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1.  Arsenic toxicity in the human nerve cell line SK-N-SH in the presence of chromium and copper 
Chemosphere  2013;91(8):1082-1087.
As, Cr, and Cu represent one potential combination of multiple metals/metalloids exposures since these three elements are simultaneously leached from chromated copper arsenate (CCA)-treated wood, a common product used for building construction, at levels that can be potentially harmful. This study investigated the neurotoxicity of As associated with CCA-treated wood when accompanied by Cr and Cu. The toxicity was evaluated on basis of a cytotoxicity model using human neuroblastoma cell line SK-N-SH. The cells were cultured with CCA-treated wood leachates or with solutions containing arsenate [As(V)], divalent copper [Cu(II)], trivalent chromium [Cr(III)] alone or in different combinations of the three elements. The toxicity was evaluated using variations in cell replication compared to controls after 96 hrs exposure. Among the three elements present in wood leachates, As played the primary role in the observed toxic effects, which exerted through multiple pathways, including the generation of oxidative stress. DOM affected the absorption of metals/metalloids into the test cells, which however did not obviously appear to impact toxicity. As toxicity was enhanced by Cu(II) and inhibited by Cr(III) at concentrations below U.S. EPA’s allowable maximum contaminant levels in drinking waters. Thus assessing As toxicity in real environments is not sufficient if based solely on the result from As.
PMCID: PMC3630255  PMID: 23473430
Arsenic; copper; chromium; neurotoxicity; combined toxic effect
2.  Synthesis, receptor binding and activity of iso and azakainoids 
Two syntheses for the production of an unsubstituted azakainoid are described. The 1,3-dipolar cycloaddition of diazomethane with trans-dibenzyl glutaconate yields a 1-pyrazoline, which may be reduced directly to the pyrazolidine. An unexpected trans-cis isomerization is observed during Hg/Al reduction of the 1-pyrazoline N=N bond. Alternatively, when TMS diazomethane is used as the dipole, the resulting 2-pyrazoline obtained after desilylation may be reduced with NaCNBH3 to provide the trans azakainate analog exclusively. The synthesis of an unsubstituted isokainoid via Michael addition is also described. Glutamate receptor binding assays revealed that the azakaniod has a moderate affinity for unspecified glutamate receptors. Membrane depolarization of Aplysia neurons upon application of the azakainoid demonstrates that it is an ionotropic glutamate receptor agonist.
PMCID: PMC3601572  PMID: 23481645
3.  Behavioral aging is associated with reduced sensory neuron excitability in Aplysia californica 
Invertebrate models have advantages for understanding the basis of behavioral aging due to their simple nervous systems and short lifespans. The potential usefulness of Aplysia californica in aging research is apparent from its long history of neurobiological research, but it has been underexploited in this model use. Aging of simple reflexes at both single sensory neuron and neural circuit levels was studied to connect behavioral aging to neurophysiological aging. The tail withdrawal reflex (TWR), righting reflex, and biting response were measured throughout sexual maturity in 3 cohorts of hatchery-reared animals of known age. Reflex times increased and reflex amplitudes decreased significantly during aging. Aging in sensory neurons of animals with deficits in measures of the TWR and biting response resulted in significantly reduced excitability in old animals compared to their younger siblings. The threshold for firing increased while the number of action potentials in response to depolarizing current injection decreased during aging in sensory neurons, but not in tail motoneurons. Glutamate receptor-activated responses in sensory neurons also decreased with aging. In old tail motoneurons, the amplitude of evoked EPSPs following tail shock decreased, presumably due to reduced sensory neuron excitability during aging. The results were used to develop stages of aging relevant to both hatchery-reared and wild-caught Aplysia. Aplysia is a viable aging model in which the contributions of differential aging of components of neural circuits may be assessed.
PMCID: PMC4023074  PMID: 24847260
L-glutamate; NMDA; D-aspartate; pleural ganglion; buccal ganglion
The marine toxin, okadaic acid (OA) is produced by dinoflagellates of the genera Prorocentrum and Dinophysis and is the causative agent of the syndrome known as diarrheic shellfish poisoning (DSP). In addition, OA acts as both a tumor promoter, attributed to OA-induced inhibition of protein phosphatases as well as an inducer of apoptosis. To better understand the potentially divergent toxicological profile of OA, the concentration dependent cytotoxicity and alterations in gene expression on the human liver tumor cell line HepG2 upon OA exposure were determined using RNA microarrays, DNA fragmentation, and cell proliferation assays as well as determinations of cell detachment and cell death in different concentrations of OA. mRNA expression was quantified for approximately 15,000 genes. Cell attachment and proliferation were both negatively correlated with OA concentration. Detached cells displayed necrotic DNA signatures but apoptosis also was broadly observed. Data suggest that OA has a concentration dependent effect on cell cycle, which might explain the divergent effects that at low concentration OA stimulates genes involved in the cell cycle and at high concentrations it stimulates apoptosis.
PMCID: PMC3500632  PMID: 23172983
5.  Unique ionotropic receptors for D-Aspartate are a target for serotonin-induced synaptic plasticity in Aplysia californica✰ 
The non-L-glutamate (L-Glu) receptor component of D-aspartate (D-Asp) currents in Aplysia californica buccal S cluster (BSC) neurons was studied with whole cell voltage clamp to differentiate it from receptors activated by other well-known agonists of the Aplysia nervous system and investigate modulatory mechanisms of D-Asp currents associated with synaptic plasticity. Acetylcholine (ACh) and serotonin (5-HT) activated whole cell excitatory currents with similar current voltage relationships to D-Asp. These currents, however, were pharmacologically distinct from D-Asp. ACh currents were blocked by hexamethonium (C6) and tubocurarine (d-TC), while D-Asp currents were unaffected. 5-HT currents were blocked by granisetron and methysergide (MES), while D-Asp currents were unaffected. Conversely, while (2S,3R)-1-(Phenanthren-2-carbonyl)piperazine-2,3-dicarboxylic acid(PPDA) blocked D-Asp currents, it had no effect on ACh or 5-HT currents. Comparison of the charge area described by currents induced by ACh or 5-HT separately from, or with, D-Asp suggests activation of distinct receptors by all 3 agonists. Charge area comparisons with L-Glu, however, suggested some overlap between L-Gluand D-Asp receptors. Ten minute exposure to 5-HT induced facilitation of D-Asp-evoked responses in BSC neurons. This effect was mimicked by phorbol ester, suggesting that protein kinase C (PKC) was involved.
PMCID: PMC3155736  PMID: 21497673
patch clamping; electrophysiology; NMDA; plasticity; 5-HT; protein kinase C
6.  Pharmacological evidence that D-aspartate activates a current distinct from ionotropic glutamate receptor currents in Aplysia californica 
Brain and Behavior  2012;2(4):391-401.
D-Aspartate (D-Asp) activates a nonspecific cation current of unknown identity independent of L-glutamate (L-Glu) in neurons of Aplysia californica. Whole-cell voltage clamp studies were conducted using primary cultures of Aplysia buccal S cluster (BSC) neurons to characterize these receptor channels pharmacologically. The N-methyl-D-aspartate receptor (NMDAR) coagonist glycine potentiated D-Asp currents only at −30 mV, while D-serine did not potentiate D-Asp currents at any amplitude. Portions of D-Asp currents were blocked by the L-Glu antagonists kynurenate, DL-2-amino-5-phosphonopentanoic acid (APV), (2S,3R)-1-(phenanthren-2-carbonyl)piperazine-2,3-dicarboxylic acid (PPDA), and 1,3-dihydro-5-[3-[4-(phenylmethyl)-1–2H-benzimidazol-2-one (TCS46b), suggesting that L-Glu channels, particularly NMDAR-like channels, may partially contribute to D-Asp whole-cell currents. In contrast, L-Glu currents were unaffected by APV, and showed greater block by kynurenate, suggesting that D-Asp and L-Glu act, in part, at different sites. The excitatory amino acid transport blocker DL-threo-b-Benzyloxyaspartic acid (TBOA) blocked a fraction of D-Asp currents, suggesting that currents associated with these transporters also contribute. Non-NMDA L-GluR antagonists that preferentially block alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA)/kainate receptors significantly increased D-Asp currents, suggesting a possible allosteric potentiating effect of these antagonists on D-Asp receptors. L-Glu-induced currents were significantly reduced in the presence of bath-applied D-Asp, whereas bath-applied L-Glu had no effect on D-Asp-induced currents. The mixed effects of these agents on D-Asp-induced currents in Aplysia illustrate that the underlying channels are not uniformly characteristic of any known agonist associated channel type.
PMCID: PMC3432962  PMID: 22950043
APV; buccal ganglion; coagonist; electrophysiology; mollusk; NMDA
7.  Changes in D-Aspartate ion currents in the Aplysia nervous system with aging 
Brain research  2010;1343:28-36.
D-Aspartate (D-Asp) can substitute for L-Glutamate (L-Glu) at excitatory Glu receptors, and occurs as free D-Asp in the mammalian brain. D-Asp electrophysiological responses were studied as a potential correlate of aging in the California sea hare, Aplysia californica. Whole cell voltage- and current clamp measurements were made from primary neuron cultures of the pleural ganglion (PVC) and buccal ganglion S cluster (BSC) in 3 egg cohorts at sexual maturity and senescence. D-Asp activated an inward current at the hyperpolarized voltage of −70 mV, where molluscan NMDA receptors open free of constitutive block by Mg2+. Half of the cells responded to both D-Asp and L-Glu while the remainder responded only to D-Asp or L-Glu, suggesting that D-Asp activated non-Glu channels in a subpopulation of these cells. The frequency of D-Asp-induced currents and their density were significantly decreased in senescent PVC cells but not in senescent BSC cells. These changes in sensory neurons of the tail predict functional deficits that may contribute to an overall decline in reflexive movement in aged Aplysia.
PMCID: PMC3062251  PMID: 20452331
A. californica; voltage clamp; D-Asp; glutamate; agonist; NMDA
8.  Novel Modulator of NaV1.1 and NaV1.2 Na+ Channels in Rat Neuronal Cells 
ACS Medicinal Chemistry Letters  2010;1(3):135-138.
A novel modulator of sodium ion currents was synthesized in 6 steps from a protected dihydroxypyrrolidine nitrone, via 1,3-dipolar cycloaddition reaction with acrylamide. Sodium ion currents in B50 cells were evaluated in comparison to saxitoxin and tetrodotoxin and revealed an IC50 of 15.7 μM. The new compound shows no evidence of binding to the C-lobe of the saxitoxin-binding protein saxiphilin.
PMCID: PMC2894714  PMID: 20607120
Nitrone; 1,3-dipolar cycloaddition; saxitoxin (STX); tetrodotoxin (TTX); sodium channel blocker; B50 cells
Earlier studies documented the loss of wood preservatives from new wood. The objective of this study was to evaluate losses from weathered treated wood under field conditions by collecting rainfall leachate from 5 different wood types, all with a surface area of 0.21 m2. Wood samples included weathered chromate copper arsenate (CCA) treated wood at low (2.7 kg/m3), medium (4.8 kg/m3) and high (35.4 kg/m3) retention levels, new alkaline copper quat (ACQ) treated wood (1.1 kg/m3 as CuO) and new untreated wood. Arsenic was found to leach at a higher rate (100 mg in 1 year for low retention) than chromium and copper (<40 mg) in all CCA treated wood samples. Copper leached at the highest rate from the ACQ sample (670 mg). Overall results suggest that metals’ leaching is a continuous process driven by rainfall, and that the mechanism of release from the wood matrix changes as wood weathers.
PMCID: PMC2847651  PMID: 20053493
ACQ; arsenic; CCA; chromium; copper; treated wood; weathered wood
10.  Novel modulator of NaV1.1 and NaV1.2 Na+ channels in rat neuronal cells 
ACS medicinal chemistry letters  2010;1(3):135-138.
A novel modulator of sodium ion currents was synthesized in 6 steps from a protected dihydroxypyrrolidine nitrone, via 1,3-dipolar cycloaddition reaction with acrylamide. Sodium ion currents in B50 cells were evaluated in comparison to saxitoxin and tetrodotoxin, and revealed an IC50 of 15.7 μM. The new compound shows no evidence of binding to the C-lobe of the saxitoxin-binding protein saxiphilin.
PMCID: PMC2894714  PMID: 20607120
Nitrone; 1,3-dipolar cycloaddition; Saxitoxin (STX); Tetrodotoxin (TTX); Sodium channel blocker; B50 cells
11.  Transport and interaction of arsenic, chromium, and copper associated with CCA-treated wood in columns of sand and sand amended with peat 
Chemosphere  2010;78(8):989-995.
Laboratory column leaching experiments were conducted to investigate the transport and interaction of As, Cr, and Cu associated with CCA-treated wood in sand with and without peat amendment. Results showed that leaching behavior of As, Cr, and Cu in these substrates were totally different. Substrate characteristics and microorganism activity posed distinct effects on the transport and transformation of these three elements. Arsenic was rapidly leached out from the columns with or without the amendment of peat, while Cr remained in all columns during the entire experimental period (215 days). Copper was leached out only in the substrate column without peat. The presence of microorganism clearly facilitated the transport of As, while it did not show obvious effects on the transport of Cr and Cu. Interactions among these three elements were observed during the processes of adsorption and transport. The adsorption of Cu on soil was enhanced with the adsorption of As, likely caused by a more negatively charged soil surface because of As adsorption. The adsorption of Cr on soil increased the adsorption of As due to the additional As binding sites induced by Cr adsorption. These results suggest that As concentrations in the soil affected by CCA-treated wood could largely exceed predictions based on soil adsorption capacity for As. The evaluation of the impact on human health associated with CCA-treated wood should take consideration of the distinct transport characteristics of three elements and their interactions in soils.
PMCID: PMC2862480  PMID: 20053417
CCA-treated wood; leachability; microorganism; sand; transport; interaction

Results 1-11 (11)