Naloxone inhibits immune cell function by suppressing superoxide production through a direct interaction with gp91 phox subunit of NADPH oxidase
© Wang et al; licensee BioMed Central Ltd. 2012
Received: 30 September 2011
Accepted: 16 February 2012
Published: 16 February 2012
Both (-) and (+)-naloxone attenuate inflammation-mediated neurodegeneration by inhibition of microglial activation through superoxide reduction in an opioid receptor-independent manner. Multiple lines of evidence have documented a pivotal role of overactivated NADPH oxidase (NOX2) in inflammation-mediated neurodegeneration. We hypothesized that NOX2 might be a novel action site of naloxone to mediate its anti-inflammatory actions.
Inhibition of NOX-2-derived superoxide by (-) and (+)-naloxone was measured in lipopolysaccharide (LPS)-treated midbrain neuron-glia cultures and phorbol myristate acetate (PMA)-stimulated neutrophil membranes by measuring the superoxide dismutase (SOD)-inhibitable reduction of tetrazolium salt (WST-1) or ferricytochrome c. Further, various ligand (3H-naloxone) binding assays were performed in wild type and gp91 phox-/- neutrophils and transfected COS-7 and HEK293 cells. The translocation of cytosolic subunit p47 phox to plasma membrane was assessed by western blot.
Both (-) and (+)-naloxone equally inhibited LPS- and PMA-induced superoxide production with an IC50 of 1.96 and 2.52 μM, respectively. Competitive binding of 3H-naloxone with cold (-) and (+)-naloxone in microglia showed equal potency with an IC50 of 2.73 and 1.57 μM, respectively. 3H-Naloxone binding was elevated in COS-7 and HEK293 cells transfected with gp91 phox ; in contrast, reduced 3H-naloxone binding was found in neutrophils deficient in gp91 phox or in the presence of a NOX2 inhibitor. The specificity and an increase in binding capacity of 3H-naloxone were further demonstrated by 1) an immunoprecipitation study using gp91 phox antibody, and 2) activation of NOX2 by PMA. Finally, western blot studies showed that naloxone suppressed translocation of the cytosolic subunit p47 phox to the membrane, leading to NOX2 inactivation.
Strong evidence is provided indicating that NOX2 is a non-opioid novel binding site for naloxone, which is critical in mediating its inhibitory effect on microglia overactivation and superoxide production.
KeywordsNeuroinflammation Microglia NADPH oxidase Opioid receptor Binding
Recent studies strongly support that neuroinflammation plays a critical role in the pathogenesis of various neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), multiple sclerosis, Huntington's disease and multiple system atrophy . Microglia, the resident immune cells of the brain, are the major players in the initiation of neuroinflammation and subsequent pathogenesis of neurodegeneration. Once activated, microglia produce and release a variety of proinflammatory factors, such as cytokines, chemokines and reactive free radicals, which contribute to the neurodegenerative process . Thus, anti-inflammatory therapy has been considered a new strategy for disease-modifying intervention.
In the course of developing new anti-inflammatory drugs for PD, we discovered that naloxone, a commonly used antagonist of opioid receptors, was highly effective in preventing dopaminergic neurodegeneration in different rodent PD models by inhibiting inflammatory responses [3–6]. The inhibitory effects of naloxone on inflammatory responses have been reported in our previous studies in vivo and in vitro. Systemic infusion of 1 mg/kg (+)- or (-)-naloxone significantly reduced intranigral LPS-induced microglial activation and neurotoxicity . In midbrain and cortical neuron-glia cultures, microglial activation and related proinflammatory cytokine production such as nitrite oxide, TNFα and IL-1β, in response to LPS stimulation, were also attenuated by naloxone at 0.1-1 micromolar concentrations [3, 4]. Further dose-response study showed a bimodal curve (effective in both micromolar and subpicomolar, but less effective in nanomolar concentrations) for both anti-inflammatory and neuroprotective effects of naloxone . Despite a huge concentration difference, mechanistic studies have revealed that inhibition of microglial superoxide production is the key event underlying naloxone-afforded neuroprotection for both micromolar and subpicomolar concentrations [3, 6, 7]. Furthermore, we have reported that the neuroprotective effect of naloxone is independent of opioid receptors, since (+)-naloxone, which is an inactive isomer in the activation of opioid receptors, exerts the same potency as the (-)-naloxone in neuroprotection [3–6]. Recent reports from several laboratories have also shown opioid receptor-independent actions of naloxone. Watkins' group reported that (+)-naltrexone, (+)-naloxone and (-)-naloxone, which they showed to be Toll-like receptor 4 (TLR4) antagonists in vitro on both stably transfected HEK293-TLR4 and microglial cell lines, suppress neuropathic pain with complete reversal upon chronic infusion [8, 9]. In a drug addiction study, Wang et al. demonstrated that the acute Gs coupling induced by morphine is completely prevented by co-treatment with both (-) and (+)-naloxone . Similarly, it has been reported that naloxone displays neuroprotective effects in a two-hit seizure model by reducing both cytokine production and microglial activation . Both (-) and (+)-naloxone have also been found to be capable of reducing the severity of aortic atherosclerosis in apolipoprotein-E (apo-E)-deficient mice through inhibition of macrophage activation and superoxide release . Taken together, the non-opioid actions of naloxone may have wide implications in therapy for a variety of diseases. Thus, it is critical to elucidate the novel non-opioid binding site(s) and action mechanisms of naloxone.
The major purpose of this study was to search for the potential site of action mediating the anti-inflammatory and neuroprotective effects of naloxone using a ligand (3H-naloxone) binding assay. We have initially attempted to determine both the low (micromloar) and high (subpicomolar) affinity sites of naloxone. However, the specific activity of 3H-naloxone is too low to study the binding at subpicomolar concentrations. Thus, the main effort of this study focused on the micromolar concentrations of naloxone. Here, for the first time, we report that gp91 phox , the catalytic subunit of microglial NOX2, is a novel non-opioid binding site of naloxone. Binding of naloxone reduced the translocation of cytosolic subunits to the plasma membrane, leading to inhibition of NOX2 and related superoxide production, which is critical in mediating the anti-inflammatory and neuroprotective effects of naloxone.
[3H]-naloxone was purchased from Perkin Elmer Life Sciences (Boston, MA, USA). Cell culture reagents were obtained from Invitrogen Life Technologies (Grand Island, NY, USA). Rabbit anti-p47 phox was purchased from Upstate (Billerica, MA, USA). FITC-conjugated goat anti-rabbit immunoglobulin G (IgG) was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Rabbit anti-β-actin was obtained from Sigma (St. Louis, MO, USA). Mouse anti-gp91 phox was purchased from BD Transduction Laboratories (San Jose, CA, USA).
NOX2-deficient (gp91 phox-/- ) and wild type (WT) C57BL/6 J (gp91phox+/+) mice were obtained from the Jackson Laboratory. All animals were housed in a pathogen free facility with a 12-hour light/12-hour dark cycle and ad libitum access to food and water. Housing, breeding and experimental use of the animals were performed in strict accordance with the National Institutes of Health guidelines.
Primary mesencephalic neuron/glia culture
Briefly, dissociated cells were seeded at 1 × 105/well in poly-D-lysine coated 96-well plates. The cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air in minimum essential medium (MEM) containing 10% heat-inactivated fetal bovine serum (FBS), 10% heat-inactivated horse serum, 1 g/L glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 μM nonessential amino acids, 50 U/ml penicillin, and 50 μg/ml streptomycin. Seven days later, cultures were used for drug treatments. At the time of treatment, immunocytochemical analysis indicated that the neuron-glia culture consisted of 10% microglia, 50% astrocytes, 40% neurons, and 1% tyrosine hydroxylase (TH)-immunoreactive neurons. For treatment, cultures were changed to treatment medium composed of MEM, 2% FBS, 2% HHS, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, and 50 μg/ml streptomycin.
The production of superoxide was assessed by measuring the superoxide dismutase (SOD) -inhibitable reduction of the tetrazolium salt WST-1. Primary neuron/glia cultures in 96-well plates were washed twice with Hanks' balanced salt solution (HBSS) without phenol red. Cells were then incubated at 37°C for 30 minutes with vehicle control or (-) and (+)-naloxone in HBSS (50 μl/well). Subsequently, 50 μl of HBSS with and without SOD (50 U/ml) was added to each well along with 50 μl of WST-1 (1 mM) in HBSS and 50 μl of vehicle or LPS. The absorbance at 450 nm was read with a SpectraMax Plus microplate spectrophotometer (Molecular Devices) every 2 minutes for 30 minutes. The difference between the absorbances in the presence and absence of SOD was considered to be the amount of produced superoxide.
Preparation, activation, and fractionation of rat blood neutrophils and cell-free assay of NADPH-dependent superoxide production by PMA-stimulated neutrophils membranes.
Neutrophils were purified from fresh blood collected from the abdominal aorta of adult Fischer 344 male rats as described before . Briefly, heparinized blood was sedimented with dextran, and the neutrophils were isolated by centrifugation through the Ficoll-Hypaque density gradient, followed by a hypotonic lysis to remove residual erythrocytes. Purified neutrophils (> 95% viable cells, trypan blue exclusion) were resuspended in warmed phenol red-free HBSS (37°C) and stimulated for 15 minutes with 100 nM PMA or vehicle with gentle agitation. The reaction was stopped by the addition of ice-cold HBSS buffer. Pelleted cells were disrupted and fractionated at 4°C by discontinuous sucrose density gradient sedimentation as described before. Superoxide generation in membrane fractions was measured by monitoring the reduction of ferricytochrome c in a spectrophotometer with the water-jacketed cuvette compartment maintained at 37°C. The reaction was initiated by the addition of 0.2 mM (final concentration) freshly prepared NADPH (tetrasodium salt, Type I, Sigma) into 1.0-ml micro-cuvettes containing detection buffer (100 μM ferricytochrome c, 10 μM FAD, 2 mM MgCl2, 2 m NaNs, and 10 mM Hepes, pH 7.4) and 10 μg of membrane. (-) or (+)-Naloxone (a final concentration of 10 μM) or vehicle was added quickly to micro-cuvettes to determine the inhibition of superoxide production. The reference cuvette was supplemented with 600 U/ml SOD (Type I, 3000 U/mg protein, Sigma). Initial rates of superoxide production were calculated from the linear segment of the increase in absorbance at 550 nm and normalized to vehicle-treated control.
Xanthine/xanthine oxidase reaction
To determine whether naloxone acts as a superoxide scavenger, the superoxide-generating xanthine/xanthine oxidase system was used. Briefly, assays were performed in the presence of indicated concentrations of (-) and (+)-naloxone isomers, 0.01 U xanthine oxidase, 50 μM xanthine, 250 μM partially acetylated WST-1 in 50 mM potassium phosphate buffer (pH 7.6) in a 96-well plate (100 μL/well final volume). Xanthine was added to initiate the reaction, and absorbance at 450 nm was continuously monitored for 5 minutes using a Synergy HT multi-detection microplate reader (Bio-Tek Instruments, Winooski, VT, USA). Results are expressed as a percentage of the increase in absorbance per minute observed with xanthine oxidase only.
HEK293 cells were maintained in DMEM supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere of 5% CO2 and 95% air. One day before transfection, 1 × 106 HEK293 cells were seeded into 6-well plates. Flag-gp91 phox and Myc/His-p22 phox plasmids were cotransfected into HEK293 cells using Lipofectamine 2000 (Invitrogen Life Technologies, Grand Island, NY) according to the manufacture's protocol. After 48 hours of expression, cells were collected for binding experiments.
Isolation of neutrophils from peritoneal cavity
Inflammation was induced by injecting 1 ml of casein solution into the peritoneal cavity of gp91 phox-/- and gp91 phox+/+ mice for overnight and repeat injection the next morning. Three hours after the second injection, mice were euthanized and 5 ml harvest solution (0.02% EDTA in PBS) was injected into the peritoneal cavity. All peritoneal fluids were transferred into 50 ml centrifuge tubes, and then centrifuged at 200 × g for 10 minutes. The pellets were washed three times using PBS, and then collected for use .
[3H]-naloxone binding assay
Binding experiments were performed according to the methods of Herren, Liu and Zhou [4, 15, 16]. Briefly, whole cell (2 × 106 or 4 × 106) or membrane (250 μg) from neutrophils, COS-7, HEK293 or BV2 microglia were incubated with [3H]-naloxone (6 nM) plus 10 μM unlabeled naloxone isomers in binding buffer (HBSS + 10% serum) for 2 hours at 4°C. Afterward, the buffer was removed and cells or membrane were washed three times with ice-cold HBSS using 1.0 μm pore size glass fiber filter (Whatman, Florham Park, NJ, USA). Then, cells or membranes were mixed with 10 ml of Ultima Gold scintillation fluid (PerkinElmer, Waltham, MA, USA) and counted for radioactivity. Experiments were performed in triplicate and results are expressed as percentage of total binding observed with corresponding WT group.
[3H]-naloxone immunoprecipitation binding
The 3H-naloxone immunoprecipitation binding study was performed by immunoprecipitation using anti-gp91 phox antibody (IP-binding). Specifically, we prepared plasma membranes from wild type COS-7 and COS-7-gp91 phox -p22 phox (COS-7 cells stably expressed with gp91 phox and p22 phox ). Membrane pellets were suspended in 0.5 ml IP buffer (10 mM HEPES (pH 7.4), 1% Triton X-100, 5 mM Mg2Cl, 10 mM KCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), 10% glycerin and protease inhibitor cocktails). After pre-incubation with 3H-labeled naloxone (6 nM) in the presence of 10 μM unlabeled naloxone for 30 minutes, membrane aliquots (0.25 mg protein) were incubated overnight with the addition of control immunoglobulin G (IgG) or anti-gp91 phox polyclonal antibody (Santa Cruz Biotech, Santa Cruz, CA) at 4°C. Immunoprecipitation was performed by protein A/G beads (Santa Cruz Biotech). After 4 hours incubation at 4°C, protein beads were washed three times by IP buffer. Then the beads were eluted twice by 0.5 ml glycine buffer (0.1 M at pH 2.5), and radioactivity was counted with a liquid scintillation counter. The binding capacity was determined by subtracting the binding of control IgG from that of gp91 phox antibody.
Membrane fractionation and western blot analysis
Membrane fractionations were extracted as described previously . BV2 microglia were lysed in hypotonic lysis buffer (1 mM Tris, 1 mM KCl, 1 mM ethylene glycol tetraacetic acid (EGTA), 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM DTT, 1 mM PMSF, and 10 μg/ml) cocktail protease inhibitor incubated on ice for 30 minutes and then subjected to Dounce homogenization (20-25 St, tight pestle A). The lysates were centrifuged at 1,600 × g for 15 minutes and the supernatant was centrifuged at 100,000 × g for 30 minutes. The pellets solubilized in 1% Nonidet P-40 hypotonic lysis buffer were used as membranous fraction. Equal amounts of protein were separated by 4% to 12% Bis-Tris Nu-PAGE gel and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% nonfat milk and incubated with rabbit anti-p47 phox and mouse anti-gp91 phox (1/1,000 dilution) and HRP-linked anti-rabbit or mouse IgG (1/3000 dilution) for 2 hours. Enhanced chemiluminescence (ECL) reagents (Amersham Biosciences, Piscataway, NJ, USA) were used as a detection system.
The data were presented as the mean ± SE. For multiple comparisons of groups, analysis of variance (ANOVA) was used. Statistical significance between groups was assessed by paired or unpaired Student's t test, with Bonferroni's correction. A value of P < 0.05 was considered.
Naloxone inhibits superoxide production through inhibition of NOX2
3H-Naloxone binding is increased in the presence of the gp91 phox membrane subunit
NOX2 activation by PMA enhances the binding capacity of naloxone
Diphenyliodonium (DPI), a NOX2 inhibitor, reduces 3H-naloxone binding
Pre-treatment of naloxone inhibits translocation of the NOX2 p47phox cytosolic subunit to membrane
Post-treatment of naloxone is still capable of inhibiting pre-existing increased binding of p47phox in membrane elicited by pre-treatment of LPS
Several reports have documented that naloxone inhibits superoxide production from microglia and neutrophils in an opioid receptor-independent manner [6, 18, 26]; however, the underlying mechanism remains unclear. Since naloxone contains a phenolic hydroxyl group, we first assessed the possibility that the reduction in superoxide production is due to its free radical scavenging effect. This possibility was ruled out since both (-)- and (+)-naloxone failed to reduce levels of superoxide produced by the xanthine/xanthine oxidase system (Figure 1). This finding led us to propose the possibility that naloxone acts by interfering with upstream events of superoxide generation. Using different types of phagocytes deficient in the gp91 phox subunit or non-phagocytes transfected with different subunits of NOX2, we provide clear evidence indicating a direct binding of naloxone to the membrane subunit, gp91 phox (Figure 3). 3H-Naloxone binding capacity was decreased in neutrophils deficient in gp91 phox . In contrast, 3H-naloxone binding in COS-7 and HEK293 cells transfected with gp91 phox was elevated compared with vehicle-transfected controls. After enrichment of gp91 phox protein from COS-7-gp91 phox -p22 phox cell membranes by immunoprecipitation using anti-gp91 phox antibody, the radiolabeled naloxone binding showed greater differences in the presence and absence of gp91 phox (Figure 3C). Overall, our data show a good correlation between the binding affinity of naloxone to the gp91 phox and its inhibition of LPS-induced superoxide production with similar IC50 values (around 2 μM, Figures 1A & 2).
It is important to note that the micromolar IC50 values reported here are much higher than the reported binding affinity of 3H-naloxone to conventional opioid receptors (in nanomolar concentrations) . The question is whether these concentrations are clinically relevant for the anti-inflammatory and neuroprotective effects of naloxone. Earlier reports indicated that micromolar concentrations can be reached both in serum and brains after naloxone treatment. Tepperman et al. showed that the peak plasma naloxone concentrations in rats were 2.1 and 17 μM after a single subcutaneous injection of naloxone with doses of 1.0 or 10 mg/kg, respectively. Furthermore, it has been reported that brain levels of naloxone are six to seven times higher than that in plasma , which might be attributed to involvement of a pH-dependent and saturable influx transport system in the blood-brain barrier transport of naloxone . Our previous report showed that systemic infusion of naloxone (0.4 to 4 mg/kg/day for two weeks with a mini-osmotic pump) dose-dependently attenuated LPS-induced nigral dopaminergic neuron loss in rats , and this further supports this contention.
Although we have identified microglial gp91 phox as a novel binding site in mediating the anti-inflammatory effect of naloxone, the underlying mechanism of how the binding affects subsequent NOX2 activation remains to be studied. Several studies have reported that naloxone could directly alter membrane characteristics. In a hemorrhagic shock rodent model, Curtis and Lefer demonstrated that naloxone increases circulating lysosomal hydrolase activities and suggested that this action results from stabilization of lysosomal membranes . Similarly, inhibition of Na+ and K+ currents in myelinated nerve fibers of sciatic nerve and membrane potential of some cells have also been reported [31, 32]. Thus, we speculated that binding of naloxone to gp91 phox might change the conformation of the protein complex and subsequently decrease the affinity of binding of the cytosolic subunits (p47 phox , p67 phox ). One common approach to determine the binding affinity between the cytosolic and membrane subunits of NOX2 is the measurement of translocation of cytosolic subunits to the membrane. Since it is known that the p67 phox is the subunit that directly binds to the gp91 phox protein, it would be desirable to measure the translocation of this subunit. However, the lack of suitable antibodies against p67 phox prevented us from performing this experiment. For this reason, an antibody against p47 phox , which is also bound to the membrane through formation of a complex with p67 phox , was used.
Our speculation is supported by the finding that naloxone pre-treatment reduces the translocation of p47 phox (Figure 8). It is of interest to note that post-treatment with naloxone was also capable of reducing the LPS-induced increase in the amount of phosphorylatd p47 phox in the cell membrane fraction (Figure 9). These results suggest that binding of naloxone to activated NOX2 may alter the conformation of gp91 phox and subsequently decrease binding affinity between cytosolic subunits and the membrane subunit. Although we do not yet have evidence showing actual conformational change of gp91 phox after naloxone binding, this finding serves to explain why post-treatment of naloxone is still effective in anti-inflammation and neuroprotection. That conformational changes can also alter the binding capacity of 3H-naloxone binding is further illustrated by the finding that binding of 3H-naloxone in NOX2-transfected COS-7 cells was greatly enhanced after NOX2 was activated by PMA (Figure 6). Taken together, our data strongly suggest that naloxone alters binding affinity between gp91 phox and the cytosolic subunits.
The non-opioid actions of naloxone have recently received wide attention mainly due to its potential new indications for a variety of diseases. One of the major advances in this research area is the recognition of an anti-nociceptive action of naloxone. Glial activation has been demonstrated to oppose opioid analgesia and enhance opioid tolerance, dependence, reward and respiratory depression by upregulating the production of proinflammatory cytokines (TNFα, IL-1), pain-relevant neurotransmitters from sensory afferent terminals and the number and/or conductivity of AMPA and NMDA glutamate receptors [33–35]. Naloxone, including both (-) and (+) isomers, has been reported to potentiate morphine analgesia equally by blocking morphine-induced glial activation and consequent increases in anti-analgesic proinflammatory cytokines [8, 9, 36]. Similarly, hyperalgesia induced by morphine-3-glucoronide (M3G), a major morphine metabolite that has little or no affinity for opioid receptors, is also blocked or reversed by (-) and (+)-naloxone . TLR4 was recently recognized to mediate such effects [38–40]. Experiments are underway in our laboratory to determine whether the anti-inflammatory effect of naloxone mentioned above is due to a direct action on the TLR-4 receptor or is indirect through the inhibition of NOX2 inhibition. We performed a radioligand binding assay with microglia prepared from TLR-4-deficient mice. Preliminary results failed to show a decrease in 3H-naloxone binding in TLR-4-deficient microglia compared with wild type controls (Wang et al., preliminary observations). Thus, the inhibitory effect shown on the TLR-4 receptor is likely due to an indirect effect through binding to NOX2. In fact, we have previously reported that naloxone inhibits LPS-induced production of proinflammatory factors, such as TNFα and prostaglandins, through inhibition of NOX2-generated superoxide release . Regardless of the site of action, the potential interactions between TLR-4 and NOX2 can be a critical pathway governing innate immunity and warrant further study.
Naloxone belongs to a class of compounds termed as morphinans, with chemical structures similar to morphine. We have previously reported that a large number of morphinan analogs, such as (-) and (+) morphine , dextromethorphan [43, 44], 3-hydroxymorphinan [45, 46] and sinomenine  display potent anti-inflammatory and neuroprotective effects similar to that of naloxone. These morphinan analogs have recently received a great deal of attention due to their new therapeutic implications for a variety of diseases, ranging from neuroprotection, anti-nociception, and bipolar depression to drug addiction. Using both in vivo and primary neuron-glia cell cultures derived from gp91 phox-/- mice, we have previously reported that NOX2 is critical in mediating this morphinans-afforded neuroprotection similar to that of naloxone. Further, our preliminary data show that dextromethorphan is able to bind similarly to what we describe for naloxone in this paper, suggesting that gp91 phox might be a common binding site for the above-mentioned morphinans in their anti-inflammatory property (unpublished data).
In summary, we provided strong evidence indicating that NOX2 is a non-opioid novel binding site for naloxone, which is critical in mediating its inhibitory effect on microglia overactivation and superoxide production. Although the new implications elicited by the mechanisms of morphinan analogs remain to be studied, the identification of this novel non-opioid binding site mediating the important anti-inflammatory action of naloxone should provide new insights into further understanding the mechanism of action of other morphinan analogs and help better design new drugs targeting this important free radical-producing enzyme.
amyotrophic lateral sclerosis
ethylene glycol tetraacetic acid
fetal bovine serum
hank's buffered salt solution
interleukin 1 β
minimum essential media
phorbol myristate acetate
sodium dodecyl sulfate polyacrylamide gel electrophoresis
Toll-like receptor 4
tumor necrosis factor α
water soluble tetrazolium salt 1.
This research was supported by the Intramural Research Program of the National Institutes of Health/National Institute of Environmental Health Sciences. We thank Dr. Saurabh Chatterjee and Baozhong Zhao at the National Institute of Environmental Health Sciences and Esteban Oyarzabal at University of North Carolina, Chapel Hill, for their helpful suggestions in editing this article.
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