Modulatory effects of <Emphasis Type="Italic">Terminalia arjuna</Emphasis> against domoic acid induced toxicity in Caco-2 cell line

Domoic acid is a potent marine algal toxin produced by diatomic genus of Pseudo-nitzschia causing amnesic shell fish poisoning. Domoic acid toxicosis mainly involves excitotoxic effects coupled with oxidative stress. The present study was aimed to evaluate the protective effects of hydro-alcoholic extract of Terminalia arjuna (TA) against domoic acid induced toxic effects in Caco-2 cell line. It was observed that the toxicity induced by domoic acid in Caco-2 cells was mediated by oxidative insult leading to morphological changes, DNA damage and apoptosis. In our study pre-treatment of the cells with TA (10, 20 and 30 μg/ml) showed significant protection against domoic acid induced morphological, oxidative and apoptotic damages in a dose dependent manner. The effect of phytocompounds present in TA viz., kaempferol and arjungenin showed significant protection against domoic acid induced toxicity in Caco-2 cell line. Hence, it could be inferred that the protective effect of TA extract against domoic acid induced toxicity could be due to the individual or synergistic effects of kaempferol and argungenin. However, further clinical studies are warranted to consider TA as a natural remedy to prevent amnesic shell fish poisoning.     

High CO2 and silicate limitation synergistically increase the toxicity of Pseudo-nitzschia fraudulenta

Anthropogenic CO(2) is progressively acidifying the ocean, but the responses of harmful algal bloom species that produce toxins that can bioaccumulate remain virtually unknown. The neurotoxin domoic acid is produced by the globally-distributed diatom genus Pseudo-nitzschia. This toxin is responsible for amnesic shellfish poisoning, which can result in illness or death in humans and regularly causes mass mortalities of marine mammals and birds. Domoic acid production by Pseudo-nitzschia cells is known to be regulated by nutrient availability, but potential interactions with increasing seawater CO(2) concentrations are poorly understood. Here we present experiments measuring domoic acid production by acclimatized cultures of Pseudo-nitzschia fraudulenta that demonstrate a strong synergism between projected future CO(2) levels (765 ppm) and silicate-limited growth, which greatly increases cellular toxicity relative to growth under modern atmospheric (360 ppm) or pre-industrial (200 ppm) CO(2) conditions. Cellular Si:C ratios decrease with increasing CO(2), in a trend opposite to that seen for domoic acid production. The coastal California upwelling system where this species was isolated currently exhibits rapidly increasing levels of anthropogenic acidification, as well as widespread episodic silicate limitation of diatom growth. Our results suggest that the current ecosystem and human health impacts of toxic Pseudo-nitzschia blooms could be greatly exacerbated by future ocean acidification and 'carbon fertilization' of the coastal ocean.     

Domoic acid, the alleged "mussel toxin," might produce its neurotoxic effect through kainate receptor activation: an electrophysiological study in the dorsal hippocampus

Domoic acid, an excitatory amino acid structurally related to kainate, was recently identified as being presumably responsible for the recent severe intoxication presented by more than 100 people having eaten mussels grown in Prince Edward Island (Canada). The amino acid kainate has been shown to be highly neurotoxic to the hippocampus, which is the most sensitive structure in the central nervous system. The present in vivo electrophysiological studies were undertaken to determine if domoic acid exerts its neurotoxic effect via kainate receptor activation. Unitary extracellular recordings were obtained from pyramidal neurons of the CA1 and the CA3 regions of the rat dorsal hippocampus. The excitatory effect of domoic acid applied by microiontophoresis was compared with that of agonists of the three subtypes of glutamatergic receptors: kainate, quisqualate, and N-methyl-D-aspartate. In CA1, the activation induced by domoic acid was about threefold greater than that induced by kainate; identical concentrations and similar currents were used. In CA3, domoic acid was also three times more potent than kainate. However, the most striking finding was that domoic acid, similar to kainate, was more than 20-fold more potent in the CA3 than in the CA1 region, whereas no such regional difference could be detected with quisqualate and N-methyl-D-aspartate. As the differential regional response of CA1 and CA3 pyramidal neurons to kainate is attributable to the extremely high density of kainate receptors in the CA3 region, these results provide the first electrophysiological evidence that domoic acid may produce its neurotoxic effects through kainate receptor activation.     

Demographics and spatio‐temporal signature of the biotoxin domoic acid in California sea lion (Zalophus californianus) stranding records

California sea lions (Zalophus californianus) in otherwise good nutritional condition have been consistently affected by the marine biotoxin domoic acid since the late 1990s. In this study we evaluated the temporal and spatial stranding patterns of suspected and confirmed cases of domoic acid intoxicated sea lions from 1998 to 2006, using records of strandings along the California coast obtained from members of the California Marine Mammal Stranding Network. The majority of domoic acid cases were adult females (47%–82% of the total annual domoic acid cases), a contrast to strandings that were not related to domoic acid, which were generally dominated by juveniles and pups. Exposure to this biotoxin led to a 6.67‐fold increase in adult female strandings in 2000, and a 5.44‐fold increase in adult female deaths in 2006, relative to strandings and deaths of adult female not affected by domoic acid. Domoic acid cases have occurred annually since 1998 (except for 1999) between April and August, with clusters centered primarily at Pismo Beach (San Luis Obispo County), as well as at other beaches in San Luis Obispo, Monterey, Santa Cruz, Santa Barbara, Orange, and San Diego counties. The larger ecological and population level implications of increased domoic acid strandings and deaths, particularly among adult female sea lions, warrant further attention and need to be investigated.     

Evaluation of the genotoxicity of domoic acid in a hepatocyte-mediated assay with V79 Chinese hamster lung cells

Domoic acid, a recognized neurotoxin derived from contaminated samples of the blue mussel (Mytilus edulis L.), was analyzed for mutagenicity at 2 loci and for 2 cytogenetic parameters in a hepatocyte-mediated assay with V79 Chinese hamster lung fibroblasts. Genetic end-points measured were: mutation to 6-thioguanine resistance at the HGPRTase locus; mutation to ouabain resistance at the Na+,K+-ATPase locus; sister-chromatid exchange (SCE) and micronucleus frequency (MN). None of these genetic end-points was significantly affected by exposure to domoic acid at dose levels of 27.2 and 54.4 micrograms/ml with or without activation by freshly isolated rat liver hepatocytes. It was concluded that, within the limits of the test system employed, domoic acid was non-genotoxic to V79 cells.     

Rapid behavioural diagnosis of domoic acid toxicosis in California sea lions

Domoic acid is a neurotoxic metabolite of widely occurring algal blooms that has caused multiple marine animal stranding events. Exposure to high doses of domoic acid, a glutamate agonist, may lead to persistent medial temporal seizures and damage to the hippocampus. California sea lions (Zalophus californianus) are among the most visible and frequent mammalian victims of domoic acid poisoning, but rapid, reliable diagnosis in a clinical setting has proved difficult owing to the fast clearance of the toxin from the blood stream. Here, we show that the behavioural orienting responses of stranded sea lions diagnosed with domoic acid toxicosis habituate more slowly to a series of non-aversive auditory stimuli than do those of sea lions with no apparent neurological deficits. A signal detection analysis based on these habituation measures was able to correctly identify 50 per cent of subjects with domoic acid toxicosis while correctly rejecting approximately 93 per cent of controls, suggesting potential diagnostic merit.     

Domoic acid production by Pseudo-nitzschia australis and Pseudo-nitzschia calliantha isolated from North Chile

The morphology and toxicity of the four ubiquitous species belonging to the genus Pseudo-nitzschia found in mixed blooms of phytoplankton from northern Chilean waters were studied. The phytoplankton samples and cultures obtained were identified by scanning electron microscopy, revealing the presence of Pseudo-nitzschia australis, P. calliantha, P. pseudodelicatissima and P. subfraudulenta. This is the first report of P. calliantha in northern Chile. Toxin analyses using the LC–MS method confirmed the presence of domoic acid in P. australis and P. calliantha. Domoic acid was not detected in cultures of P. subfraudulenta. This study therefore confirms P. australis and P. calliantha as an unequivocal source of domoic acid in Chilean waters. P. australis is probably the most important producer of amnesic shellfish toxin in view of its domoic acid content. However, more research is needed to evaluate the potential for toxin production in P. pseudodelicatissima.     

Domoic acid production by Pseudo-nitzschia australis and Pseudo-nitzschia calliantha isolated from North Chile

The morphology and toxicity of the four ubiquitous species belonging to the genus Pseudo-nitzschia found in mixed blooms of phytoplankton from northern Chilean waters were studied. The phytoplankton samples and cultures obtained were identified by scanning electron microscopy, revealing the presence of Pseudo-nitzschia australis, P. calliantha, P. pseudodelicatissima and P. subfraudulenta. This is the first report of P. calliantha in northern Chile. Toxin analyses using the LC–MS method confirmed the presence of domoic acid in P. australis and P. calliantha. Domoic acid was not detected in cultures of P. subfraudulenta. This study therefore confirms P. australis and P. calliantha as an unequivocal source of domoic acid in Chilean waters. P. australis is probably the most important producer of amnesic shellfish toxin in view of its domoic acid content. However, more research is needed to evaluate the potential for toxin production in P. pseudodelicatissima.     

Reduced neuroexcitatory effect of domoic acid following mossy fiber denervation of the rat dorsal hippocampus: further evidence that toxicity of domoic acid involves kainate receptor activation

Domoic acid, an excitatory amino acid structurally related to kainic acid, has been shown to be responsible for the severe intoxication presented, in 1987, by more than one hundred and fifty people having eaten mussels grown in Prince Edward Island (Canada). Unitary extracellular recordings were obtained from pyramidal neurons of the CA3 region of the rat dorsal hippocampus. The excitatory effects of microiontophoretic applications of domoic acid and of the agonists of the two other subtypes of glutamatergic receptors, quisqualate and N-methyl-D-aspartate, were compared on intact and colchicine-lesioned sides. Similar to what has been previously found for kainate, the colchicine lesion of the mossy fiber projections induced a 95% decrease of the neuronal responsiveness to domoic acid, whereas the effect of quisqualate was unchanged and that of N-methyl-D-aspartate was only slightly decreased. These results provide further electrophysiological evidence that domoic acid is a potent agonist of kainate receptors and that it may produce its neuroexcitatory and neurotoxic effects, in the hippocampal CA3 region, through activation of kainate receptors located on the mossy fiber terminals.     

Domoic acid neurotoxicity in hippocampal slice cultures

Summary.  The neurotoxicity of domoic acid was studied in 2–3 week old rat hippocampal slice cultures, derived from 7 day old rat pups. Domoic acid 0.1–100 μM was added to the culture medium for 48 hrs, alone or together with the glutamate receptor antagonists NS-102 (5-Nitro-6,7,8,9-tetrahydrobenzo[G]indole-2,3-dione-3-oxime), NBQX (2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline) or MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate), followed by transfer of the cultures to normal medium for additional 48 hrs. Neuronal degeneration in the fascia dentata (FD), CA3 and CA1 hippocampal subfields was monitored and EC₅₀ values estimated by densitometric measurements of the cellular uptake of propidium iodide (PI). The CA1 region was most sensitive to domoic acid, with an EC₅₀ value of 6 μM domoic acid, estimated from the PI-uptake at 72 hrs. Protective effects of 10 μM NBQX against 3 and 10 μM domoic acid were observed for both dentate granule cells and CA1 and CA3c pyramidal cells. NS102 and MK 801 only displayed protective effects when combined with NBQX. MK801 significantly increased the combined neuroprotective effect of NBQX and NS102 against 10 μM domoic acid in both CA1 and FD, but not in CA3. We conclude, that domoic acid neurotoxicity in CA3 and in hippocampal slice cultures in general primarily involves AMPA/kainate receptors. At high concentrations (10 μM domic acid) NMDA receptors are, however, also involved in the toxicity in CA1 and FD. Received June 29, 2001 Accepted August 6, 2001 Published online June 3, 2002     

Weak acid permeability through lipid bilayer membranes. Role of chemical reactions in the unstirred layer

The premeabilities of planar lipid bilayer (egg phosphatidylcholine- decane) membranes to butyric and formic acids were measured by tracer and pH electrode techniques. The purposes of the study were (a) to establish criteria for the applicability of each method and (b) to resolve a discrepancy between previously published permeabilities determined using the different techniques. Tracer fluxes of butyric acid were measured at several concentrations and pH's. Under symmetrical conditions the one-way flux of butyric acid(J) is described by 1/J = 1/Pul ([HA] + [A-]) + 1/Pm([HA]), where Pul and Pm are the unstirred layer and membrane permeability coefficients. Pm determined in this manner is 950 x 10(4) cm s-1. Published values for the butyric acid permeability for egg phosphatidylcholine-decane bilayers are 11.5 x 10(-4) (Wolosin and Ginsburg, 1975) and 640 x 10(-4) cm s-1 (Orbach and Finkelstein, 1980). Wolosin and Ginsburg measured net fluxes from a solution of pH = Pka into an unbuffered solution containing a pH electrode. Orbach and Finkelstein measured tracers fluxes under symmetrical conditions at pH 7.4. We reproduced the results of Wolosin and Ginsburg and showed that their apparently low Pm was caused by unstirred layer effects in their poorly buffered solutions. The permeability to formic acid (pKa = 3.75) measured by both tracer and pH electrode techniques was approximately 10(-2) cm s-1. However, if pm greater than Pul, the pH electrode technique cannot be used for measuring the permeabilities of weak acids with pKa's greater than approximately 4.     

FACTORS AFFECTING THE TOXICITY OF SEVERAL LICHEN ACIDS: EFFECT OF PH AND LICHEN ACID CONCENTRATION

The effect of pH 5–8 and lichen acid concentration gradients (2.7 times 10⁻² - 2.7 times 10⁻⁶ m ) on the toxicity of the following lichen acids: usnic, lecanoric, evernic, vulpinic, stictic, fumarpro-tocetraric, psoromic, and atranorin, on spores of Funaria hygrometrica was tested. Percent germination and sporeling growth were used as indicators of toxicity. None of the lichen acids were significantly toxic, for either percent germination or sporeling growth at concentrations equal to or below 2.7 times 10⁻⁵ m at pH 7.0, but many of the lichen acids which increased in toxicity at values different from pH 7 may have been toxic at lower concentrations if a different pH was used for the assay. Lichen acid toxicity showed a good correlation with pH for the parameter of spore germination, or sporeling growth, or both. Some lichen acids did not inhibit germination but were effective in retarding sporeling growth, or vice versa. This observation is discussed in relation to changing fatty acids and other lipid composition as germination occurs. Two of the three O-methylated lichen acids (evernic and psoromic) were among the most effective in inhibiting growth over all, but at lower pH values these were less effective than non-O-methylated lichen acids. Stictic, which is also an O-methylated lichen acid, was the least effective inhibitor over all the pH values for both parameters, while vulpinic was the most toxic over all the pH values. The order of relative toxicity for the lichen acids is different, depending on the pH and concentration at which they are tested and depending on the parameter measured. Thus, in an ecological sense, it is difficult to evaluate the adaptive significance of a particular compound or group of compounds without knowing what factors influence the toxicity of those compounds and how these factors vary in the organism's habitat.     

Effect of environmental pH on adenovirus-associated virus.

The influence of environmental pH on AAV was studied in infectious virus titrations, induction of CF antigens production of infectious virus, induction of immunofluorescent stainable antigen, and aggregation of the viral particles. The pH of the medium was found to influence the titer of virus stocks in that less virus was registered at acid pH's, giving differences of up to 105 TCID50 in HEK and HEp-2 cells. Less infectious virus was produced in KB cells, and decreased amounts of CF antigen appeared at acid pH's. However, increased levels of detectable intracellular FA antigen appeared at acid pH's. Electron microscopic examination of AAV particles negatively stained at various pH's showed increasingly large aggregates of particles as the pH was lowered. Under the acid conditions studied, the adenovirus helper and cell activities were only slightly suppressed, with the greatest effect due to aggregation of the virus particles.     

A comparison of the effect of kainate and some related amino acids on locomotor activity in cockroaches and electrical activity recorded from locust ventral nerve cord

The ED50 for loss of righting behaviour of cockroaches induced by kainate (43 mumol/kg body weight) indicated the toxicity of kainate to be much greater than would have been predicted from the excitatory action of this amino acid at insect skeletal muscle fibres. N-Methyl-D-aspartate had little effect on righting behaviour (ED50 greater than 3500 mumol/g body weight). Electrical recordings from the locust ventral nerve cord showed kainate (0.1-2 mM) to have a depolarizing action on neurons within the metathoracic ganglion. The depolarizing action of kainate was partially resistant to tetrodotoxin. The kainate-induced abolition of rostrally evoked potentials recorded in the abdominal connectives from the metathoracic ganglion suggests that the giant fibres are sensitive to kainate. Domoic acid was 46 times more potent than kainate. The lack of effect of N-methyl-D-aspartate (2 mM), dihydrokainate (2 mM), quisqualate (2 mM) and L-glutamate (20 mM) on nerve cords in the present experiments suggests that the kainate receptors in this preparation show a chemical selectivity comparable to that observed at vertebrate central neurones.     

Dynamics of the phycotoxin domoic acid: accumulation and excretion in two commercially important bivalves

Batch cultures of the toxigenic diatomNitzschia pungens Grunow f.multiseries Hasle were fed to blue mussels (Mytilus edulis) and deep sea Atlantic scallops (Placopecten magellanicus) to elucidate conditions under which domoic acid (DA) was accumulated and excreted (depurated). Mussels accumulated the toxin to a maximum level of 13 g g^-1, at rates of 0.21 to 3.7 g h^-1 g^-1, dry weight. Accumulation efficiency (the proportion of accumulated DA to estimated net uptake) ranged from 1–5%. The highest filtration rate of 1.71 h^-1 occurred at concentrations of 4–8 × 10⁶ Nitzschia cells 1^-1 with no formation of pseudofeces. Depuration rates between fed and starved mussels over a 2 h test period were the same. The depuration rate of domoic acid was about 17% d^-1 and did not account for the low uptake efficiencies, so it is suggested that most of the DA is lost from mussels in the solution during the feeding process. Domoic acid accumulation in mussels was dependent on the amount of toxin available, which in turn was a function of the density and growth phase of theNitzschia population. Changes in filtration rate withNitzschia concentration and depuration rate with time can account for the DA levels of mussels collected during toxic episodes in Cardigan Bay, Prince Edward Island, Canada in 1988 and 1989.Scallops accumulated DA (0.39–1.3 g h^-1 g^-1, more slowly than mussels, however, accumulation efficiencies ranged from 5–100%. Filtration rates remained relatively low and constant at 0.081 h^-1. Scallops retained domoic acid longer than mussels, a fact which must be considered in the marketing of whole scallops for human consumption.     

A novel antibody-based biomarker for chronic algal toxin exposure and sub-acute neurotoxicity

The neurotoxic amino acid, domoic acid (DA), is naturally produced by marine phytoplankton and presents a significant threat to the health of marine mammals, seabirds and humans via transfer of the toxin through the foodweb. In humans, acute exposure causes a neurotoxic illness known as amnesic shellfish poisoning characterized by seizures, memory loss, coma and death. Regular monitoring for high DA levels in edible shellfish tissues has been effective in protecting human consumers from acute DA exposure. However, chronic low-level DA exposure remains a concern, particularly in coastal and tribal communities that subsistence harvest shellfish known to contain low levels of the toxin. Domoic acid exposure via consumption of planktivorous fish also has a profound health impact on California sea lions (Zalophus californianus) affecting hundreds of animals yearly. Due to increasing algal toxin exposure threats globally, there is a critical need for reliable diagnostic tests for assessing chronic DA exposure in humans and wildlife. Here we report the discovery of a novel DA-specific antibody response that is a signature of chronic low-level exposure identified initially in a zebrafish exposure model and confirmed in naturally exposed wild sea lions. Additionally, we found that chronic exposure in zebrafish caused increased neurologic sensitivity to DA, revealing that repetitive exposure to DA well below the threshold for acute behavioral toxicity has underlying neurotoxic consequences. The discovery that chronic exposure to low levels of a small, water-soluble single amino acid triggers a detectable antibody response is surprising and has profound implications for the development of diagnostic tests for exposure to other pervasive environmental toxins.     

Copper(II) complexes of some N-substituted bis(aminomethyl)phosphinate ligands. An integrated EPR study of microspeciation and coordination modes by the two-dimensional simulation method

Copper(II) complexes of bis(aminomethyl)phosphinic acid (L1), bis(N-glycino-N-methyl)phosphinic acid (L2), bis(N-benzylglycino-N-methyl)phosphinic acid (L3), bis(l-prolino-N-methyl)phosphinic acid (L4) and bis(iminodicarboxymethyl-N-methyl)phosphinic acid (L5) were studied in aqueous solution by pH-potentiometric and electron paramagnetic resonance (EPR) spectroscopic methods. The EPR spectrum packages recorded at various ligand-to-metal concentration ratios and pH's were analyzed (after matrix rank analysis by the method of residual intensities as a complementary method) by the two-dimensional computer simulation method, which simultaneously determines the formation constants and the EPR parameters of the various (micro)species. L1 forms mono and bis complexes in different protonation states; for the other ligands, the mono complexes are always prevalent. For steric reasons, the formation of CuL is shifted to increasingly higher pH regions in the sequence L2, L3 and L4. CuLH was identified for L3, L4 and L5, and also CuLH(2) for L4 and L5. Cu(2)L(2) was found in small amounts for L3 and L4, while it predominates at pH>4 for L5. For L5, Cu(2)L(2)H(2) was also detected. For the ligands that form dimeric metal complexes in equimolar solution or at a ligand excess, Cu(2)L is formed at a metal ion excess. Ligation of the phosphinate O was suggested by indirect proofs in the protonated complexes of L1. For the ligands L2, L3 and L4, the copper(II) coordination in various species in different protonation states is reminiscent of that in the mono and bis complexes of simple amino acids. For the bis(aminomethyl)phosphinates, however, the cis positions of the amino groups in CuL are ensured by the structure of the ligand, and the isomers differ from each other in the (equatorial or axial) position of the second carboxylate group.     

Hydrogen-bond network and pH sensitivity in transthyretin: Neutron crystal structure of human transthyretin

Transthyretin (TTR) is a tetrameric protein associated with human amyloidosis. In vitro, the formation of amyloid fibrils by TTR is known to be promoted by low pH. Here we show the neutron structure of TTR, focusing on the hydrogen bonds, protonation states and pH sensitivities. A large crystal was prepared at pD 7.4 for neutron protein crystallography. Neutron diffraction studies were conducted using the IBARAKI Biological Crystal Diffractometer with the time-of-flight method. The neutron structure solved at 2.0 Å resolution revealed the protonation states of His88 and the detailed hydrogen-bond network depending on the protonation states of His88. This hydrogen-bond network is composed of Thr75, Trp79, His88, Ser112, Pro113, Thr118-B and four water molecules, and is involved in both monomer–monomer and dimer–dimer interactions, suggesting that the double protonation of His88 by acidification breaks the hydrogen-bond network and causes the destabilization of the TTR tetramer. In addition, the comparison with X-ray structure at pH 4.0 indicated that the protonation occurred to Asp74, His88 and Glu89 at pH 4.0. Our neutron model provides insights into the molecular stability of TTR related to the hydrogen-bond network, the pH sensitivity and the CH···O weak hydrogen bond.