Search

The age-associated decline in cellular antioxidant defenses and resultant accumulation of DNA damage in central nervous system has been mechanistically implicated in the etiology and pathogenesis of neurodegenerative diseases. Neurons possess a high metabolic activity and are especially vulnerable to the long-term effects of continuous exposure to endogenous reactive oxygen species. It is well recognized that adequate availability of essential nutrients involved in cellular one-carbon metabolism is essential for normal brain development and function. Additionally, the synthesis of the primary low-molecular cellular antioxidant glutathione is inter-dependently linked to one-carbon metabolic pathway. Thus, any aberrant disruptions in one-carbon metabolism can result in potentially deleterious effects including cell death as a result of an imbalance in the cellular redox state. Hence, in the present study, we examined the long-term effects of a folate/methyl-deficient (FMD) diet on cellular antioxidant defenses and DNA damage in the rat brain. Feeding male Fisher 344 rats a FMD diet resulted in perturbations in the levels of one-carbon metabolites along with induction of oxidative stress and oxidative DNA damage in the brain. This was evidenced by a decrease in the reduced oxidized/glutathione ratio, imbalance of cellular antioxidant defense system; specifically, altered activity and expression of antioxidant enzymes Mn-containing superoxide dismutase (Mn-SOD), catalase, and glutathione peroxidase (GPX), increased accumulation of oxidative DNA lesions, 8-hydroxydeoxyguanosine (8-OH-dG) and DNA single-strand breaks, even in the presence of increased expression of critical DNA repair genes apurinic/apyrimidinic endonuclease 1 (Apex1) and DNA polymerase beta (Polbeta), and apoptosis in the brains of folate/methyl-deficient rats. These results indicate that chronic methyl group deficiency leads to an imbalance in cellular antioxidant defense systems, increased oxidative stress, and apoptosis. Any of these events may compromise normal central nervous system function and contribute to the development of various neurological, behavioral, and neurocognitive dysfunctions.

Over the past decade diatom blooms of domoic acid (DA)-producing Pseudo-nitzschia spp. have been responsible for numerous marine mammal and bird mortalities in Monterey Bay, CA. One possible toxin vector is the market squid, Loligo opalescens , a small pelagic mollusk that plays an important role in the near-shore food web of the California Current ecosystem as a favored vertebrate prey species. This study examined the trophic link between toxic Pseudo-nitzschia and L. opalescens using toxin and stomach content analyses of animals collected from Monterey Bay, CA in 2000. Receptor binding assay data (confirmed by tandem mass spectrometry), demonstrated the presence of DA in squid during a toxic Pseudo-nitzschia event, with P. australis frustules observed in stomach samples. Though DA levels were low ( −1 tissue) in L. opalescens during the study period, it is now clear that this potent neurotoxin can occur in squid and is likely delivered through its krill prey species, which are known to retain DA after feeding on toxic Pseudo-nitzschia . Our findings suggest that further study of the relationship between Pseudo-nitzschia blooms and DA contamination of squid is warranted to better evaluate the potential health risk to humans and wildlife associated with this major commercial seafood species and important prey item.

The transfer of marine algal toxins involving a range of phytoplanktivorous vectors is well documented as a means of exposing organisms at higher trophic levels (including humans) to these naturally occurring yet harmful compounds. While previous studies have examined the potential for, and dynamics of, algal toxin accumulation by individual zooplankton species, few have attempted to distinguish the contribution of various grazer size classes to toxin trophic transfer in natural communities and characterize some of the factors that can influence this process. The current investigation was aimed at describing the size-fractioned (64–100, 100–200, 200–500, >500 μm) accumulation of paralytic shellfish poisoning (PSP) toxins by zooplankton in Casco Bay and the adjacent coastal waters of the Gulf of Maine during a series of cruises from April to June 1998. Several variables, including the abundance of PSP toxin-producing Alexandrium fundyense , in-water toxin concentrations associated with this dinoflagellate, and algal toxin cell quotas, were measured and their relationship to zooplankton toxin accumulation assessed. A principal finding of this work was the ability of any grazer size class examined (including grazers present in the 20–64 μm A. fundyense -containing fraction) to serve as an initial vector for introducing PSP toxins into the Casco Bay food web at various times during the sampling period, thereby providing multiple potential routes of toxin trophic transfer. In addition, trends observed in the coincident mapping of A. fundyense cells and their associated toxin were generally in agreement, yet did not remain closely coupled at all times. Therefore, although A. fundyense abundance can be a reasonable indicator of PSP toxin presence in the phytoplankton, this relationship can vary considerably and lead to situations where elevated toxin levels occur at low cell concentrations and vice versa. The uncoupling of A. fundyense cell and in-water toxin concentrations in the 20–64 μm, A. fundyense -containing size fraction implied fluctuations in the algal toxin cell quota, which ranged from ca. 10 to 2000 fmol STX equiv. cell −1 . Some of this variability may reflect the changing presence in this size fraction of grazers (e.g., tintinnids) capable of toxin accumulation, causing an upward bias in A. fundyense toxin cell quota estimates. Overall, the extent of PSP toxin transfer into zooplankton will be determined by a complex interaction among several factors, including A. fundyense and grazer abundance, algal toxin cell quota, and zooplankton community composition. An ability to predict zooplankton toxin accumulation will require further investigation of the relationships between these and other factors, aimed specifically at modeling the process of toxin trophic transfer to grazers and ultimately to their predators.

Saxitoxins, the etiological agent of paralytic shellfish poisoning, are synthesized by dinoflagellates and cyanobacteria. Several reports indicate that bacteria are capable of saxitoxin synthesis. Two bacterial strains were isolated from saxitoxin-producing dinoflagellates, Alexandrium tamarense and A. lusitanicum (=Alexandrium minutum), and grown under a variety of culture conditions including those previously reported to induce saxitoxin synthesis in bacteria. Five fluorescent compounds were accumulated by the bacteria that had HPLC-FLD retention times similar to a reference standard of GTX(4), one of the saxitoxin congeners. However, we were unable to detect GTX(1), the epimeric partner of GTX(4), in the bacterial samples. The GTX(4) standard was hydrolyzed by NaOH/heat treatment but four of the bacterial compounds were stable. Unlike GTX(4), none of the five bacterial compounds were detectable by HPLC-FLD following electrochemical oxidation. The fluorescence emission spectrum of each of the five bacterial compounds was unique and readily discernable from the spectrum of GTX(4). None of the samples containing the putative GTX(4) toxin yielded positive results when analyzed by a 3H-saxitoxin receptor-binding assay for saxitoxin-like activity. We cannot rule out the possibility that these bacteria produce saxitoxins, however, our data clearly demonstrate that they accumulate at least five different fluorescent compounds that could be easily mistaken for GTX(4). We conclude that these five fluorescent compounds are GTX(4) imposters and that fluorescence scanning and chemical/heat stability should, at a minimum, be incorporated into HPLC-FLD protocols for identification of saxitoxins.

Blooms of domoic acid (DA) synthesizing diatoms (Pseudo-nitzschia spp.) have been associated with the death and injury of hundreds of marine shorebirds and mammals, exposed humans to potentially serious health risks, and threatened to significantly impact coastal fisheries and commerce dependent on marine resources. While indicator organisms are widely utilized to monitor for marine biotoxins like paralytic shellfish poisoning toxins, a reliable intertidal indicator species to monitor DA remains to be identified. Here we evaluate and confirm the utility of the common sand crab (Emerita analoga) as an indicator for DA in comparison with sea mussels (Mytilus californianus). Mussels and sand crabs, collected from natural populations in Santa Cruz, California (April 1999-February 2000), were tested for DA using the HPLC-UV method. Toxin loads in sand crabs ranged from below detectable limits to 13.4 micro g DA g(-1) and coincided with the abundance of DA producing Pseudo-nitzschia species nearshore. Toxin levels in mussels collected during the study period were below HPLC-UV detectable limits. The rise and fall of DA in sand crabs in synchrony with Pseudo-nitzschia abundance, combined with this common intertidal species' accessibility and ease of DA extraction, clearly indicate the utility of sand crabs as a reliable, cost-effective monitoring tool for DA in the nearshore coastal environment.

In August and September 2001, Kuwait Bay, a semi-enclosed embayment of the Arabian Gulf, experienced a massive fish kill involving over >2500 metric tons of wild mullet ( Liza klunzingeri ), due to the bacterium Streptococcus agalactiae . In the Bay, this event was preceded by a small fish kill (100–1000 dead fish per day) of gilthead sea bream ( Sparus auratus ) in aquaculture net pens associated with a bloom of the dinoflagellate Ceratium furca . Sea bream were found to be culture positive for S. agalactiae , but did not show any visible signs of disease. Unusually warm temperatures (up to 35 °C) and calm conditions prevailed during this period. As the wild fish kill progressed, various harmful algae were observed, including Gymnodinium catenatum , Gyrodinium impudicum , and Pyrodinium bahamense var. compressum . Cell numbers of G. catenatum and G. impudicum exceeded 10 6 l −1 in some locations. All fish tested below the limits of detection for paralytic shellfish poisoning (PSP) and brevetoxins. Clams ( Circe callipyga ) were positive for PSP but at levels below regulatory limits. Nutrient concentrations, both inorganic and organic, were highly variable with time and from site to site, reflecting inputs from sewage outfalls, the aquaculture operations, a high biomass of decomposing fish, and other sources. It is hypothesized that many factors contributed to the initial outbreak of the bacterial disease, including unusual warm and calm conditions. The same factors, as well as enriched nutrient conditions, also apparently were conducive to the subsequent HAB outbreaks. The detection of PSP, while below regulatory limits, warrants further monitoring to protect human health.

This report describes the preparative scale production of 11-[3H]-tetrodotoxin (TTX) and its evaluation as a substitute for [3H]-saxitoxin (STX) as the radioligand in a receptor binding assay for paralytic shellfish poisoning (PSP) toxins. Restrictions on the world-wide distribution of [3H]-STX imposed by the international Chemical Weapons Convention served as the primary impetus for this study. We have incorporated on a preparative scale, a nonexchangeable tritium label into the TTX molecule at a specific activity of 12.90 Ci/mmol and recovered material of high radiochemical purity (98%). The resulting 11-[3H]-TTX was found to exhibit site-specific binding characteristics in the receptor assay ( dissociation constant (K d )=4.77±1.54 nM ; maximum binding (B max )=1.62±0.24 pmol / mg of synaptosomal protein ). The inhibition constant (Ki) for the assay was 1.46±0.28 nM STX equiv. ( n=6 ), with an estimated detection limit of ca. 2–4 ng STX equiv./ml in a sample extract. Moreover, quantitative comparisons indicated that 11-[3H]-TTX could be used interchangeably with [3H]-STX in the receptor assay for determination of PSP toxicity in shellfish and algal extracts without compromising assay performance. We conclude that the 11-[3H]-TTX produced and evaluated herein exhibits physical, chemical and biological characteristics suitable not only for use in the PSP receptor binding assay, but likely for other applications employing [3H]-STX as the radioligand.

The neurotoxin domoic acid (DA) was identified as the probable cause of death and neurological illness for approximately 70 California sea lions, Zalophus californianus, along the central California coast in May 1998. The apparent vector for sea lion intoxication was the filter-feeding northern anchovy, Engraulis mordax, via transfer of DA from the diatom, Pseudo-nitzschia australis, a natural source of this toxin. Both DA and siliceous frustules of P. australis were found in anchovy gut contents and in fecal material collected from sea lions exhibiting seizures. Anchovies contained 200.29±3.34 μg DA/g in viscera samples, 49.69±0.79 μg DA/g in whole body samples and 35.14±0.47 μg DA/g in body tissue samples. DA concentrations in sea lion fecal material ranged from 1.31 to 182.01 μg DA/g. This is the first documented case of DA toxicity in a marine mammal species.