Environmental and occupational exposure to heavy metals remains one of the major concerns in public health. Increased levels of manganese (Mn) pollution are associated with profound neurotoxic effects, including neurobehavioral deficits and disturbances resembling Parkinson’s disease. While Mn absorption is in part mediated by iron transporters, recent studies have shown that the levels of iron transporters are modified by alcohol and that chronic alcohol consumption increases body iron stores. However, it is largely unexplored whether alcohol exposure influences the transport and neurotoxicity of Mn. To address this question, we exposed mice to ethanol (10%; v/v) by drinking water for 4 weeks, during which period MnCl2 (5 mg/kg) or saline solutions were administered daily by intranasal instillation. Ethanol consumption in mice increased brain Mn levels in a dose-dependent manner after Mn instillation, determined by inductively-coupled plasma mass spectrometry, which was accompanied by up-regulation of iron transporters, as assessed by western blotting and qPCR. In addition, alcohol drinking increased hypoxic response and decreased hepcidin expression, providing the molecular mechanism of increased iron transporters and Mn uptake upon alcohol consumption. Moreover, brain dopamine levels, analyzed by HPLC, were decreased after intranasal Mn instillation, which was worsened by alcohol. Likewise, alcohol-Mn co-exposure synergistically altered dopaminergic protein expression. Finally, alcohol binge-drinking, which resembles alcohol drinking manner in humans, increased brain Mn content along with upregulation of iron transporters. Our study suggests that individuals who consume alcohol may have a higher risk of Mn neurotoxicity upon Mn exposure.

A rat model of iron loading anemia resulting in hypertrophic cardiomyopathy was used to assess the composition of cardiac isomyosin by ELISAs and immunohistochemistry relative to the chronological age of these animals and their normal controls

Iron chelators have been widely used to remove excess toxic iron from patients with secondary iron overload. However, small molecule-based iron chelators can cause adverse side effects such as infection, gastrointestinal bleeding, kidney failure, and liver fibrosis. Here we report renal clearable nanochelators for iron overload disorders. First, after a singledose intravenous injection, the nanochelator shows favorable pharmacokinetic properties, such as kidney-specific biodistribution and rapid renal excretion (>80% injected dose in 4 h), compared to native deferoxamine (DFO). Second, subcutaneous (SC) administration of nanochelators improves pharmacodynamics, as evidenced by a 7-fold increase in efficiency of urinary iron excretion compared to intravenous injection. Third, daily SC injections of the nanochelator for 5 days to iron overload mice and rats decrease iron levels in serum and liver. Furthermore, the nanochelator significantly reduces kidney damage caused by iron overload without demonstrating DFO's own nephrotoxicity. This renal clearable nanochelator provides enhanced efficacy and safety.

Iron (Fe) is an essential metal, but high Fe stores are toxic and cause heart and liver failures, arthritis, and diabetes. Since there is no recognized active pathway for Fe excretion, disposing extra Fe from the body is the primary therapeutic goal of treating patients with iron overload (IO). While the chelation therapy has been widely used to alleviate IO, Fe chelators have serious adverse effects, such as hypotension, gastrointestinal bleeding, kidney failure and liver fibrosis, likely due to non‐specific distribution in off‐target tissues. Consequently, there is an unmet need for a new therapeutic strategy for IO. Here we developed an ultrasmall nanoparticle (NP) that covalently binds to an FDA‐approved Fe chelator deferoxamine (DFO), which limits the distribution into off‐target tissues, while efficiently capturing excess Fe. We first generated renally‐clearable ɛ‐poly‐L‐lysine (EPL) conjugated with zwitterionic near‐infrared fluorophore ZW800. We then produced DFO‐coated NP by conjugating four DFO molecules on the surface of each EPL with balanced charges (DFO4‐NP). The ferrozine competition assay demonstrated that the association constant of DFO4‐NP to Fe was greater by a 3.5‐fold (p<0.001) compared to native DFO, indicating improved Fe binding affinity after conjugation. Next, in vivo chelation efficacy of DFO4‐NP (2 μmol as NP/kg) was determined in IO mice induced by intraperitoneal injection of Fe dextran (100 mg/kg). Urinary Fe content collected over 4 h post‐dose was increased by a 2.5‐fold (p<0.001) and by an 8.6‐fold (p<0.001) after intravenous (i.v.) and subcutaneous (s.c.) injection of DFO4‐NP, respectively, compared to blank NP. Similarly, renal clearance of Fe was increased by a 2.8‐fold (p<0.001) and a 9.5‐fold (p<0.001) after i.v. and s.c. of DFO4‐NP, respectively, compared to NP alone. Moreover, when compared to equimolar dose of native DFO, DFO4‐NP showed increased urinary recovery and renal clearance of Fe by a 2.8‐fold (p=0.003) and by a 2.5‐fold (p=0.003), respectively. These suggest the greater chelation efficiency and urinary excretion of Fe by s.c. DFO‐NP administration than by native DFO or blank NP. We further evaluated the efficacy of DFO4‐NP after s.c. doses daily for 5 d in dietary IO mice. DFO4‐NP decreased non‐heme Fe content in serum (22%, p=0.010), liver (26%, p=0.011) and spleen (26%, p=0.011) compared to blank NP. Consistently, DFO4‐NP decreased the status of an Fe‐storage protein ferritin (Ftn), a reliable marker of body Fe stores, in both liver (28%, p=0.041) and spleen (35%, p=0.024), compared to blank NP. These results indicate that DFO‐NPs efficiently remove excess Fe stores from plasma and tissues after repeated administrations. Moreover, H&E staining showed a decreased density of tubular cells in the kidney after native DFO treatment, while no kidney damage was observed in mice treated with NP alone or DFO4‐NP. Taken together, the renally‐clearable DFO‐NPs provide improved therapeutic efficacy against physiological complications resulting from IO disorders, while minimizing the inherent toxicity of small molecule chelators.Support or Funding InformationNIH U54 HL119145, American Heart Association17GRNT33460134This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nucleic acid-based therapeutics has the potential for treating numerous diseases by correcting abnormal expression of specific genes. Lack of safe and efficacious delivery strategies poses a major obstacle limiting clinical advancement of nucleic acid therapeutics. Oral route of drug administration has greater delivery challenges, because the administered genes or oligonucleotides have to bypass degrading environment of the gastrointestinal (GI) tract in addition to overcoming other cellular barriers preventing nucleic acid delivery. For efficient oral nucleic acid delivery, vector should be such that it can protect encapsulated material during transit through the GI tract, facilitate efficient uptake and intracellular trafficking at desired target sites, along with being safe and well tolerated. In this review, we have discussed multicompartmental systems for overcoming extracellular and intracellular barriers to oral delivery of nucleic acids. A nanoparticles-in-microsphere oral system-based multicompartmental system was developed and tested for in vivo gene and small interfering RNA delivery for treating colitis in mice. This system has shown efficient transgene expression or gene silencing when delivered orally along with favorable downstream anti-inflammatory effects, when tested in a mouse model of intestinal bowel disease. WIREs Nanomed Nanobiotechnol 2018, 10:e1478. doi: 10.1002/wnan.1478 This article is categorized under: Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Emerging Technologies.

Quantitative characterization of pharmacokinetics (PK) and pharmacodynamics (PD) of drugs is an essential component in pharmacology/toxicology education as well as drug discovery/development. PK determines the relationship between the dose of the administered drug and the concentration measured in the body, whereas PD characterizes the extent of pharmacological effect induced by drug concentrations. Simulation and modeling of such properties is a powerful tool to predict drug's efficacy and toxicity with the limited dose‐concentration‐response data. Several software tools, such as WinNonlin, Simcyp, GastroPlus and SAAM II, are traditionally used for PK/PD modeling and simulation. However, these tools have not demonstrated the dynamic flexibility in the generation of novel modeling approaches in both academic and research settings. While MATLAB has been extensively used in the field of Science and Engineering with a variety of applications, SimBiology provides an app to model, simulate, and analyze dynamic systems, focusing on PK/PD and quantitative systems pharmacology (QSP) applications. It also provides a block diagram editor for building models, or the user can create models programmatically using the MATLAB language. Moreover, SimBiology includes a library of common PK models, which one can customize and integrate with mechanistic systems biology models. Consequently, MATLAB/SimBiology provides a comprehensive tool that can help to train young scientists in the field of pharmaceutical sciences, including pharmacology and toxicology. While our graduate program at Northeastern University offers several important features of fundamental PK/PD principles and advanced PK/PD analyses, an opportunity to have a hands‐on experience on PK/PD simulation and modeling has not been provided. In Spring 2016, we incorporated SimBiology as a modeling and simulation tool to our graduate Advanced Pharmacokinetics and Toxicology course through a collaboration with MathWorks. We then assessed the benefits of SimBiology in students' learning based on class participation and problem sets/exams, as well as students' survey through Teacher Rating and Course Evaluation (TRACE). The results demonstrated that SimBiology improved students' learning in several TRACE sections, including “The classroom technology helped me to learn”, “I found this course intellectually challenging”, and “I learned a lot in this course”. Our results also indicate the benefits and importance of hands‐on experience in modeling software to better understand PK/PD in pharmacology. Support or Funding Information This project was supported by MathWorks Curriculum Development Grant.

Cardiac damage associated with iron overload is the most common cause of morbidity and mortality in patients with hereditary hemochromatosis, but the precise mechanisms leading to disease progression are largely unexplored. Here we investigated the effects of iron overload and age on cardiac hypertrophy using 1-, 5- and 12-month old Hfe-deficient mice, an animal model of hemochromatosis in humans. Cardiac iron levels increased progressively with age, which was exacerbated in Hfe-deficient mice. The heart/body weight ratios were greater in Hfe-deficient mice at 5- and 12-month old, compared with their age-matched wild-type controls. Cardiac hypertrophy in 12-month old Hfe-deficient mice was consistent with decreased alpha myosin and increased beta myosin heavy chains, suggesting an alpha-to-beta conversion with age. This was accompanied by cardiac fibrosis and up-regulation of NFAT-c2, reflecting increased calcineurin/NFAT signaling in myocyte hypertrophy. Moreover, there was an age-dependent increase in the cardiac isoprostane levels in Hfe-deficient mice, indicating elevated oxidative stress. Also, rats fed high-iron diet demonstrated increased heart-to-body weight ratios, alpha myosin heavy chain and cardiac isoprostane levels, suggesting that iron overload promotes oxidative stress and cardiac hypertrophy. Our findings provide a molecular basis for the progression of age-dependent cardiac stress exacerbated by iron overload hemochromatosis.

The divalent metal transporter 1 (DMT1) is a major iron transporter required for iron absorption and erythropoiesis. Loss of DMT1 function results in microcytic anemia. While iron plays an important role in neural function, the behavioral consequences of DMT1 deficiency are largely unexplored. The goal of this study was to define the neurobehavioral and neurochemical phenotypes of homozygous Belgrade (b/b) rats that carry DMT1 mutation and explore potential mechanisms of these phenotypes. The b/b rats (11-12weeks old) and their healthy littermate heterozygous (+/b) Belgrade rats were subject to elevated plus maze tasks. The b/b rats spent more time in open arms, entered open arms more frequently and traveled more distance in the maze than +/b controls, suggesting increased impulsivity. Impaired emotional behavior was associated with down-regulation of GABA in the hippocampus in b/b rats. Also, b/b rats showed increased GABA(A) receptor 1 and GABA transporter, indicating altered GABAergic function. Furthermore, metal analysis revealed that b/b rats have decreased total iron, but normal non-heme iron, in the brain. Interestingly, b/b rats exhibited unusually high copper levels in most brain regions, including striatum and hippocampus. Quantitative PCR analysis showed that both copper importer copper transporter 1 and exporter copper-transporting ATPase 1 were up-regulated in the hippocampus from b/b rats. Finally, b/b rats exhibited increased 8-isoprostane levels and decreased glutathione/glutathione disulfide ratio in the hippocampus, reflecting elevated oxidative stress. Combined, our results suggest that copper loading in DMT1 deficiency could induce oxidative stress and impair GABA metabolism, which promote impulsivity-like behavior.Iron-copper model: Mutations in the divalent metal transporter 1 (DMT1) decrease body iron status and up-regulate copper absorption, which leads to copper loading in the brain and consequently increases metal-induced oxidative stress. This event disrupts GABAergic neurotransmission and promotes impulsivity-like behavior. Our model provides better understanding of physiological risks associated with imbalanced metal metabolism in mental function and, more specifically, the interactions with GABA and redox control in the treatment of emotional disorders.

While nutritional and neurobehavioral problems are associated with both iron deficiency during growth and overload in the elderly, the effect of iron loading in growing ages on neurobehavioral performance has not been fully explored. To characterize the role of dietary iron loading in memory function in the young, weanling rats were fed iron-loading diet (10,000 mg iron/kg diet) or iron-adequate control diet (50 mg/kg) for one month, during which a battery of behavioral tests were conducted. Iron-loaded rats displayed elevated non-heme iron levels in serum and liver, indicating a condition of systemic iron overload. In the brain, non-heme iron was elevated in the prefrontal cortex of iron-loaded rats compared with controls, whereas there was no difference in iron content in other brain regions between the two diet groups. While iron loading did not alter motor coordination or anxiety-like behavior, iron-loaded rats exhibited a better recognition memory, as represented by an increased novel object recognition index (22% increase from the reference value) than control rats (12% increase; P=0.047). Western blot analysis showed an up-regulation of dopamine receptor 1 in the prefrontal cortex from iron-loaded rats (142% increase; P=0.002). Furthermore, levels of glutamate receptors (both NMDA and AMPA) and nicotinic acetylcholine receptor (nAChR) were significantly elevated in the prefrontal cortex of iron-loaded rats (62% increase in NR1; 70% increase in Glu1A; 115% increase in nAChR). Dietary iron loading also increased the expression of NMDA receptors and nAChR in the hippocampus. These results support the idea that iron is essential for learning and memory and further reveal that iron supplementation during developmental and rapidly growing periods of life improves memory performance. Our investigation also demonstrates that both cholinergic and glutamatergic neurotransmission pathways are regulated by dietary iron and provides a molecular basis for the role of iron loading in improved memory.

Title: Effect of iron overload on spatial memory and acetylcholine receptor expression in the hippocampus of rats Department of Pharmaceutical Sciences, Northeastern University, Boston, MA, USA Author: Murui Han, Jonghan Kim Iron plays an essential role in cognition and memory. While iron deficiency is associated with reduced memory capacity, we have previously demonstrated that iron overload improved a recognition memory performance in growing rats. We here characterized the effect of iron overload on special memory and explored molecular mechanisms involved in memory. Barnes maze test was conducted using rats fed iron overload diet (10,000 mg iron/kg diet) or control diet (50 mg/kg) for 4 weeks. Compared with controls, iron overload (IO) rats spent less time (57% decrease; p<0.001) and poked less errors (13% decrease; p=0.042) to find the target hole during the first training day (n=8/group), which indicates a better spatial memory upon iron loading. Western blotting was employed to examine memory‐related proteins in the hippocampus. IO rats up‐regulated nicotinic acetylcholine receptor alpha‐7 (nAChR; p=0.045; n=3‐4/group). Activities of superoxide dismutase (anti‐oxidant enzyme) or levels of malondialdehyde (lipid peroxidation marker) were not altered in IO rats. In addition, ICP‐MS results showed that brain metal levels (iron, zinc, manganese) were not altered in IO rats, indicating no evidence that IO promotes oxidative stress. Collectively, increasing memory performance under iron loading in growing rats could be due to up‐regulation of nAChR with brain metals unaffected. Supported by NIH R00 ES017781.Grant Funding Source: NIH R00 ES017781

Our recent study demonstrated that manganese exposure corrected neurobehavioral problems resulting from iron deficiency in young rats, suggesting a beneficial role of metal loading in brain function in growing children. To characterize the effect of dietary iron supplementation on behavioral performance, weanling rats were fed iron‐loading diet (10,000 mg iron/kg diet) or iron‐adequate control diet (50 mg/kg) for 4 weeks and a battery of behavioral tests (elevated plus maze, novel object recognition, rotarod) was conducted. Iron‐supplemented (IS) rats displayed higher non‐heme iron levels in serum (1.8‐fold) and liver (10.6‐fold) compared with controls, reflecting iron loading condition. Although iron loading did not affect anxiety determined by elevated plus maze, IS rats exhibited a better short‐term memory function (2‐hour) as assessed by an increased novel object recognition index (IS 22% change vs. control 12%; P=0.047; n=11/group). Iron loading did not alter overall activity (open field) or motor performance (rotarod test), indicating that increased memory functions are not due to altered mobile activity. Our investigation suggests that iron supplementation improves memory performance during developmental and rapidly growing periods of life. Supported by NIH ES017781.