Role of excretion in manganese homeostasis and neurotoxicity: a historical perspective.

The essential metal manganese (Mn) induces incurable neurotoxicity at elevated levels that manifests as parkinsonism in adults and fine motor and executive function deficits in children. Studies on Mn neurotoxicity have largely focused on the role and mechanisms of disease induced by elevated Mn exposure from occupational or environmental sources. In contrast, the critical role of excretion in regulating Mn homeostasis and neurotoxicity has received less attention although 1) studies on Mn excretion date back to the 1920s; 2) elegant radiotracer Mn excretion assays in the 1940s to 1960s established the routes of Mn excretion; and 3) studies on patients with liver cirrhosis in the 1990s to 2000s identified an association between decreased Mn excretion and the risk of developing Mn-induced parkinsonism in the absence of elevated Mn exposure. Notably, the last few years have seen renewed interest in Mn excretion largely driven by the discovery that hereditary Mn neurotoxicity due to mutations in SLC30A10 or SLC39A14 is caused, at least in part, by deficits in Mn excretion. Quite remarkably, some of the recent results on SLC30A10 and SLC39A14 provide explanations for observations made ∼40-50 years ago. The goal of the current review is to integrate the historic studies on Mn excretion with more contemporary recent work and provide a comprehensive state-of-the-art overview of Mn excretion and its role in regulating Mn homeostasis and neurotoxicity. A related goal is to discuss the significance of some of the foundational studies on Mn excretion so that these highly consequential earlier studies remain influential in the field.

Sex-dependent Cav2.3 channel contribution to the secondary hyperalgesia in a mice model of central sensitization.

Central sensitization (CS) is characteristic of difficult to treat painful conditions, such as fibromyalgia and neuropathies and have sexual dimorphism involved. The calcium influx in nociceptive neurons is a key trigger for CS and the role of Cav2.1 and Cav2.2 voltage gated calcium channels (VGCC) in this role were evidenced with the use of ω-agatoxin IVA and ω-agatoxin MVIIA blockers, respectively. However, the participation of the α1 subunit of the voltage-gated channel Cav2.3, which conducts R-type currents, in CS is unknown. Furthermore, the role of sexual differences in painful conditions is still poorly understood. Thus, we investigated the role of Cav2.3 in capsaicin-induced secondary hyperalgesia in mice, which serve as a CS model predictive of the efficacy of novel analgesic drugs. Capsaicin injection in C57BL/6 mice caused secondary hyperalgesia from one to five hours after injection, and the effects were similar in male and female mice. In female but not male mice, intrathecal treatment with the Cav2.3 inhibitor SNX-482 partially and briefly reversed secondary hyperalgesia at a dose (300 pmol/site) that did not cause adverse effects. Moreover, Cav2.3 expression in the dorsal root ganglia (DRG) and spinal cord was reduced by intrathecal treatment with an antisense oligonucleotide (ASO) targeting Cav2.3 in female and male mice. However, ASO treatment was able to provide a robust and durable prevention of secondary hyperalgesia caused by capsaicin in female mice, but not in male mice. Thus, our results demonstrate that Cav2.3 inhibition, especially in female mice, has a relevant impact on a model of CS. Our results provide a proof of concept for Cav2.3 as a molecular target. In addition, the result associated to the role of differences in painful conditions linked to sex opens a range of possibilities to be explored and needs more attention. Thus, the relevance of testing Cav2.3 inhibition or knockdown in clinically relevant pain models is needed.

Reversal of Peripheral Neuropathic Pain by the Small-Molecule Natural Product Physalin F via Block of CaV2.3 (R-Type) and CaV2.2 (N-Type) Voltage-Gated Calcium Channels.

No universally efficacious therapy exists for chronic pain, a disease affecting one-fifth of the global population. An overreliance on the prescription of opioids for chronic pain despite their poor ability to improve function has led to a national opioid crisis. In 2018, the NIH launched a Helping to End Addiction Long-term plan to spur discovery and validation of novel targets and mechanisms to develop alternative nonaddictive treatment options. Phytochemicals with medicinal properties have long been used for various treatments worldwide. The natural product physalin F, isolated from the Physalis acutifolia (family: Solanaceae) herb, demonstrated antinociceptive effects in models of inflammatory pain, consistent with earlier reports of its anti-inflammatory and immunomodulatory activities. However, the target of action of physalin F remained unknown. Here, using whole-cell and slice electrophysiology, competition binding assays, and experimental models of neuropathic pain, we uncovered a molecular target for physalin F's antinociceptive actions. We found that physalin F (i) blocks CaV2.3 (R-type) and CaV2.2 (N-type) voltage-gated calcium channels in dorsal root ganglion (DRG) neurons, (ii) does not affect CaV3 (T-type) voltage-gated calcium channels or voltage-gated sodium or potassium channels, (iii) does not bind G-protein coupled opioid receptors, (iv) inhibits the frequency of spontaneous excitatory postsynaptic currents (EPSCs) in spinal cord slices, and (v) reverses tactile hypersensitivity in models of paclitaxel-induced peripheral neuropathy and spinal nerve ligation. Identifying CaV2.2 as a molecular target of physalin F may spur its use as a tool for mechanistic studies and position it as a structural template for future synthetic compounds.

Cyanidin Increases the Expression of Mg2+ Transport Carriers Mediated by the Activation of PPARα in Colonic Epithelial MCE301 Cells.

Mg2+ deficiency may be involved in lifestyle-related diseases, including hypertension, cardiovascular diseases, and diabetes mellitus. Dietary Mg2+ is absorbed in the intestine mediated through transcellular and paracellular pathways. However, there is little research into what factors upregulate Mg2+ absorption. We searched for food constituents that can increase the expression levels of Mg2+ transport carriers using mouse colonic epithelial MCE301 cells. Cyanidin, an anthocyanidin found in black beans and berries, increased the mRNA levels of Mg2+ transport carriers including transient receptor potential melastatin 6 (TRPM6) channel and cyclin M4 (CNNM4). The cyanidin-induced elevation of Mg2+ transport carriers was blocked by GW6471, a peroxisome proliferator-activated receptor α (PPARα) inhibitor, but not by PPARγ, PPARδ, and protein kinase A inhibitors. Cyanidin-3-glucoside showed similar results to cyanidin. Cyanidin increased the protein levels of TRPM6 and CNNM4, which were distributed in the apical and lateral membranes, respectively. The nuclear localization of PPARα and reporter activities of Mg2+ transport carriers were increased by cyanidin, which were inhibited by GW6471. The cyanidin-induced elevation of reporter activity was suppressed by a mutation in a PPAR-response element. Fluorescence measurements using KMG-20, an Mg2+ indicator, showed that Mg2+ influx and efflux from the cells were enhanced by cyanidin, and which were inhibited by GW6471. Furthermore, cyanidin increased paracellular Mg2+ flux without affecting transepithelial electrical resistance. We suggest that cyanidin increases intestinal Mg2+ absorption mediated by the elevation of TRPM6 and CNNM4 expression, and may constitute a phytochemical that can improve Mg2+ deficiency.

Down-regulation of natural resistance-associated macrophage protein-1 (Nramp1) is associated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)/1-methyl-4-phenylpyridinium (MPP+ )-induced α-synuclein accumulation and neurotoxicity.

AIMS: The accumulation of α-synuclein is a hallmark in the pathogenesis of Parkinson's disease (PD). Natural resistance-associated macrophage protein-1 (Nramp1) was previously shown to contribute to the degradation of extracellular α-synuclein in microglia under conditions of iron overload. This study was aimed at investigating the role of Nramp1 in α-synuclein pathology in the neurone under 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)/1-methyl-4-phenylpyridinium (MPP+ ) treatment. METHODS: The expression of Nramp1 and pathological features (including iron and α-synuclein accumulation) were examined in the dopaminergic neurones of humans (with and without PD) and of mice [with and without receiving chronic MPTP intoxication]. The effects of Nramp1 expression on low-dose MPP+ -induced α-synuclein expression and neurotoxicity were determined in human dopaminergic neuroblastoma SH-SY5Y cells. RESULTS: Similar to the findings in the substantia nigra of human PD, lower expression of Nramp1 but higher levels of iron and α-synuclein were identified in the dopaminergic neurones of mice receiving chronic MPTP intoxication, compared to controls. In parallel to the loss of dopaminergic neurones, the numbers of glial fibrillary acidic protein- and ionized calcium-binding adapter molecule-1-positive cells were significantly increased in the substantia nigra of MPTP-treated mice. Likewise, in human neuroblastoma SH-SY5Y cells exposed to low-dose MPP+ , Nramp1 expression and cathepsin D activity were decreased, along with an increase in α-synuclein protein expression and aggregation. Overexpression of functional Nramp1 restored cathepsin D activity and attenuated α-synuclein up-regulation and neuronal cell death caused by MPP+ treatment. CONCLUSIONS: These data suggest that the neuronal expression of Nramp1 is important for protecting against the development of MPTP/MPP+ -induced α-synuclein pathology and neurotoxicity.

Reciprocal modulation of Cav 2.3 voltage-gated calcium channels by copper(II) ions and kainic acid.

Kainic acid (KA) is a potent agonist at non-N-methyl-D-aspartate (non-NMDA) ionotropic glutamate receptors and commonly used to induce seizures and excitotoxicity in animal models of human temporal lobe epilepsy. Among other factors, Cav 2.3 voltage-gated calcium channels have been implicated in the pathogenesis of KA-induced seizures. At physiologically relevant concentrations, endogenous trace metal ions (Cu2+ , Zn2+ ) occupy an allosteric binding site on the domain I gating module of these channels and interfere with voltage-dependent gating. Using whole-cell patch-clamp recordings in human embryonic kidney (HEK-293) cells stably transfected with human Cav 2.3d and ß3 -subunits, we identified a novel, glutamate receptor-independent mechanism by which KA can potently sensitize these channels. Our findings demonstrate that KA releases these channels from the tonic inhibition exerted by low nanomolar concentrations of Cu2+ and produces a hyperpolarizing shift in channel voltage-dependence by about 10 mV, thereby reconciling the effects of Cu2+ chelation with tricine. When tricine was used as a surrogate to study the receptor-independent action of KA in electroretinographic recordings from the isolated bovine retina, it selectively suppressed a late b-wave component, which we have previously shown to be enhanced by genetic or pharmacological ablation of Cav 2.3 channels. Although the pathophysiological relevance remains to be firmly established, we speculate that reversal of Cu2+ -induced allosteric suppression, presumably via formation of stable kainate-Cu2+ complexes, could contribute to the receptor-mediated excitatory effects of KA. In addition, we discuss experimental implications for the use of KA in vitro, with particular emphasis on the seemingly high incidence of trace metal contamination in common physiological solutions.

Molecular cloning and expression analysis of ammonium transporters in tea plants (Camellia sinensis (L.) O. Kuntze) under different nitrogen treatments.

Ammonium is a major inorganic nitrogen source for tea plant growth and is mainly taken up and transported by ammonium transporters (AMTs). Here, we analyzed the NH4+ uptake kinetics of three tea cultivars, Longjing43 (LJ43), Zhongcha108 (ZC108) and Zhongcha302 (ZC302). The results revealed that ZC302 had a higher NH4+ uptake efficiency than the other two cultivars. The full CDS sequences of three Camellia sinensis ammonium transporter (CsAMT) genes, i.e., CsAMT1.1, CsAMT1.2 and CsAMT3.1, were cloned. Analysis of tissue-specific expression showed that CsAMT1.2 followed a root-specific expression pattern, while transcripts of CsAMT1.1 and CsAMT3.1 were mainly accumulated in leaves. The temporal course experiment on gene expression levels showed CsAMT1.1 and CsAMT3.1 followed a reciprocal expression pattern in leaves as CsAMT1.1 was up-regulated by a short time (2 h, 6 h) nitrogen (N) supply both in the leaves and buds of LJ43 and ZC108; and the expression of CsAMT3.1 in leaves was increased by a long time (72 h) N supply, particularly in ZC302. Therefore, we inferred that CsAMT1.1 and CsAMT3.1 might play important roles in photorespiratory ammonium metabolism. The expression of CsAMT1.2 was extremely high in roots and can be greatly induced by N over a short period of time, especially in ZC302; thus, we concluded CsAMT1.2 might play an important role in ammonium uptake from soils in tea plant roots.

Targeting Zinc(II) Signalling to Prevent Cancer.

Zinc is an important element that is gaining momentum as a potential target for cancer therapy. In recent years zinc has been accepted as a second messenger that is now recognized to be able to activate many signalling pathways within a few minutes of an extracellular stimulus by release of zinc(II) from intracellular stores. One of the major effects of this store release of zinc is to inhibit a multitude of tyrosine phosphatases which will prevent the inactivation of tyrosine kinases and hence, encourage further activation of tyrosine kinasedependent signalling pathways. Most of these signalling pathways are not only known to be involved in driving aberrant cancer growth, they are usually the main driving force. All this data together now positions zinc and zinc signalling as potentially important new targets to prevent aggressive cancer growth.

Changes in the Expression of Various Transporters as Influencing Factors of Resistance to Cisplatin.

BACKGROUND: Changes in the expression of transporters have been reported as factors in resistance to cisplatin (CDDP). This study was designed to clarify whether CDDP-resistant strains isolated from a cell line had the same characteristics, and whether these characteristics could be therapeutic targets. MATERIALS AND METHODS: Intracellular platinum levels were determined by the inductively-coupled plasma method. mRNA expression levels were determined using the real-time polymerase chain reaction. RESULTS: Some CDDP-resistant HepG2 cell lines exhibited changes in the expression of copper transporter 1, multidrug resistant protein (MRP)2, and/or MRP3, resulting in decreased intracellular platinum amounts, while others showed no change in platinum accumulation. Expression of these transporters was not necessarily maintained in a constant direction within the cell population isolated from the same origin. CONCLUSION: These results suggest that the CDDP-resistant tumors caused by a decrease in intracellular platinum content consist of a heterogeneous cell population showing expression changes of several transporters.

Inhibitors of Acetylcholinesterase Derived from 7-Methoxytacrine and Their Effects on the Choline Transporter CHT1.

BACKGROUND: Reversible acetylcholinesterase inhibitors are used in Alzheimer disease therapy. However, tacrine and its derivatives have severe side effects. Derivatives of the tacrine analogue 7-methoxytacrine (MEOTA) are less toxic. METHODS: We evaluated new derivatives of 7-MEOTA (2 homodimers linked by 2 C4-C5 chains and 5 N-alkylated C4-C8 side chain derivatives) in vitro, using the rat hippocampal choline transporter CHT1. RESULTS: Some derivatives were effective inhibitors of rat acetylcholinesterase and comparable with 7-MEOTA. All derivatives were able to inhibit CHT1, probably via quaternary ammonium, and this interaction could be involved in the enhancement of their detrimental side effects and/or in the attenuation of their promising effects. Under conditions of disrupted lipid rafts, the unfavorable effects of some derivatives were weakened. Only tacrine was probably able to stereospecifically interact with the naturally occurring amyloid-ß isoform and to simultaneously stimulate CHT1. Some derivatives, when coincubated with amyloid ß, did not influence CHT1. All derivatives also increased the fluidity of the cortical membranes. CONCLUSION: The N-alkylated derivative of 7-MEOTA bearing from C4 side chains appears to be the most promising compound and should be evaluated in future in vivo research.

Gadolinium toxicity: Iron and ferroportin as central targets.

Gadolinium-based magnetic resonance (MR) contrast agents (GBCM) causes a devastating systemic fibrosing illness, nephrogenic systemic fibrosis (NSF), in patients with reduced kidney function. GBCM targets iron-recycling CD163- and ferroportin-expressing macrophages to release labile iron that mediates gadolinium toxicity and NSF. GBCA might similarly target iron-rich, ferroportin-expressing structures such as globus pallidus and cerebellar dentate nucleus in the brain to result in metal accumulation and potential toxicity.

Dietary supplementation with ipriflavone decreases hepatic iron stores in wild type mice.

Hepcidin, a peptide produced in the liver, decreases intestinal iron absorption and macrophage iron release by causing degradation of the iron exporter, ferroportin. Because its levels are inappropriately low in patients with iron overload syndromes, hepcidin is a potential drug target. We previously conducted a chemical screen that revealed ipriflavone, an orally available small molecule, as a potent inducer of hepcidin expression. To evaluate ipriflavone's effect on iron homeostasis, we placed groups of 5-week old wild type or thalassemia intermedia (Hbb(Th3+/-)) mice on a soy-free, iron-sufficient diet, AIN-93G containing 220mg iron and 0-750mgipriflavone/kg of food for 50days. Ipriflavone 500mg/kg significantly reduced liver iron stores and intestinal ferroportin expression in WT mice, while increasing the ratio of hepcidin transcript levels to liver iron stores. Ipriflavone supplementation in Hbb(Th3+/-) mice failed to alleviate iron overload and was associated with a milder reduction in intestinal ferroportin and a failure to alter the ratio of hepcidin transcript levels to liver iron stores or splenic expression of the hepcidin-regulatory hormone, erythroferrone. These data suggest that dietary supplementation with ipriflavone alone would not be sufficient to treat iron overload in thalassemia intermedia.

Influence of Iron Supplementation on DMT1 (IRE)-induced Transport of Lead by Brain Barrier Systems in vivo.

OBJECTIVE: To investigate the potential involvement of DMT1 (IRE) protein in the brain vascular system in vivo during Pb exposure. METHODS: Three groups of male Sprague-Dawley rats were exposed to Pb in drinking water, among which two groups were concurrently administered by oral gavage once every other day as the low and high Fe treatment group, respectively, for 6 weeks. At the same time, the group only supplied with high Fe was also set as a reference. The animals were decapitated, then brain capillary-rich fraction was isolate from cerebral cortex. Western blot method was used to identify protein expression, and RT-PCR to detect the change of the mRNA. RESULTS: Pb exposure significantly increased Pb concentrations in cerebral cortex. Low Fe dose significantly reduced the cortex Pb levels, However, high Fe dose increased the cortex Pb levels. Interestingly, changes of DMT1 (IRE) protein in brain capillary-rich fraction were highly related to the Pb level, but those of DMT1 (IRE) mRNA were not significantly different. Moreover, the consistent changes in the levels of p-ERK1/2 or IRP1 with the changes in the levels of DMT1 (IRE). CONCLUSION: These results suggest that Pb is transported into the brain through DMT1 (IRE), and the ERK MAPK pathway is involved in DMT1 (IRE)-mediated transport regulation in brain vascular system in vivo.

Role of platinum DNA damage-induced transcriptional inhibition in chemotherapy-induced neuronal atrophy and peripheral neurotoxicity.

Platinum-based anticancer drugs cause peripheral neurotoxicity by damaging sensory neurons within the dorsal root ganglia (DRG), but the mechanisms are incompletely understood. The roles of platinum DNA binding, transcription inhibition and altered cell size were investigated in primary cultures of rat DRG cells. Click chemistry quantitative fluorescence imaging of RNA-incorporated 5-ethynyluridine showed high, but wide ranging, global levels of transcription in individual neurons that correlated with their cell body size. Treatment with platinum drugs reduced neuronal transcription and cell body size to an extent that corresponded to the amount of preceding platinum DNA binding, but without any loss of neuronal cells. The effects of platinum drugs on neuronal transcription and cell body size were inhibited by blocking platinum DNA binding with sodium thiosulfate, and mimicked by treatment with a model transcriptional inhibitor, actinomycin D. In vivo oxaliplatin treatment depleted the total RNA content of DRG tissue concurrently with altering DRG neuronal size. These findings point to a mechanism of chemotherapy-induced peripheral neurotoxicity, whereby platinum DNA damage induces global transcriptional arrest leading in turn to neuronal atrophy. DRG neurons may be particularly vulnerable to this mechanism of toxicity because of their requirements for high basal levels of global transcriptional activity. Findings point to a new stepwise mechanism of chemotherapy-induced peripheral neurotoxicity, whereby platinum DNA damage induces global transcriptional arrest leading in turn to neuronal atrophy. Dorsal root ganglion neurons may be particularly vulnerable to this neurotoxicity because of their high global transcriptional outputs, demonstrated in this study by click chemistry quantitative fluorescence imaging.

Hippocampal Gene Expression Is Highly Responsive to Estradiol Replacement in Middle-Aged Female Rats.

In the hippocampus, estrogens are powerful modulators of neurotransmission, synaptic plasticity and neurogenesis. In women, menopause is associated with increased risk of memory disturbances, which can be attenuated by timely estrogen therapy. In animal models of menopause, 17ß-estradiol (E2) replacement improves hippocampus-dependent spatial memory. Here, we explored the effect of E2 replacement on hippocampal gene expression in a rat menopause model. Middle-aged ovariectomized female rats were treated continuously for 29 days with E2, and then, the hippocampal transcriptome was investigated with Affymetrix expression arrays. Microarray data were analyzed by Bioconductor packages and web-based softwares, and verified with quantitative PCR. At standard fold change selection criterion, 156 genes responded to E2. All alterations but 4 were transcriptional activation. Robust activation (fold change > 10) occurred in the case of transthyretin, klotho, claudin 2, prolactin receptor, ectodin, coagulation factor V, Igf2, Igfbp2, and sodium/sulfate symporter. Classification of the 156 genes revealed major groups, including signaling (35 genes), metabolism (31 genes), extracellular matrix (17 genes), and transcription (16 genes). We selected 33 genes for further studies, and all changes were confirmed by real-time PCR. The results suggest that E2 promotes retinoid, growth factor, homeoprotein, neurohormone, and neurotransmitter signaling, changes metabolism, extracellular matrix composition, and transcription, and induces protective mechanisms via genomic effects. We propose that these mechanisms contribute to effects of E2 on neurogenesis, neural plasticity, and memory functions. Our findings provide further support for the rationale to develop safe estrogen receptor ligands for the maintenance of cognitive performance in postmenopausal women.

Dysregulation of hepatic zinc transporters in a mouse model of alcoholic liver disease.

Zinc deficiency is a consistent phenomenon observed in patients with alcoholic liver disease, but the mechanisms have not been well defined. The objective of this study was to determine if alcohol alters hepatic zinc transporters in association with reduction of hepatic zinc levels and if oxidative stress mediates the alterations of zinc transporters. C57BL/6 mice were pair-fed with the Lieber-DeCarli control or ethanol diets for 2, 4, or 8 wk. Chronic alcohol exposure reduced hepatic zinc levels, but increased plasma and urine zinc levels, at all time points. Hepatic zinc finger proteins, peroxisome proliferator-activated receptor-α (PPAR-α) and hepatocyte nuclear factor 4α (HNF-4α), were downregulated in ethanol-fed mice. Four hepatic zinc transporter proteins showed significant alterations in ethanol-fed mice compared with the controls. ZIP5 and ZIP14 proteins were downregulated, while ZIP7 and ZnT7 proteins were upregulated, by ethanol exposure at all time points. Immunohistochemical staining demonstrated that chronic ethanol exposure upregulated cytochrome P-450 2E1 and caused 4-hydroxynonenal accumulation in the liver. For the in vitro study, murine FL-83B hepatocytes were treated with 5 µM 4-hydroxynonenal or 100 µM hydrogen peroxide for 72 h. The results from in vitro studies demonstrated that 4-hydroxynonenal treatment altered ZIP5 and ZIP7 protein abundance, and hydrogen peroxide treatment changed ZIP7, ZIP14, and ZnT7 protein abundance. These results suggest that chronic ethanol exposure alters hepatic zinc transporters via oxidative stress, which might account for ethanol-induced hepatic zinc deficiency.

Drug delivery of zinc to Barrett's metaplasia by oral administration to Barrett's esophagus patients.

BACKGROUND: Delivery of a pharmacologically effective drug dosage to a target tissue is critical. Barrett's epithelia are a unique challenge for drug delivery of orally administered zinc due to rapid transit down the esophageal lumen, incomplete absorptive differentiation of these epithelia, and the use of proton-pump inhibitor drugs abrogating intestinal uptake of supplemental zinc. METHODS: Barrett's esophagus patients were administered oral zinc gluconate (26 mg zinc twice daily) for 14 days prior to biopsy procurement. Barrett's biopsies were analyzed for total zinc content by atomic absorption spectroscopy and by western immunoblot for cellular proteins known to be regulated by zinc. RESULTS: Cellular levels of both the Znt-1 transport protein and the alpha isoform of PKC were over 50% lower in the zinc treatment group. CONCLUSION: Oral zinc administration can result in effective delivery of zinc to Barrett's epithelia with resulting effects on intracellular signal transduction.

Involvement of CTR1 and ATP7A in lead (Pb)-induced copper (Cu) accumulation in choroidal epithelial cells.

The blood-cerebrospinal fluid barrier (BCB) plays a key role in maintaining copper (Cu) homeostasis in the brain. Cumulative evidences indicate that lead (Pb) exposure alters cerebral Cu homeostasis, which may underlie the development of neurodegenerative diseases. This study investigated the roles of Cu transporter 1 (CTR1) and ATP7A, two Cu transporters, in Pb-induced Cu accumulation in the choroidal epithelial cells. Pb exposure resulted in increased intracellular (64)Cu retention, accompanying with up-regulated CTR1 level. Knockdown of CTR1 using siRNA before Pb exposure diminished the Pb-induced increase of (64)Cu uptake. The expression level of ATP7A was down-regulated following the Pb exposure. ATP7A siRNA knockdown, or PCMB treatment, inhibited the (64)Cu efflux from the cells, while the following additional incubation with Pb failed to further increase the intracellular (64)Cu retention. Cu exposure, or intracellular Cu accumulation following the tetracycline (Tet)-induced overexpression of CTR1, did not result in significant change in ATP7A expression. Taken together, these data indicate that CTR1 and ATP7A play important roles in Cu transport in choroidal epithelial cells, and the Pb-induced intracellular Cu accumulation appears to be mediated, at least in part, via the alteration of CTR1 and ATP7A expression levels following Pb exposure.

[Effects of lead exposure on copper and copper transporters in choroid plexus of rats].

OBJECTIVE: To investigate the effects of lead exposure on the copper concentration in the brain and serum and the expression of copper transporters in the choroid plexus among rats. METHODS: Sixty specific pathogen-free Sprague-Dawley rats were randomly divided into a control group and three lead-exposed groups, with 8 mice in each group. The lead-exposed groups were orally administrated with 500 (low-dose group)), 1 000 (middle-dose group), and 2 000 mg/L (high-dose group) lead acetate in drinking water for eight weeks. And the rats in control group were given 2 000 mg/L sodium acetate in drinking water. The content of lead and copper in the serum, hippocampus, cortex, choroid plexus, bones, and cerebrospinal fluid (CSF) was determined by inductively coupled plasma-mass spectrometry (ICP-MS). Confocal and real-time PCR methods were applied to measure the expression of copper transporters including copper transporter 1 (Ctr1), antioxidant protein 1 (ATX1), and Cu ATPase (ATP7A). RESULTS: Compared with the control group, the lead-exposed groups showed significantly higher lead concentrations in the serum, cortex, hippocampus, choroid plexus, CSF, and bones (P < 0.05) and significantly higher copper concentrations in the CSF, choroid plexus, serum, and hippocampus (P < 0.05). Confocal images showed that Ctr1 protein was expressed in the cytoplasm and cell membrane of choroid plexus in control group. However, Ctr1 migrated to CSF surface microvilli after lead exposure. Ctr1 fluorescence intensity gradually increased with increasing dose of lead, except that the middle-dose group had a higher Ctr1 fluorescence intensity than the high-dose group. In addition, the middle- and high-dose groups showed a lower ATX1 fluorescence intensity compared with the control group. Real-time PCR data indicated that the three lead-exposed groups showed significantly higher mRNA levels of Ctr1 and ATP7A compared with the control group (P < 0.05). CONCLUSION: Copper homeostasis in the choroid plexus is affected by lead exposure to induce copper homeostasis disorders in brain tissue, which may be one of the mechanisms of lead neurotoxicity.

Peroxides with antiplasmodial activity inhibit proliferation of Perkinsus olseni, the causative agent of Perkinsosis in bivalves.

Perkinsus olseni, the causative agent of Perkinsosis, can drastically affect the survival of target marine mollusks, with dramatic economic consequences for aquaculture. P. olseni is a member of the Alveolata group, which also comprises parasites that are highly relevant for medical and veterinary sciences such as Plasmodium falciparum and Toxoplasma. P. olseni shares several unique metabolic pathways with those pathological parasites but is not toxic to humans. In this work, six antimalarially active peroxides, derived from the natural product artemisinin or synthetic trioxolanes, were synthesized and tested on P. olseni proliferation and survival. All peroxides tested revealed an inhibitory effect on P. olseni proliferation at micromolar concentrations. The relevance of the peroxide functionality on toxicity and the effect of Fe(II)-intracellular concentration on activity were also evaluated. Results demonstrated that the peroxide functionality is the toxofore and intracellular iron concentration also proved to be a crucial co-factor on the activation of peroxides in P. olseni. These data points to a mechanism of bioactivation in P. olseni sharing similarities with the one proposed in P. falciparum parasites. Preliminary studies on bioaccumulation were conducted using fluorescent-labeled peroxides. Results show that synthetic trioxolanes tend to accumulate on a vacuole while the labeled artemisinin accumulates in the cytoplasm. Preliminary experiments on differential genes expression associated to Fe(II) transport protein (Nramp) and calcium transport protein (ATP6/SERCA) were also conducted by qPCR. Results point to a fourfold increase in expression of both genes upon exposure to trioxolanes and approximately twofold upon exposure to artemisinin derivatives. Data obtained in this investigation is relevant for better understanding of the biology of Perkinsus and may also be important in the development of new strategies for Perkinsosis prevention and control.