated groups (n = 8). Tyrosine hydroxylase immunofluorescence of substantia nigra pars compacta (SNpc). Scale bar = 250 µm. We found that there were fewer TH positive neurons in the similar anatomic level sections of SN in the MPTP mice than hydralazine-treated mice, MPTP mice with hydralazine and control mice, compared to the MPTP mice group, MPTP mice with hydralazine group significantly attenuated the loss of dopaminergic neurons in SNpc (E,F). *p < 0.05, **p < 0.01, ***p < 0.001, n = 6, very significant difference between the two indicated groups. The western blotting of TH in SN further reconfirmed the findings (G and H). *p < 0.05, ***p < 0.001 n = 6, significant difference between the two indicated groups. The MPTP neurotoxicity also resulted the decrease of the TH positive DA terminals in the striatal region of the MPTP mice group compared with MPTP mice with hydralazine group. (I,J). Scale bar = 500 µm. **p < 0.01, ***p < 0.001, n = 6, very significant difference between the two indicated groups. Immunoblotting also validated this observation (K,L). **p < 0.01, ***p < 0.001, n = 6, compared with the two indicated groups. Data were presented as mean ± SEM. FIGURE 4 | Hydralazine alleviate oxidative stress and activates Nrf2-triggered gene expression in vivo. Hydralazine significantly decreased MDA in the substantia nigra (A) and striatum(B) of MPTP mice treated with hydralazine compared with MPTP mice. *p < 0.05, n = 5, significant difference between the two indicated groups. MPTP administration showed a remarkable decrease in GSH levels and increase in GSSH that was inhibited by hydralazine in the SNpc, corresponding increase in the ratio of GSH/GSSG (C,D). *p < 0.05, **p < 0.01, n = 5, significant difference between the two indicated groups. Hydralazine treatment leads to a shift in Nrf2 migration and increases nuclear Nrf2 Protein levels, in the meantime, cytosolic Nrf2 had corresponding decrease in the substantia nigra (E-H) and striatum (I-K) of (Continued) FIGURE 4 | MPTP-treated mice. **p < 0.01, ***p < 0.001. n = 6, significant difference between the two indicated groups. Nrf2 colocalized with TH in the SNpc, not with IBA1, GFAP by double immunofluorescence (L) in the SNpc of hydralazine-treated mice with MPTP, hydralazine can increase nuclear translocation of Nrf2 in the dopaminergic neuron (L). Protein levels of Nrf2 were analyzed by Western blot. β-actin and Histonen H3 are used as markers for cytosolic and nuclear fractions, respectively. Hydralazine treatment increased the expression of Nrf2 downstream target in the substantia nigra (M,N) and striatum (O,P) of MPTP-treated mice. *p < 0.05, **p < 0.01, ***p < 0.001, n = 6, significant difference between the two indicated groups. Data were presented as mean ± SEM.
neuroblastoma SH-SY5Y cells (Figure 1). The cell viability was increased following hydralazine treatment when exposed to MPP + or H 2 O 2 . Among the key Nrf2-regulated genes stimulated by hydralazine were NQO1, HMOX1, GCLM, GCLC. It was reported NQO1 ameliorated the baleful effects of DA quinines, and had various antioxidant properties (46,47). HMOX1 has been implicated in the metabolism of the pro-oxidant heme to form the antioxidant pigment biliverdin which confers resistance to chronic oxidative stress and apoptosis in dopaminergic neurons (48,49). GCLC and GCLM regulates the production of cellular antioxidant glutathione (50). These proteins are involved in oxidative stress and redox homeostasis. Nrf2 SiRNA transfection was performed to explore the function of Nrf2 in the protective effect of hydralazine against neurotoxicity MPP + induced in SHSY5Y cells. Nrf2 SiRNA2 transfection treatment abolished the hydralazine-induced ARE genes expression and increased the vulnerability to MPP + neurotoxicity (Figure 2). However, we lacked Nrf2 knockout mice and Nrf2 knockin mutant mice to perform more confirmatory experiments. A noteworthy phenomenon is that hydralazine treatment alone increased nuclear translocation of Nrf2 and elevated the Nrf2-mediated ARE gene transcription in vitro and in vivo (Figures 1, 2, 4). But the mechanism for this effect remains elusive. It is possible that Nrf2 was modified by hydralazine in a way that disrupted the Nrf2:Keap1 interaction, which then allowed it to translocate to the nucleus where it binds the antioxidant response elements (AREs), a consensus gene sequence present in the promoter region of a large number of genes encoding antioxidant enzymes (11). In the future, we will further explore this concept. In addition, we found that hydralazine increased the migration of Nrf2 to the nucleus in dopaminergic neurons, induced the expression of its downstream antioxidative genes, although we did not observe this phenomenon in microglial cells and astrocytes. Collectively, our experimental results demonstrate the ability of hydralazine to modulate key antioxidant genes which are crucial to the survival of SH-SY5Y cells and dopaminergic neurons in PD model.
Hydralazine has been known to act as an antioxidant which eliminates acrolein, an agent that transduces oxidative stress signals (51, 52) and a potent Nrf2 activator (30). Here, we demonstrate that Nrf2 activation is a novel mechanism by which hydralazine exerts protection in PD. However, additional investigations are required to explain how this nucleophilic drug activates the Nrf2 pathway. One possibility is that hydralazine directly disrupts the interaction between Keap-1 and Nrf2 (53). It is also likely that hydralazine activates Nrf2 pathway through other pathways e.g., Notch or AMP kinase pathways, nuclear factor-kappa B (NF-κB), synoviolin, the GSK-3-β-TrCP, and PI3K/Akt pathway (54), which are reported to interact with Nrf2 elements. Another possibility is that the "adduct-trapping" properties of hydralazine which involves the classic electrophile-mediated pathway to increase the degradation of Keap1, thus accelerate Nrf2 liberation (55). Certainly, there is little evidence that hydrazaline crosses the Blood-Brain Barrier, However, this drug has been studied in the Alzheimer's disease mouse model (33). Together with the findings obtained in this study, we speculate that hydrazaline has some degree of permeability through the Blood-Brain Barrier. To improve its clinical application, its oral bioavailability, in vivo pharmacokinetic profiles, permeability through Blood-Brain Barrier and the potential side effects need to be explored.

CONCLUSION
This study shows that hydralazine as a potent Nrf2 activator, increases the expression of Nrf2 downstream ARE target genes and confers strong neuroprotection in model of PD both in vitro and vivo in a Nrf2 -independent manner. These datasets provide novel insights that have high therapeutic value for Parkinson's disease.

ETHICS STATEMENT
All animal procedures were in strict accordance with the National Institutes of Health's Guidelines for Care and were approved by the Animal Care and Use Committees of Huazhong University of Science and Technology (HUST).


Lifetime Pesticide Use and Antinuclear Antibodies in Male Farmers From the Agricultural Health Study

Farming and pesticide use have been associated with systemic autoimmune diseases, and while certain organochlorine insecticides and other pesticides are suspected to influence risk, the role of specific pesticides in the development of systemic autoimmunity is not known. We measured serum antinuclear autoantibodies (ANA) by immunofluorescence on Hep-2 cells in 668 male farmers in the study of Biomarkers of Exposure and Effect in Agriculture (BEEA; 2010–2013), an Agricultural Health Study (AHS) subcohort. We examined ANA in relation to lifetime use of 46 pesticides first reported at AHS enrollment (1993–1997) and updated at intervals through BEEA enrollment. Odds ratios (OR) and 95% confidence intervals (CI) were estimated after adjusting for age, state, education, season of blood draw, current pesticide use, and correlated pesticides. Having ANA antibodies (3 or 4+ intensity at a 1:80 dilution, 21% of study participants) was associated with a reported history of seeking medical care due to exposure to pesticides (OR 2.15; 95%CI 1.17, 3.95), use of the fumigant methyl bromide (OR 3.16; 95%CI 1.05, 9.5), and use of petroleum oil/distillates (OR 1.50; 95%CI 1.00, 2.25). Using a higher threshold (3 or 4+ at a 1:160 dilution, 9%) ANA positivity was associated with the carbamate insecticide aldicarb (OR 4.82; 95%CI 1.33, 17.5) and greater combined use of four cyclodiene organochlorine insecticides (top tertile of intensity-weighted lifetime days vs. no use; OR T3 3.20; 95%CI 1.10, 9.27). By contrast, greater use of non-cyclodiene organochlorine insecticides was inversely associated with ANA (1:80 dilution 3 or 4+, OR T3 0.24; 95%CI 0.08, 0.72). Specific autoantibodies (to extractable nuclear antigens and anti-dsDNA), measured on those with ANA detected at the 1:80 dilution 3 or 4+, were seen in 15 individuals (2%), and were associated with use of two or more cyclodiene organochlorine insecticides and several other pesticides (e.g., carbofuran, ethylene dibromide). These findings suggest that specific pesticide exposures may have long-term effects on ANA prevalence and support the hypothesis that certain organochlorine insecticides may increase the risk of developing systemic autoimmunity.

INTRODUCTION
Farming occupation and pesticide use have been associated with elevated risk of systemic autoimmune diseases (1)(2)(3)(4)(5)(6)(7)(8)(9), but the role of specific pesticide exposures in the development of systemic autoimmunity is not known. Systemic autoimmunity, as measured by anti-nuclear antibodies (ANA; based on an intensity of 3+ or 4+ at the 1:80 titer), is estimated to occur in 12 to 16% of the US population ages 12 and older, with higher rates in women and older adults (10). Overall ANA positivity, including lower levels of ANA (i.e., at the 1:40 titer), is even more common, seen in almost a third of healthy adults (11). By contrast, higher level ANA (i.e., 1:160 titer and higher) are considered more likely to be clinically relevant; elevated ANA are typically present in patients with systemic lupus erythematosus, an uncommon autoimmune disease, and ANA prevalence is often elevated in family members and patients with other systemic autoimmune diseases (12,13).
Various pesticides have immunotoxic effects and case series have suggested associations with autoantibodies (14). Only a few epidemiologic studies have examined ANA in relation to specific agricultural pesticides. In a cross-sectional study of Canadian farmers, ANA prevalence was significantly higher in association with use of some, but not all organochlorine insecticides (15). In another similar study from the same region, both positive and inverse associations were seen for ANA with the recent use of different herbicides and fungicides, though estimates were imprecise due to small sample size (16). Other studies have examined serum levels of organochlorine pesticides in relation to ANA, with inconsistent findings (17)(18)(19).
In the current study, we examined associations of lifetime pesticide use with ANA in a sample of male farmers from the Biomarkers of Exposure and Effect in Agriculture (BEEA) study in the Agricultural Health Study (AHS) (20), a large prospective cohort that includes farmers in Iowa and North Carolina (21). Cross-sectional studies show that ANA prevalence rises slowly with age over several decades (10,22,23). In theory, the loss of immune tolerance enabling the production of antibodies to self-antigens may occur at any time in the past, thereby conferring life-long potential to produce ANA. Therefore, our analyses focused on the association of ANA with lifetime exposures to pesticides, with a specific focus on the organochlorine insecticides.

Study Population and Sample
The design and methodology for enrollment and specimen collection in the AHS and BEEA study have been described (20,21). BEEA participants were recruited from among the male private pesticide applicators in the AHS who were ≥50 years of age, resided in Iowa or North Carolina, had completed the AHS enrollment questionnaire as well as two follow-up interviews (in 1999-2003 and 2005-2010), and had never been diagnosed with cancer (other than non-melanoma skin cancer). In the current investigation, we assessed ANA and other markers of autoimmunity in 699 participants enrolled in BEEA between June 2010 and September 2013. Because a diagnosis of autoimmune disease might have influenced pesticide use or reporting, we excluded 31 individuals who reported a doctor diagnosis of systemic autoimmune disease at AHS enrollment, for a final analysis sample of 668 participants. The study was approved by Institutional Review Boards at the National Cancer Institute and other participating institutions, and all participants provided written informed consent.

Questionnaire Data
Lifetime history of 50 specific pesticides was assessed based on participant report at AHS enrollment in 1993-1997. Pesticide use was updated in two follow-up surveys in 1999-2003 and 2005-2010, and at BEEA enrollment. All participants completed these four questionnaires, and 62% (N = 410) also completed a "takehome" questionnaire at AHS enrollment that solicited additional detailed information on the use of 28 of the pesticides, including organochlorine insecticides. The enrollment questionnaire also ascertained information about days per year and years of use pesticides overall, along with mixing and application practices, use of protective equipment, and repairing equipment. Data were updated in the follow-up surveys and used to derive intensity-weighted lifetime days of use (24). In the enrollment questionnaire, participants were also asked whether they had ever sought medical care or been hospitalized due to pesticide exposure, and in the take home questionnaire they were asked about high pesticide exposure events (HPEE) and whether they ever had ever been diagnosed with pesticide poisoning (25). Data on HPEE, medical treatment and pesticide poisoning were updated in follow-up surveys and combined as a single variable with three levels: (1) any medical treatment due to pesticides or pesticide poisoning diagnosis, (2) HPEE without medical treatment, and (3) neither. A health history, including diagnosis with rheumatoid arthritis and systemic lupus erythematosus, was collected at enrollment and throughout follow-up. BEEA enrollment questionnaires also collected data on current medication use, which were used to identify use of disease modifying antirheumatic drugs (DMARDs) and non-steroidal anti-inflammatory drugs. Questionnaires are available at https:// aghealth.nih.gov/collaboration/questionnaires.html; the primary DMARD medications reported included methotrexate and hydroxychloroquine, as well as leflunomide, sulfasalazine, azathioprine, mycophenolate mofetil, cyclosporine, and biologics, but not steroids.

Assays
Non-fasting blood specimens were collected in the participant's home and transported and shipped cold via overnight delivery before processing, aliquoting, and storage at −80 • C. Antinuclear antibodies (ANA) were measured by the gold standard immunofluorescence assay for serum samples under a standardized protocol in a clinical rheumatology laboratory with prior experience with high-throughput testing in epidemiologic studies (26); in brief, ANA were detected using HEp-2 cell slides (Kallestad, Bio-Rad Laboratories, Hercules, CA), incubated with a 1:80 dilution of sera, then washed and incubated with the burro anti-human polyvalent immunoglobulin FITC conjugate (Kallestad) read using fluorescent microscopy (Leitz Fluorescence Scope, 50/1.0 magnification). Samples with a positive ANA (3 or 4+ at the 1:80 dilution) were subsequently tested for extractable nuclear autoantibodies (ENA), including RNP, SM, SSA (combined 52 and 60 kDa) and SSB (QUANTA Lite, Inova Diagnostics, San Diego, CA; RNP/SM/SSBochterlony; SSA-ELISA) and anti-dsDNA antibodies (EIA, Kallenstad). Anti-citrullinated peptide (anti-CCP) antibodies, specific for rheumatoid arthritis (27), were measured by secondgeneration ELISA; any detectable anti-CCP were seen in 2.3% of the study sample and <1% were positive based on a clinical cut-point of 25 AU/ml, within the expected prevalence in the general population (27). Testing for anti-TPO antibodies was previously described, with antibodies seen in 9% of the study sample (28). Coefficients of variation were <10% for all assays. Frequencies are shown in Supplemental Table 1.

Analyses
Three cut-points for ANA positivity were considered: first, ANA prevalence was based on a rating of 3+ or 4+ at 1:80 dilution, similar to recently published population-level data on ANA (10). We then lowered the cut-point, by including lower-level ANA, i.e., those with a rating of 2+ at 1:80 dilution, similar to a positive reading at the 1:40 dilution used in prior studies. Finally, we raised the cut-point in analyses limited to those with higher titer ANA based on having 3+ or 4+ intensity rating at the 1:160 dilution. Only five individuals had a positive reading (3+) at the 1:320 level (one with anti-dsDNA antibodies), so these were not considered separately. These groups are labeled as "Any" (≥1:80 2+), "Moderate-higher" (≥1:80 3+) and Higher (≥1:160 3+), to indicate the increasing threshold for positivity. Three sets of models were run comparing the different definitions of ANA positives to those with no detectable ANA, using logistic regression to calculate odds ratios (OR) and 95% confidence intervals (CIs), adjusting for age and other covariates at BEEA enrollment (i.e., BMI, state, education, season of blood draw, and use of agricultural pesticides in the past 12 months). We considered a p-value of <0.05 to be statistically significant, and a value of 0.05 to <0.10 as suggestive. Associations were estimated for lifetime use of pesticides (overall, and for use of 46 specific pesticides with at least five ANA positive participants exposed). We also adjusted for correlated pesticides (Spearman's rho ≥0.40 in the sample, Supplemental Table 2) when confounding was observed, i.e., inclusion in the model changed the effect estimate by at least 10%.
We explored intensity-weighted lifetime days for individual organochlorine insecticides (Supplemental Table 3), but numbers were smaller due to a lack of data on those who did not complete the take-home questionnaire, and we saw no clear exposure-response patterns. To increase statistical power, we also grouped the OCs into two groups based on similarities in structure and function. We then tested the hypothesis that ANA was related to use of certain organochlorine insecticides, examining exposure-response relationships across cumulative intensity-weighted lifetime days for two groups of insecticides, summing the intensity-weighted lifetime days for each individual pesticide in the group: cyclodienes (aldrin, dieldrin, chlordane, and heptachlor) and other organochlorines (DDT, toxaphene, and lindane). These two groups were correlated (rho = 0.53 for any use), so they were run in a mutually adjusted model. Finally, we explored the frequency of disease-specific autoantibodies (i.e., ENA and anti-dsDNA autoantibodies) by pesticide use, including combined organochlorine variables (e.g., none, 1, or 2 or more cyclodienes used), and estimated age-adjusted ORs to describe these associations. Sensitivity analyses excluded potential incident cases of autoimmune disease, i.e., based on self-reported rheumatoid arthritis or other autoimmune disease, DMARD use, or the presence of anti-thyroid or anti-citrullinated peptide autoantibodies identified during AHS follow-up or at BEEA enrollment.

RESULTS
Of 668 samples tested, 282 (42%) had detectable ANA: 143 (21%) had any detectable ANA (i.e., ≥1:80 dilution at 2+ intensity reading), 139 (21%) had ANA detected at ≥1:80 3 or 4+ and 60 (9%) had ANA detectable at 1:160 3 or 4+. Sample characteristics by ANA status are shown in Table 1. Participants with ANA were older: those over age 60 years had two to three times the odds of having moderate to higher-levels of ANA (at ≥1:80 3 or 4+) compared with those ages 50-55 years. After adjusting for age, no associations were noted for smoking status, BMI, state, current farming status and season at the time of blood draw. During follow-up or at BEEA enrollment, 44 participants reported an incident diagnosis of an autoimmune disease, which was associated with overall ANA (OR 3.62; 95%CI 1.87, 6.98) (Supplemental Table 1). Among those with moderate to high level ANA, 15 (2% of the total sample) had disease-specific autoantibodies, i.e., ENA (n = 6) or anti-dsDNA (n = 11).
Neither lifetime years nor average days per year of any pesticide use at AHS enrollment was associated with ANA positivity ( Table 2). However, having sought medical care due to pesticide exposure was associated with having moderate to high level ANA (OR 2.15; 95%CI 1.17, 3.95). In analyses of specific pesticides ( We examined the relationship of ANA with intensityweighted lifetime days of use for organochlorines grouped as cyclodienes (heptachlor, chlordane, aldrin, and dieldrin) and non-cyclodienes (DDT, lindane, toxaphene), in a mutually adjusted model ( Table 4). Prevalence of high ANA was associated NC, North Carolina; NA, not applicable due to fewer than five exposed cases. 1 ANA Level shows four exclusive categories of ANA positivity based on highest reading observed; Negative, none detected at 1:80 dilution. 2 Odds Ratios (ORs) and 95% Confidence Intervals (CI) were calculated by logistic regression models adjusted for age at interview; "Any ANA," "Moderate-higher ANA," and "Higher ANA" indicate increasing thresholds for positivity used for modeling. 3 All were current at BEEA enrollment, except for state at AHS study enrollment. 4 Blood collected in spring or summer, compared to fall or winter.
with being in the top tertile of intensity-weighted lifetime days of combined cyclodiene use vs. no use (OR T3 3.20; 95%CI 1. 10, 9.27). By contrast, greater use of non-cyclodiene organochlorines was inversely associated with moderate to high ANA (OR T3 0.24; 95%CI 0.08, 0.72), which was similar to the association with greater DDT use (OR 0.35; 95%CI 0.13, 0.99, above the median intensity-weighted lifetime days; Supplemental Table 3).
In age adjusted models, having specific ENA or anti-dsDNA autoantibodies was associated with using two or more cyclodiene organochlorine insecticides, and with fonofos, cyanazine, and ethylene dibromide ( Table 5). Because these comparisons are based on only a small number of participants with ENA or anti-dsDNA autoantibodies, confidence limits were wide, and we did not adjust these models for other covariates or correlated pesticides. All but one of the participants with diseasespecific autoantibodies used carbofuran (p = 0.002). We also saw suggestive associations of disease-specific autoantibodies with aldicarb and methyl bromide, and an inverse association with malathion. Sensitivity analyses showed no substantial impact of excluding participants with a potential autoimmune disease during follow-up or at BEEA enrollment, i.e., who reported an incident diagnosis of an autoimmune disease since enrollment, or used disease-specific antirheumatic drugs, or were positive for thyroid or anti-CCP autoantibodies (not shown).

DISCUSSION
Our findings provide evidence that moderate to higher level ANA are associated with past use of specific pesticides and a history of seeking medical care for pesticide exposures in male farmers. Further, our results support the hypothesis that certain types of organochlorine insecticides may increase risk of developing autoimmunity. Two other pesticides, the carbamate insecticide aldicarb, and fumigant methyl bromide, were associated with increased odds of having moderate or higher-level ANA. By contrast, two of more commonly used organophosphate insecticides (in this sample), malathion and fonofos, and the herbicide butylate, were related to lower overall ANA. Together, these results suggest complex and lasting effects of some pesticides on autoimmunity.
Systemic autoimmune diseases are more common in reproductive age women, so past studies have been interested in the potential endocrine-disrupting effects of organochlorine pesticides on autoimmunity. Experimental and epidemiologic studies suggest a nuanced relationship. For example, the insecticide chlordecone (a cyclodiene) accelerated in a dose-dependent fashion the development of disease-specific anti-dsDNA autoantibodies in ovariectomized female mice in the NZB X NZW(F1) model of lupus (29). However, these effects did not appear to be mediated by estrogen-related mechanisms and did not extend to DDT. A recent study of a U.S. population NA, not applicable due to fewer than five ANA positive individuals who were exposed. 1 [1999][2000][2001][2002][2003][2004] reported no significant associations of measured organochlorines with moderate or higher ANA (3+ or 4+ at the 1:80 titer) after adjusting for multiple comparisons (17); in men, however, ANA was negatively associated with the DDT metabolite, DDE, and oxychlordane (a chlordane metabolite) at the p < 0.05 level. By contrast, in a small sample of African-American male farmers, higher DDE levels were suggestively (but non-significantly) associated with ANA (1:40 titer, equivalent to "overall" in the current study) (19). This later sample was selected from the population of rural eastern North Carolina as an add-on study to the AHS (non-overlapping with the current study sample); participants had an average of 2.2 (SD 5.5) years of DDT use and 12.3 (SD 13.4) years of overall pesticide use. In the current study sample, prevalence of high or moderate ANA was associated with greater intensity-weighted lifetime days using any of the four cyclodiene organochlorine insecticides (i.e., aldrin, dieldrin, chlordane, and heptachlor), and inversely associated with greater use of the non-cyclodiene organochlorine insecticides (especially DDT). Notably, DDT and the cyclodiene insecticides are no longer in use in the U.S., but our results suggest persistent effects on immunity decades later. Our analyzing cyclodienes separately from the other organochlorines had both empirical and theoretical rationale. In analyses of ever-use of the individual organochlorine insecticides, we saw suggestive positive associations with heptachlor and chlordane, but not with DDT. This was consistent with results by Rosenberg et al. who reported that ANA prevalence was associated with lifetime organochlorine use, excluding DDT and methoxychlor in a sample of 322 rural adults (15). Organochlorine insecticides share a chlorinated hydrocarbon structure and physical properties such as lipid solubility and environmental persistence. Their toxic effects on insects are due to hyperexcitation of nerve cells. The DDT-type chemicals work by perturbing sodium gates, while the cyclodiene insecticides impact the γ-aminobutyric acid (GABA) receptor, impairing chlorine influx (30). While GABA is best known as a neurotransmitter, emerging evidence suggests an important role in immune function and innate immunity (31,32). The closely related cyclodienes, chlordane, and heptachlor, have been associated with ANA in case series (33). Similar studies of aldrin and its breakdown product, dieldrin, were not identified in the autoimmunity literature, but there is some evidence of immunosuppressive effects of aldrin/dieldrin across different studies (34)(35)(36). In our data, having used two or more of these pesticides was also associated with having any of the disease-specific autoantibodies (e.g., anti-dsDNA autoantibodies). Together, these findings suggest these pesticides may increase the risk of developing clinically relevant autoimmunity. Lindane, a cyclohexane, also impacts GABA receptor function, but we saw limited evidence of an association with ANA. Besides their neurotoxicity, organochlorine pesticides can have other effects, for example endocrine disruption through estrogen or pregnane X receptors (37,38), so targeted research is needed to identify which pathways may impact autoimmunity.   4 Mutually adjusted for correlated pesticides (see Supplemental Table 2).
The carbamate insecticide aldicarb was the only other insecticide associated with high ANA; although it has a high acute toxicity, its immune effects are not well-known (39)(40)(41). The other carbamate insecticides, carbaryl and carbofuran, were not associated with elevated ANA. However, all but one of the participants with disease-specific autoantibodies had used carbofuran. High ANA was suggestively associated with the organophosphate insecticide diazinon, and we noted increased ORs for the two other phosphorothioates, chlorpyrifos, and coumaphos, though neither was statistically significant. These chemicals have documented immunotoxic effects (42)(43)(44)(45). Conversely, the phosphorodithioate insecticides fonofos and malathion were inversely associated with overall ANA, and the other phosphorodithioate insecticides also showed inverse, though not statistically significant, associations with overall ANA. There is some evidence of immunosuppression by malathion (46), while other studies (including in a MRL-lpr lupus mouse model) suggest that malathion may stimulate and accelerate the onset and intensity of the autoimmune response (47,48). Immune effects of fonofos are not described in the literature, and our findings seem somewhat paradoxical; while inversely associated with overall ANA, fonofos was positively associated with disease-specific autoantibodies in those with moderate to high ANA compared with no ANA detected. The primary mechanism of action for organophosphate and carbamate insecticides is blocking acetylcholinesterase enzyme. Given cholinergic receptors on human lymphocytes, pesticides may influence the immune response through acetylchlolinergic pathways, though the direction of effects may depend on the activated pathway (49,50). The phosphorodithioates require oxidative activation in vivo. However, it is unclear how this would differentially influence immune-related mechanisms of action compared with the other organophosphates and carbamates.
We also saw an association of high or moderate level ANA with the fumigant methyl bromide. Ethylene dibromide was also associated with disease-specific autoantibodies. Immunerelated mechanisms of these highly toxic chemicals are not well-described in the literature, although one study suggested a similar threshold for immune modulation and the level at which acute toxicity occurred (51,52). Along with aldicarb and carbofuran, methyl bromide and ethylene dibromide are often used against nematodes. Many participants with elevated ANA and disease-specific autoantibodies used more than one of these chemicals, but we did not have sufficient sample size to examine them independently. Use of petroleum oil or distillates was also associated with high or moderate ANA. Petroleum oil is a plausible candidate for inducing systemic autoimmunity, given the model of pristane-induced lupus (53). Pristane is a byproduct of petroleum distillation and induces lupus-like autoantibodies and clinical disease in mice. Petroleum distillates in the past may also have included other contaminants such as benzene or xylene. Overall ANA prevalence was inversely associated with butylate, a thiocarbamate herbicide, linked to lymphohematopoetic cancers and NHL in the AHS (54), but immune effects are not described in the literature. Interestingly, past use of butylate and recent use of malathion were associated with shorter leukocyte telomere length, a potential marker of immune aging, in a different subsample of AHS participants (55). NA, not applicable due to fewer than five exposed cases. 1 ANA Level shows four exclusive categories of ANA positivity based on highest reading observed. 2 Limited to applicators who completed the take-home questionnaire with information on frequency and duration of use. 3  Our results also suggest that seeking medical care due to pesticide exposure (i.e., seeing a doctor or being hospitalized due to pesticide exposure, or being diagnosed with pesticide poisoning) was associated with having moderate or high ANA. The reasons for seeking medical care for pesticide exposure included high pesticide exposure events (HPEE) and presumably pesticide-related symptoms, however we did not analyze ANA in relation to symptoms data. We also saw an elevated OR for overall ANA with HPEE in those who experienced an event but did not seek medical care (OR 1.37). Prior AHS research suggests those reporting high pesticide exposure events used pesticides for more days per year and had certain practices that might increase personal exposures, such as storing pesticides in the home or spraying pesticides with open cab windows, and that many do not seek medical care for HPEE and exposure-related symptoms (56). In our study, among ANA positive individuals, only 21 (3% of the total) reported both HPEE and seeking medical care, while 39 (6%) sought medical care without reporting HPEE, and 88 (13%) reported only HPEE (not shown in tables). Thus, the actual exposures underlying our findings remain undetermined. The AHS collected data on specific pesticides involved in some high pesticide exposure events, but we had insufficient sample size to take advantage of these data.
Diverse and interrelated pathways lead to dysregulation of the immune response and development of autoimmunity. In healthy individuals, ANA may reflect increased exposure to self-antigens and dysregulation of various pathways, some of which are shared and others distinctive from patterns seen in autoimmune disease patients (57,58). Pesticides may influence both the generation of autoimmunity and the strength of the immune response. Our study used the gold standard assay, which is sensitive to lower levels of ANA at the 1:40 dilution level while providing increased specificity at higher titers (e.g., 1:160). ANA are thought to arise as an immune response to nuclear antigens released from apoptotic cells. The interpretation of ANA detection at different thresholds for positivity varies by context: although detection at the 1:80 dilution of 3 or 4+ intensity has recently been specified among the constellation of clinical criteria for systemic lupus erythematosus, higher levels may be seen in lupus and sometimes associated with other clinical autoimmune diseases (59). Interestingly, we only saw positive associations for moderate or higher-level ANA, while inverse associations were seen only with having any detectable ANA (including lower levels, at least 2+ intensity at the 1:80 dilution).
A positive response to the HEp-2 ANA assay does not imply an association with clinical disease. The pattern of nuclear antibody staining is emerging as a key means to differentiate the origins and relevance of ANA in clinical and healthy populations, especially the dense fine speckled pattern and the associated anti-DFS70 antibody (60)(61)(62). Our analyses did not consider the fluorescence pattern, as the vast majority of ANA were identified as fine speckled, which at the time of the study was not differentiated from dense fine speckled and speckled. Antibodies to DFS70 may be a promising marker, as they appear to be more common in the general population and may even help to rule-out specific autoimmune diseases (60); had use of this marker been more common and validated at the time of the original testing, it would have been considered. We focused on specific disease-associated autoantibodies commonly examined in clinical assessments for SLE, but our results support further investigation of other types of antibodies as well, such as antihistone and chromatin, which have been linked with certain  drug-and xenobiotic exposures and may help to identify the underlying role of specific exposures (63)(64)(65)(66).
Half of the ANA-positive individuals in our study sample were positive only at the lowest-level (2+ at a 1:80 dilution); this threshold was used to enable comparisons with studies using a lower dilution (i.e., 1:40). The implications of having lower-level ANA are not well-understood. Most research on autoimmunity in the absence of autoimmune diseases has been based on cross-sectional and clinical samples; however, a few longitudinal studies showing that individuals with low level ANA are more likely to revert to seronegative status over time compared to those at lower titers (22,62,67). Notably, in a sample of rural residents and farmers, Semchuk observed that ANA prevalence at higher titers (3+ or 4+ at 1:80 and 1:160) was similar in the summer and winter, with similar rates of seroconversion and reversion across the seasons (16). On the other hand, lower level ANA (at the at 1:40 dilution, but not higher) were elevated in summer, supporting the idea that changing exposures by season may account for transient expression of ANA in some individuals. Although we adjusted for season in our statistical models, we saw no strong or consistent differences in ANA frequency for participants sampled at different times of year. Smoking history and BMI at the time of sampling were non-significantly associated with ANA and were included in our multivariable models along with other covariates as they generally increased the precision of observed associations. The causal relationship of specific pesticides with BMI is unknown; however, adjusting for BMI did not appear to explain any of the observed pesticide associations. Odds ratios approximate the relative risk for rare outcomes (<10%), for example with high ANA and ENA, but overestimate the relative risk for more common outcomes (i.e., overall ANA or high/moderate ANA). Our estimated ORs, therefore, may most accurately reflect the relative risks for high ANA. On the other hand, small numbers may inflate the OR and confidence limits, for example, in the analyses of uncommon pesticides and diseasespecific autoantibodies.
We excluded prevalent cases of autoimmune diseases (e.g., rheumatoid arthritis and lupus) to reduce potential recall or reporting bias, or changes in exposures due to health conditions; in follow-up, some participants reported incident autoimmune diseases, which were associated with ANA as expected (Supplemental Table 1). Overall ANA (i.e., including low level ANA) was also associated with anti-TPO autoantibodies. Autoimmune thyroid diseases occur more often in those with systemic autoimmune diseases (68), and anti-TPO autoantibodies have been previously associated with ANA in healthy controls (57). Prevalence of anti-CCP autoantibodies, specific for rheumatoid arthritis, was not associated with ANA in our sample, and levels were similar to the general population (69)(70)(71), despite evidence that rheumatoid arthritis may be associated with agricultural work and pesticide use in the AHS and other studies (1)(2)(3)(4)(5)(6)(7)(8)(9). Sensitivity analyses showed no substantial changes after excluding possible new cases of autoimmune diseases and those specific autoantibodies (i.e., anti-CCP, anti-TPO, and disease specific ENA and anti-dsDNA autoantibodies) (not shown).
The BEEA study provides an opportunity to investigate the long-term health effects of pesticides and other agricultural exposures on autoimmunity. The study sample is representative of men who were actively farming at AHS enrollment in the mid-1990s, most of whom did not have a diagnosis of RA or other autoimmune diseases. Women in the AHS, mostly spouses of the licensed applicators, were not included in the BEEA sample. Spouses reported less pesticide use than the applicators, but potential associations of pesticides with ANA in spouses may be relevant given the higher prevalence of ANA in women generally. Farmers are a unique population, with diverse exposures to agricultural pesticides and other physical or chemical factors, so results may not be generalizable to the general population. Further research is warranted to investigate ANA in relation to other agricultural factors, for example to solvents and fertilizers, recently associated with rheumatoid arthritis in the AHS (72). Our study addresses several limitations of prior studies on pesticides and ANA, with data on lifetime exposure to over 40 specific agricultural pesticides. However, despite having a larger sample than most studies, we had low statistical power to examine associations with uncommon pesticides and disease-specific autoantibodies, and for testing exposure-response gradients. Non-differential exposure misclassification is a possibility due to errors in recall of past exposures. Evaluation of self-reported data in the AHS has established the reliability of recall for specific pesticides; further, an intensity-weighting algorithm developed for the study (integrating across multiple determinants of exposure intensity) has shown good correlation with measurement data in an exposure sub-study, and may reduce the potential for non-differential misclassification to bias results toward the null in analyses of dose-response (24,73,74).
Our analyses are based on prospectively collected exposure data, but temporal ambiguity relative to ANA incidence is inherent to our study design focused on lifetime exposures. We cannot know whether exposures occurred prior to or after the development of ANA, or whether ANA appeared in the past and became non-detectable at the time they were measured. While the natural history of ANA in the general population is not well-understood, ANA prevalence typically increases with age, suggesting increasing incidence of ANA over time. Presumably, pesticides may influence two processes that contribute to the observed ANA at a single time point: (1) the breach of immune tolerance and initial production of autoantibodies, and (2) external factors impacting the production of measurable levels at the time of sampling. Further research is needed to investigate the associations of ANA with contemporary use of specific pesticides and the time since last use. Autoantibodies can precede the development of autoimmune diseases for a decade or longer (75), so other factors are likely to contribute to the development of pathology and disease.
Altogether our findings provide evidence that past pesticide use may influence the development of autoimmunity in middle age and older farmers, consistent with prior findings of elevated ANA with some organochlorine insecticides. The presence of disease-specific autoantibodies was also related to certain organochlorines, supporting the need to further explore these persistent pesticides as potential causal agents in development of autoimmune diseases. Future research should focus on the role of recent exposure and the evaluation of less common pesticides in larger studies or in targeted populations with greater use of these chemicals.

DATA AVAILABILITY
Due to ethical restrictions imposed in the interest of protecting participant confidentiality, the datasets for this study are available upon request to interested, qualified researchers. Requests to access the datasets should be directed to JH (hofmannjn@mail.nih.gov).

ETHICS STATEMENT
The study was approved by Institutional Review Boards at the National Cancer Institute and other participating institutions, and all participants provided written informed consent.

AUTHOR CONTRIBUTIONS
CP conceived of the research question, obtained funding for assays, performed analyses, evaluated results, and wrote the manuscript. AS assisted with planning analyses, evaluated results, and contributed to the manuscript. SB, MW, and CL assisted with planning analyses, evaluating results, and editing the manuscript. JH, DS, MA, CD, and LB obtained funding and generated data for the AHS and BEEA sub-cohort, assisted with planning analyses, evaluated results, and edited the manuscript.