Peter D. R. Moeller

Detection of domoic acid in northern anchovies and california sea lions associated with an unusual mortality event

The occurrence of an unusual mortality event involving California sea lions (Zalophus californianus) along the central California coast in May 1998 was recently reported. The potent neurotoxin domoic acid (DA), produced naturally by the diatom Pseudo-nitzschia australis and transmitted to the sea lions via planktivorous northern anchovies (Engraulis mordax), was identified as the probable causative agent. Details of DA analyses for anchovy tissues and sea lion feces are described. Domoic acid levels were estimated in anchovy samples by HPLC-UV, and in sea lion feces using the same method as well as a microplate receptor binding assay, with absolute confirmation by tandem mass spectrometry. The highest DA concentrations in anchovies occurred in the viscera (223 +/- 5 microg DA g(-1)), exceeding values in the body tissues by seven-fold and suggesting minimal bioaccumulation of DA in anchovy tissue. HPLC values for DA in sea lion fecal material (ranging from 152 to 136.5 microg DA g(-1)) required correction for interference from an unidentified compound. Inter-laboratory comparisons of HPLC data showed close quantitative agreement. Fecal DA activity determined using the receptor binding assay corresponded with HPLC values to within a factor of two. Finally, our detection of P. australis frustules, via scanning electron microscopy, in both anchovy viscera and fecal material from sea lions exhibiting seizures provides corroborating evidence that this toxic algal species was involved in this unusual sea lion mortality event.

Identification of a P2X7 receptor in GH(4)C(1) rat pituitary cells: a potential target for a bioactive substance produced by Pfiesteria piscicida.

We examined the pharmacologic activity of a putative toxin (pPfTx) produced by Pfiesteria piscicida by characterizing the signaling pathways that induce the c-fos luciferase construct in GH(4)C(1) rat pituitary cells. Adenosine-5-triphosphate (ATP) was determined to increase and, at higher concentrations, decrease luciferase activity in GH(4)C(1) rat pituitary cells that stably express c-fos luciferase. The inhibition of luciferase results from cytotoxicity, characteristic of the putative P. piscicida toxin (pPfTx). The actions of both pPfTx and ATP to induce c-fos luciferase were inhibited by the purinogenic receptor antagonist pyridoxalphosphate-6-azophenyl-2,4-disulfonic acid (PPADS). Further characterization of a P2X receptor on the GH(4)C(1) cell was determined by the analog selectivity of P2X agonists. The P2X1/P2X3 agonist alpha,beta-methylene ATP (alpha,beta-MeATP) failed to increase or decrease c-fos luciferase. However, the P2X7 agonist 2,3-(4-benzoyl)benzoyl ATP (BzATP), which had a predominant cytotoxic effect, was more potent than ATP. Immunoblot analysis of GH(4)C(1) cell membranes confirmed the presence of a 70-kDa protein that was immunoreactive to an antibody directed against the carboxy-terminal domain unique to the P2X7 receptor. The P2X7 irreversible antagonist oxidized-ATP (oxATP) inhibited the action of ATP, BzATP, and pPfTx. These findings indicate that GH(4)C(1) cells express purinogenic receptors with selectivity consistent with the P2X7 subtype and that this receptor pathway mediates the induction of the c-fos luciferase reporter gene by ATP and the putative Pfiesteria toxin

Neuroexcitatory actions of ciguatoxin on brain regions associated with thermoregulation

We have investigated the action of ciguatoxin (CTX) in the mouse following i.p. administration. CTX (0.5 mouse units) induced a rapid (10-20 min) decrease in body temperature that persisted for several hours. This corresponded closely with a neuroexcitatory action of ciguatoxin on c-fos mRNA in the brain. We identified the neuronal pathways activated by CTX action in the mouse brain by immunostaining of c-fos translational product, a biomarker for neuroexcitability. c-fos-like immunoreactivity was prominent in the hypothalamus, including the medial preoptic and supraoptic nuclei. Immunostaining was also evident in certain regions of the brain stem, including the locus coeruleus, dorsolateral parabranchial nucleus, area postrema and the nucleus of the solitary tract. These studies indicate that CTX has neuroexcitatory actions on brain stem regions receiving vagal afferents and ascending pathways associated with visceral and thermoregulatory responses.

Morphology and toxicology of Prorocentrum arabianum sp. nov., (dinophyceae) a toxic planktonic dinoflagellate from the Gulf of Oman, Arabian Sea ☆

Abstract A new species of planktonic dinoflagellate, Prorocentrum arabianum isolated from the Gulf of Oman, is described using both scanning electron microscopy (SEM) and light microscopy. This clonal isolate has the following morphological characteristics: (1) cell shape is asymmetric; (2) thecal surface is rugose, covered with small poroids; (3) periflagellar area is unornamented, and (4) intercalary band is horizontally striated. Analysis of P. arabianum confirms the production of one cytotoxic compound and one ichthyotoxic compound. P. arabianum is the second known toxic planktonic Prorocentroid dinoflagellate.

Learning impairment caused by a toxin produced by Pfiesteria piscicida infused into the hippocampus of rats.

Pfiesteria piscicida, an estuarine dinoflagellate, which has been shown to kill fish, has also been associated with neurocognitive deficits in humans. With a rat model, we have demonstrated the cause-and-effect relationship between Pfiesteria exposure and learning impairment. In several studies, we have replicated the finding in Sprague-Dawley rats that exposure to fixed acute doses of Pfiesteria cells or filtrates caused radial-arm maze learning impairment. Recently, this finding of Pfiesteria-induced learning impairment in rats has been independently replicated in another laboratory as well. We have demonstrated significant Pfiesteria-induced learning impairment in both the win-shift and repeated-acquisition tasks in the radial-arm maze and in reversal learning in a visual operant signal detection task. These learning impairments have been seen as long as 10 weeks after a single acute exposure to Pfiesteria. In the current study, we used a hydrophilic toxin isolated from clonal P. piscicida cultures (PfTx) and tested its effect when applied locally to the ventral hippocampus on repeated acquisition of rats in the radial-arm maze. Toxin exposure impaired choice accuracy in the radial-arm maze repeated acquisition procedure. The PfTx-induced impairment was seen at the beginning of the session and the early learning deficit was persistent across 6 weeks of testing after a single administration of the toxin. Eventually, with enough practice, in each session, the PfTx-exposed rats did learn that sessions problem as did control rats. This model has demonstrated the cause-and-effect relationship between exposure to a hydrophilic toxin produced by P. piscicida and learning impairment, and specifically that the ventral hippocampus was critically involved.

First report of goniodomin A production by the dinoflagellate Alexandrium pseudogonyaulax developing in southern Mediterranean (Bizerte Lagoon, Tunisia).

The dinoflagellate Alexandrium pseudogonyaulax is widely distributed around the world including the Mediterranean waters. The objectives of this study were to determine the morphology and phylogenic affiliation of A. pseudogonyaulax strain isolated from Bizerte Lagoon (Mediterranean waters, Tunisia) and investigate its toxicity. Molecular analyses confirmed the morphological identification of the isolated strain (APBZ12) as A. pseudogonyaulax. Moreover, it showed that it is 100% identical with strains of this species found in New Zealand, Japan, China and North Sea (Norway and Denmark) suggesting that this species is cosmopolitan. Until now, no toxin studies have been conducted on fully characterized (morphologically and molecularly) A. pseudogonyaulax. Cellular toxin production was determined using high pressure liquid chromatography coupled to mass spectrometry (HPLC/MS). Results showed for the first time that A. pseudogonyaulax contains goniodomin A (GDA), a highly toxic macrolide polyether previously shown to be produced by two other dinoflagellate species Alexandrium monilatum (Hsia et al., 2006) and Alexandrium hiranoi (erroneously identified as A. pseudogonyaulax in Murakami et al., 1988) in American and Japanese waters, respectively. This biologically active toxin has been associated over decades with fish mortality. Our study showed that the cell extracts of APBZ12 showed an important bioactivity using GH4C1 rat pituitary cytotoxicity bioassay.

The role of 2,4-dihydroxyquinoline (DHQ) in Pseudomonas aeruginosa pathogenicity.

Bacteria synchronize group behaviors using quorum sensing, which is advantageous during an infection to thwart immune cell attack and resist deleterious changes in the environment. In Pseudomonas aeruginosa, the Pseudomonas quinolone signal (Pqs) quorum-sensing system is an important component of an interconnected intercellular communication network. Two alkylquinolones, 2-heptyl-4-quinolone (HHQ) and 2-heptyl-3-hydroxy-4-quinolone (PQS), activate transcriptional regulator PqsR to promote the production of quinolone signals and virulence factors. Our work focused on the most abundant quinolone produced from the Pqs system, 2,4-dihydroxyquinoline (DHQ), which was shown previously to sustain pyocyanin production and antifungal activity of P. aeruginosa. However, little is known about how DHQ affects P. aeruginosa pathogenicity. Using C. elegans as a model for P. aeruginosa infection, we found pqs mutants only able to produce DHQ maintained virulence towards the nematodes similar to wild-type. In addition, DHQ-only producing mutants displayed increased colonization of C. elegans and virulence factor production compared to a quinolone-null strain. DHQ also bound to PqsR and activated the transcription of pqs operon. More importantly, high extracellular concentration of DHQ was maintained in both aerobic and anaerobic growth. High levels of DHQ were also detected in the sputum samples of cystic fibrosis patients. Taken together, our findings suggest DHQ may play an important role in sustaining P. aeruginosa pathogenicity under oxygen-limiting conditions.

Induced thyme product prevents VEGF-induced migration in human umbilical vein endothelial cells.

Compounds with anti-angiogenic properties are useful in combating cancer by preventing new blood vessel formation to support the tumor. In this report we introduce a rapid method for screening potential anti-angiogenic compounds in a model system that stimulates the production of secondary defense chemicals in plants. This methodology identified an inducible vascular factor (IVF3), which was found to be inhibitory in all of the model systems tested. Thyme plants were exposed to highly vascular mint plants and the methanol extracts were analyzed by reverse phase HPLC. The thyme compounds induced by the invading mint tissue, and not present in the thyme plants grown alone, were tested in a vertical plate assay measuring root length as a quantitative assay for drug sensitivity. The HPLC-purified extract, referred to as IVF3, reduced the growth of root vascular tissue compared to the control and vehicle control, and 50% as well as known angiogenesis inhibitors, VEGF receptor tyrosine kinase inhibitor and amiloride hydrochloride. Extracted compounds that were effective inhibitors of plant roots were assayed in Madin Darby canine kidney epithelial cells (MDCK) for toxicity, and in human umbilical vein endothelial cells (HUVEC) for their effect on migration. IVF3 was effective at limiting HUVEC migration in VEGF-stimulated cultures. In vivo video capture of intersegmental vessel circulation between 48 and 72 h post fertilization in the developing vasculature of zebrafish embryos showed IVF3 also significantly reduced ISV functional circulation. This report demonstrates the anti-angiogenic effects of IVF3 extract in endothelial cells and in an intact vertebrate model for angiogenesis.

Aflatoxin-Exposure of Vibrio gazogenes as a Novel System for the Generation of Aflatoxin Synthesis Inhibitors.

Aflatoxin is a mycotoxin and a secondary metabolite, and the most potent known liver carcinogen that contaminates several important crops, and represents a significant threat to public health and the economy. Available approaches reported thus far have been insufficient to eliminate this threat, and therefore provide the rational to explore novel methods for preventing aflatoxin accumulation in the environment. Many terrestrial plants and microbes that share ecological niches and encounter the aflatoxin producers have the ability to synthesize compounds that inhibit aflatoxin synthesis. However, reports of natural aflatoxin inhibitors from marine ecosystem components that do not share ecological niches with the aflatoxin producers are rare. Here, we show that a non-pathogenic marine bacterium, Vibrio gazogenes, when exposed to low non-toxic doses of aflatoxin B1, demonstrates a shift in its metabolic output and synthesizes a metabolite fraction that inhibits aflatoxin synthesis without affecting hyphal growth in the model aflatoxin producer, Aspergillus parasiticus. The molecular mass of the predominant metabolite in this fraction was also different from the known prodigiosins, which are the known antifungal secondary metabolites synthesized by this Vibrio. Gene expression analyses using RT-PCR demonstrate that this metabolite fraction inhibits aflatoxin synthesis by down-regulating the expression of early-, middle-, and late- growth stage aflatoxin genes, the aflatoxin pathway regulator, aflR and one global regulator of secondary metabolism, laeA. Our study establishes a novel system for generation of aflatoxin synthesis inhibitors, and emphasizes the potential of the under-explored Vibrio’s silent genome for generating new modulators of fungal secondary metabolism.

Complementary feeding may pose a risk of simultaneous exposures to aflatoxin M1 and deoxynivalenol in Indian infants and toddlers: Lessons from a mini-survey of food samples obtained from Kolkata, India

A mini-survey of 29 different foods produced by 21 different Indian manufacturers was conducted for the presence of aflatoxins B1, B2, G1 and G2, aflatoxin M1 and deoxynivalenol. The products were purchased from local markets in Kolkata, India and commonly used in the complementary feeding of infants and toddlers in India. Using a previously established direct competitive enzyme-linked immunoassay for this analysis we show that 100% of the samples contained aflatoxin M1 at levels exceeding the recommended European Union levels of 25u202fngu202fkg-1 by more than an order of magnitude. Also, several (66%) of them contained detectable concentrations of deoxynivalenol with two samples (6.9%) exceeding European Union guidelines for baby food products (200u202fμgu202fkg-1) and 51.7% samples with DON levels that can lead to dietary intake higher than 1u202fu202fμgu202fkg-1 recommended by the joint FAO/WHO expert committee on food additives. None of the samples contained aflatoxins B1, B2, G1 and G2. The results, therefore, suggest that complementary feeding can put Indian infants and toddlers at risk of simultaneous exposures to deoxynivalenol and aflatoxin M1 and warrant an urgent in-depth research to track, increase surveillance and reduce mycotoxin contamination of baby foods manufactured in India.