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Alcohol-induced liver injury (ALI) has been associated with, among other molecular changes, abnormal hepatic methionine metabolism, resulting in decreased levels of S-adenosylmethionine (SAM). Dietary methyl donor supplements such as SAM and betaine mitigate ALI in animal models; however, the mechanisms of protection remain elusive. It has been suggested that methyl donors may act via attenuation of alcohol-induced oxidative stress. We hypothesized that the protective action of methyl donors is mediated by an effect on the oxidative metabolism of alcohol in the liver. Male C57BL/6J mice were administered a control high-fat diet or diet enriched in methyl donors with or without alcohol for 4 weeks using the enteral alcohol feeding model. As expected, attenuation of ALI and an increase in reduced glutathione:oxidized glutathione ratio were achieved with methyl donor supplementation. Interestingly, methyl donors led to a 35% increase in blood alcohol elimination rate, and while there was no effect on alcohol metabolism in the stomach, a profound effect on liver alcohol metabolism was observed. The catalase-dependent pathway of alcohol metabolism was induced, yet the increase in CYP2E1 activity by alcohol was blunted, which may be mitigating production of oxidants. Additional factors contributing to the protective effects of methyl donors in ALI were increased activity of low- and high-K(m) aldehyde dehydrogenases leading to lower hepatic acetaldehyde, maintenance of the efficient mitochondrial energy metabolism, and promotion of peroxisomal beta-oxidation. Profound changes in alcohol metabolism represent additional important mechanism of the protective effect of methyl donors in ALI.

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.

Toxicogenomics provides the ability to examine in greater detail the underlying molecular events that precede and accompany toxicity, thus allowing prediction of adverse events at much earlier times compared to classical toxicological end points. Acetaminophen (APAP) is a pharmaceutical that has similar metabolic and toxic responses in rodents and humans. Recent gene expression profiling studies with APAP found an oxidative stress signature at a subtoxic dose that we hypothesized can be phenotypically anchored to conventional biomarkers of oxidative stress. Liver tissue was obtained from experimental animals used to generate microarray data, where male rats were given APAP at subtoxic (150 mg/kg) or overtly toxic (1500 and 2000 mg/kg) doses and sacrificed at 6, 24, or 48 h. Oxidative stress in liver was evaluated by a diverse panel of markers that included assessing expression of base excision repair (BER) genes, quantifying oxidative lesions in genomic DNA, and evaluating protein and lipid oxidation. A subtoxic dose of APAP produced significant accumulation of nitrotyrosine protein adducts. Both subtoxic and toxic doses caused a significant increase in 8-hydroxy-deoxyguanosine (8-OH-dG) as well as a significant decrease in glutathione (GSH) content. Only toxic doses of APAP significantly induced expression levels of BER genes. None of the doses examined resulted in a significant increase in the number of abasic sites or in the amount of lipid peroxidation. The accumulation of nitrotyrosine and 8-OH-dG adducts along with reduced GSH content in the liver phenotypically anchors the oxidative stress gene expression signature observed with a subtoxic dose of APAP, lending support to the validity of gene expression studies as a sensitive and biologically meaningful end point in toxicology.

Hepatocellular carcinoma (HCC) is the terminal event in chronic liver diseases with repeated cycles of cellular injury and regeneration. Although much is known about the cellular pathogenesis and etiological agents leading to HCC, the molecular events are not well understood. The choline-deficient (CD) model of rodent HCC involves the consecutive emergence of a fatty liver, apoptosis, compensatory proliferation, fibrosis, and cirrhosis that is markedly similar to the sequence of events typified by human HCC. Moreover, oxidative stress is thought to play a pivotal role in the progression of the disease. Here, we hypothesize that gene expression profiling can temporally mirror the histopathology and oxidative DNA damage observed with this model. We show that clusters of highly co-regulated genes representing distinct cellular pathways for lipid biosynthesis and metabolism, apoptosis, cell proliferation, and tissue remodeling temporally correlate with the well-defined sequential emergence of pathological alterations in the progression of liver disease. Additionally, an oxidative stress signature was observed that was corroborated in a time-dependent manner with increases in oxidized purines and abasic sites in DNA. Collectively, expression patterns were strongly driven by pathology, demonstrating that patterns of gene expression in advanced stages of liver disease are primarily driven by histopathological changes and to a much lesser degree by the original etiological agent. In conclusion, gene expression profiling coupled with the CD model of HCC provides a unique opportunity to unveil the molecular events associated with various stages of liver injury and carcinogenesis and to distinguish between causal and consecutive changes.

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.

A receptor binding assay (RBA) for detection of paralytic shellfish poisoning (PSP) toxins was formatted for use in a high throughput detection system using microplate scintillation counting. The RBA technology was transferred from the National Ocean Service, which uses a Wallac TriLux 1450 MicroBeta microplate scintillation counter, to the California Department of Health Services, which uses a Packard TopCount scintillation counter. Due to differences in the detector arrangement between these 2 counters, markedly different counting efficiencies were exhibited, requiring optimization of the RBA protocol for the TopCount instrument. Precision, accuracy, and sensitivity [limit of detection = 0.2 μg saxitoxin (STX) equiv/100 g shellfish tissue] of the modified protocol were equivalent to those of the original protocol. The RBA robustness and adaptability were demonstrated by an interlaboratory study, in which STX concentrations in shellfish generated by the TopCount were consistent with MicroBeta-derived values. Comparison of STX reference standards obtained from the U.S. Food and Drug Administration and the National Research Council, Canada, showed no observable differences. This study confirms the RBA's value as a rapid, high throughput screen prior to testing by the conventional mouse bioassay (MBA) and its suitability for providing an early warning of increasing PSP toxicity when toxin levels are below the MBA limit of detection.

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.

Over the past decade, blooms of the domoic acid (DA) producing diatom Pseudo- nitzschia have been responsible for numerous deaths of marine mammals and birds in Monterey Bay, California. Euphausiids (krill) are important members of the local zooplankton grazer community and comprise the primary diet of squid, baleen whales, and many seabirds. Krill are thus a key potential vector for the transfer of DA to higher trophic level organisms in Monterey Bay. A better understand- ing of the quantitative trophic interactions and body burden of DA in krill is required to predict whether they can act as an effective vector for this neurotoxin. Here we report results of toxin analy- ses and gut content examinations of krill Euphausia pacifica collected from Monterey Bay in 2000. Corresponding counts of toxic Pseudo-nitzschia species in the water and their cellular DA concen- trations were also obtained at the collection sites. Toxin analysis by receptor binding assay demon- strated that DA in krill tissue varied between 0.1 to 44 µg DA equiv. g -1 tissue (confirmed by tandem mass spectrometry), with levels corresponding to the abundance of toxic Pseudo-nitzschia species present in the water. The occurrence of Pseudo-nitzschia australis frustules in the digestive tract of E. pacifica verified that a toxic species of this diatom was an important part of their diet and thus impli- cated this phytoplankter as the source of DA. These findings provide compelling evidence for the role of krill as a potential transfer agent of the phycotoxin DA to higher trophic levels in marine food web.

Domoic acid (DA), a potent neurotoxin, is synthesized by certain members of the ubiq- uitous marine diatom genus Pseudo-nitzschia. We recently detected elevated concentrations of DA in phytoplankton field samples from the northern Gulf of Mexico. In searching for a possible source of the toxin, we used a receptor-binding assay to detect DA activity in cultures of P. sp. cf. pseudodeli- catissima (Hasle) isolated from this region and confirmed its presence in 2 of 7 clones using liquid chromatography coupled with tandem mass-spectrometric detection (LC-MS/MS). Unlike other toxic Pseudo-nitzschia species examined previously (e.g., P. multiseries, P. australis), cellular levels and net production of DA in these clones were highest in the early exponential phase, while the population growth rate was high and cell concentration was low. There was a negative correlation between cel- lular DA and cell concentration. The maximum cellular DA activity in cultures was 36 fg DA equiv. cell -1 . No net toxin production was evident in the stationary phase, yet extracellular DA levels increased markedly during this period to as much as 88% of the total DA in the cultures. Interest- ingly, these 2 toxic clones were able to enlarge their cell size after the apical axes declined to 15 to 25 µm, and these larger cells had considerably higher levels of DA than the original small cells. This study unequivocally establishes P. sp. cf. pseudodelicatissima as a source of DA in the northern Gulf of Mexico. Moreover, our work suggests that rapidly growing, rather than nutrient-limited, popula- tions of this diatom should yield maximum net DA production rates and DA cell quotas. Thus, the presence of P. sp. cf. pseudodelicatissima cells, even at the low levels of early, rapidly growing bloom stages, can potentially lead to toxic events.

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.

Phytoplankton toxins undergo trophic transport and accumulation in marine food webs, causing vectorial intoxication of upper-level consumers such as fishes, seabirds, and marine mam- mals. An entry point for phytoplankton toxins into these pelagic trophic pathways is frequently the herbivorous zooplankton. During the 1995 spring-summer red tide season in Massachusetts Bay, we examined accumulation of paralytic shellfish poisoning (PSP) toxins from the dinoflagellate Alexan- drium spp. in various plankton size fractions (20-64, 64-100, 100-200, 200-500, and >500 µm), and identified the relative composition of the zooplankton in these size fractions. Toxin levels were esti- mated by both high-performance liquid chromatography (HPLC) and a receptor-binding assay, the latter based on sample toxic potency. Although no PSP toxicity was detected in nearshore shellfish by routine monitoring programs using the mouse bioassay, positive responses were detected in zoo- plankton size fractions with the more sensitive HPLC and the receptor assay methods. The toxin signal was disproportionately concentrated in the larger zooplankton size fraction, frequently domi- nated by large copepods such as Calanus finmarchicus and Centropages typicus, which comprised only a small portion of total zooplankton abundance in quantitative samples obtained with 100 µm mesh nets. By comparison, signal levels were low or undetectable in the smaller size fraction, which contained the overwhelmingly most-abundant zooplankters such as protists, copepod nauplii and copepodites and adults of small copepods such as Oithona similis, Paracalanus parvus, and Pseudo- calanus spp. The larger toxin-accumulating copepods could provide a direct trophic linkage for vectorial intoxication of baleen whales that are known to feed upon such copepods.

Large-subunit ribosomal RNA-targeted probes for Pseudo-nitzschia australis Frenguelli, P. multiseries (Hasle) Hasle, P. pseudodelicatissima (Hasle) Hasle, and P. pungens (Grunow) Hasle were applied to cultured and natural samples using whole-cell and sandwich hybridization. Testing of the latter method is emphasized here, and technique refinements that took place during 1996–1997 are documented. Application of the sandwich hybridization test showed that the signal intensity obtained for a given number of target cells remained constant as batch cultures of these organisms progressed from active through stationary growth phases. This suggests that cellular rRNA content for each target species remained relatively stable despite changes in growth state. Application of whole-cell and sandwich hybridization assays to natural samples showed that both methods could be used to detect wild P. australis, P. pseudodelicatissima, and to a lesser degree P. multiseries, but detection of P. pungens was prone to error. A receptor-binding assay for domoic acid (DA) enabled detection of this toxin activity associated with a particulate fraction of the plankton and provided a context in which to view results of the rRNA probe tests. In one case, the probe for P. australis cross-reacted with P. cf. delicatissima. The sample that contained the latter species also contained a low amount of DA activity. Under certain field conditions, results of whole-cell and sandwich hybridization tests disagreed. Detailed analysis of selected field samples illustrates how such situations arose. Collectively, the rRNA probe and toxin analyses suggest that manifestation of DA in the environment is possible in the absence of readily recognizable intact cells.

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.

Lipid peroxidation and the accompanying translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the lipid bilayer have recently been identified as key components of a signaling pathway for phagocytosis of apoptotic cells by macrophages. Drug-induced hemolytic anemia has long been known to be caused by an accelerated uptake of damaged (but intact) erythrocytes by macrophages in the spleen, and this process has been associated with enhanced formation of reactive oxygen species (ROS). However, the role of lipid peroxidation in hemolytic injury has remained unclear, and the effect of hemolytic agents on the distribution of PS in the erythrocyte membrane is unknown. The present studies were undertaken to determine whether lipid peroxidation and PS translocation could be detected in rat and human erythrocytes by three types of direct-acting hemolytic agents--dapsone hydroxylamine, divicine hydroquinone, and phenylhydrazine. 2',7'-Dichlorodihydrofluorescein diacetate was employed as a probe for intracellular ROS formation; lipid peroxidation was assessed by GC/MS analysis of F2-isoprostanes; and PS externalization was measured by annexin V labeling and the prothrombinase assay. The data confirmed that all three hemolytic agents generate ROS within erythrocytes under hemolytic conditions; however, no evidence for lipid peroxidation or PS translocation was detected. Instead, ROS production by these hemolytic agents was associated with extensive binding of oxidized and denatured hemoglobin to the membrane cytoskeleton. The data suggest that the transmembrane signal for macrophage recognition of hemolytic injury may be derived from oxidative alterations to erythrocyte proteins rather than to membrane lipids.

Oxidative stress induced DNA damage is considered to be the most common insult affecting the genome. Moreover, it is recognized as a common pathway to mutations and is suggested to play a major role in the development of chronic diseases such as cancer. However, current analytical methods used to detect oxidative DNA damage have been hampered by both technical and biological obstacles. These include spurious oxidation during DNA isolation and processing, and the inherent removal of damaged bases by numerous operating DNA repair systems. The removal of oxidized bases is performed predominantly by the base excision repair (BER) pathway and it has been shown that induction of DNA repair genes occurs in response to oxidative stress. Here, we demonstrate the utility of measuring changes in expression of BER genes as a sensitive in vivo biomarker for oxidative DNA damage.

A receptor binding assay (RBA) for detection of paralytic shellfish poisoning (PSP) toxins was formatted for use in a high throughput detection system using microplate scintillation counting. The RBA technology was transferred from the National Ocean Service, which uses a Wallac TriLux 1450 MicroBeta microplate scintillation counter, to the California Department of Health Services, which uses a Packard TopCount scintillation counter. Due to differences in the detector arrangement between these 2 counters, markedly different counting efficiencies were exhibited, requiring optimization of the RBA protocol for the TopCount instrument. Precision, accuracy, and sensitivity [limit of detection = 0.2 microg saxitoxin (STX) equiv/100 g shellfish tissue] of the modified protocol were equivalent to those of the original protocol. The RBA robustness and adaptability were demonstrated by an interlaboratory study, in which STX concentrations in shellfish generated by the TopCount were consistent with MicroBeta-derived values. Comparison of STX reference standards obtained from the U.S. Food and Drug Administration and the National Research Council, Canada, showed no observable differences. This study confirms the RBA's value as a rapid, high throughput screen prior to testing by the conventional mouse bioassay (MBA) and its suitability for providing an early warning of increasing PSP toxicity when toxin levels are below the MBA limit of detection.

The aim of this study was to begin evaluating the utility of sand crabs (Emerita analoga) as an indicator species for the algal neurotoxin, domoic acid (DA), in Monterey Bay, California, USA, a site of recurrent blooms of the DA-producing diatom, Pseudo-nitzschia. One of the current sentinel organisms, the sea mussel (Mytilus californianus), has shown minimal or undetectable toxicity during some local bloom events. As a critical step in assuring the accuracy of DA determinations in E. analoga, we have developed and validated a highly efficient extraction protocol that yields toxin recoveries of 97+/-2.9%. We also determined by HPLC-UV and receptor binding assay, with confirmation by LC-MS/MS, that sand crabs accumulated measurable amounts of DA during toxic Pseudo-nitzschia blooms, while the sea mussel showed no detectable toxin. In addition, a comparison of inter-animal variability in DA content revealed values ranging from ca. 0.5 to 5 microg DAg(-1) tissue and no consistent trend with size class, based on either animal weight or length. These data on the toxicity of individual animals will be useful in designing an appropriate sampling strategy for monitoring DA and, importantly, indicate that sand crabs do not appear to progressively bioaccumulate DA with age.

Over 400 California sea lions (Zalophus californianus) died and many others displayed signs of neurological dysfunction along the central California coast during May and June 1998. A bloom of Pseudo-nitzschia australis (diatom) was observed in the Monterey Bay region during the same period. This bloom was associated with production of domoic acid (DA), a neurotoxin1 that was also detected in planktivorous fish, including the northern anchovy (Engraulis mordax), and in sea lion body fluids. These and other concurrent observations demonstrate the trophic transfer of DA resulting in marine mammal mortality. In contrast to fish, blue mussels (Mytilus edulus) collected during the DA outbreak contained no DA or only trace amounts. Such findings reveal that monitoring of mussel toxicity alone does not necessarily provide adequate warning of DA entering the food web at levels sufficient to harm marine wildlife and perhaps humans.

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.