Total Toxin Research Articles

First paralytic shellfish poison (PSP) infestation of bivalves due to toxic dinoflagellate Alexandrium tamiyavanichii, in the southeast coasts of the Seto Inland Sea, Japan.

The mussel Mytilus edulis and the cultured ark shell Anadara broughtonii in the southeast coasts of the Seto Inland Sea were contaminated with paralytic shellfish poison (PSP) following the appearance of the dinoflagellate Alexandrium tamiyavanichii in early December 1999. A. tamiyavanichii plankton collected around the Straits of Naruto on December 3, 1999 showed PSP toxicity, of which 83 mol% was accounted for by GTX2, GTX3 and GTX4. Its specific toxicity was 112.5 fmol/cell, and one MU was equivalent to 7,200 cells. Toxicity values at the beginning of toxification were 4.7 MU/g for the ark shell and 7.3 MU/g for the mussel. In the former, the value remained at almost 4 MU/g, resulting in prohibition of marketing for about two months. In the latter, it sharply decreased to less than 4 MU/g. These bivalves collected during the toxification period were dissected into five tissues, mantle, adductor muscle, hepatopancreas, gills and "others", and submitted to high-performance liquid chromatography (HPLC). The cultured ark shell accumulated GTX2, GTX3 and STX as major components and GTX1, GTX4, GTX5, neoSTX, dcSTX and PX1-3 (C1-C3) as minor ones. The amount of GTX3 decreased with time, while STX tended to increase. At the early stage of PSP toxification, toxins were accumulated in the gills and "others", most of which were quickly detoxified. On the other hand, PSP of the toxified mussel consisted of GTX4 as a main component, and GTX1, GTX2, GTX3, GTX5, STX and PX1-2 (C1-C2) as minor ones. Its toxin composition pattern was similar to that of the ingested causative plankton. Its total toxin decreased soon after disappearance of the dinoflagellate. During the decrease of toxicity, PSP tended to be retained in the hepatopancreas, resulting in accumulation of 50 mol% of total toxin.

Utilization efficiency of nitrogen associated with riverine dissolved organic carbon (&GT1 kDa) by two toxin-producing phytoplankton species

Riverine high molecular weight dissolved organic material (HMWDOM) >1 kDa contains varying amounts of nitrogen. This nitrogen is partly available for growth of phytoplankton and can therefore contribute to algal bloom formation. However, there is a lack of knowledge on species-specific utilization efficiency of this HMWDOM-bound nitrogen. A specific question is whether this nitrogen can be used by toxic or otherwise harmful species and, thus, contribute to unwanted eutrophication effects in coastal waters. In this context, 2 phytoplankton species and known toxic bloom-formers in marine coastal waters were cultured in nitrogen-limited semi-continuous cultures with river extracted HMWDOM and nitrate as nitrogen source in the following proportions (%): 0:100, 25:75, 50:50, 75:25 and 100:0 (HMWDOM-bound nitrogen). Prymnesium parvum f. patelliferum (Green, Hibberd et Pienaar) A. Larsen, an ichthyotoxic prymnesiophyte, was not able to utilize the nitrogen bound to HMWDOM. The toxicity of this species, measured as hemolytic activity, did not show any significant correlation with HMWDOM concentration. In contrast, Alexandrium tamarense, a toxin-producing dinoflagellate responsible for paralytic shellfish poisoning (PSP), was able to use nitrogen from riverine HMWDOM as efficiently as nitrate, when the HMWDOM fraction was up to 75 % of the total dissolved nitrogen. When HMWDOM was supplied as the sole nitrogen source, the growth efficiency was reduced by approximately half. At steady state, the total cellular toxin content and toxicity of A. tamarense was negatively correlated with the fraction of HMWDOM. The use of organic nitrogen sources by marine dinoflagellates is well known. Here, we show that nitrogen from a terrestrial origin can be very efficiently used by toxic A. tamarense and could therefore contribute to unwanted eutrophication effects. (Less)

Microcystins in Natural Blooms and Laboratory Cultured Microcystis aeruginosa from Laguna de Bay, Philippines

Laguna de Bay, the largest freshwater lake in the Philippines, experiences periodic blooms of the cyanobacteria Microcystis aeruginosa. Blooms of these cyanobacteria in 1996, 1998 and 1999 were sampled. HPLC and MALDI-TOF mass spectrometry were used to analyze for microcystins. A total of 16 structural variants of the toxin were isolated from the samples with microcystin LR (MC-LR) as the most abundant variant in the samples from 1996 and 1999 making up 77 to 85% of the total, respectively. MC-RR was the dominant variant in the 1998 bloom making up 38%. The samples from 1996 had the highest total toxin concentration (4049 microg g(-1)) followed by those from 1998 (1577 microg g(-1)) and 1999 (649 microg g(-1)). A strain of M. aeruginosa previously isolated from the lake was also cultured in the laboratory under different nitrogen concentrations (1, 3 and 6 mg L(-1)) and elevated phosphorus concentration (0.5 mg L(-1)) to determine the influence of these factors on toxin production. A total of 9 different structural variants of microcystin were isolated from the laboratory cultures with MC-LR consisting more than 75% of the total in all treatments. No significant differences in the total toxin concentration as well as the % distribution of the different variants among treatments were observed. However, the strain of M. aeruginosa cultured in the laboratory had from 3 to 20 times higher total microcystin than those harvested from the lake.

Fate of paralytic shellfish poisoning toxins ingested by the copepod Acartia clausi

The fate of paralytic shellfish poisoning (PSP) toxins ingested by the copepod Acartia clausi was studied in unialgal and mixed cultures of the toxic dinoflagellate Alexandrium minutum and the non-toxic dinoflagellate Prorocentrum micans. Acartia clausi fed actively on Alexandrium minutum, but feeding pressure diminished over time. This reduced feeding upon toxic phytoplank- ton seems to be due to behavioural rejection, since feeding pressure on the non-toxic dinoflagellate did not diminish over time. The assimilation efficiency of toxins ingested by copepods was 3.8%. Some of these toxins assimilated by copepods were redirected to the eggs, but the daily total toxin output in the eggs was only 0.98% of the daily toxins assimilated by the copepods. This small amount of toxins in the eggs had no effect on the fate of the toxins in the copepods, but did affect copepod reproductive success, since reduced egg hatching was observed with increasing toxin accumulation in the copepod tissues. The amount of toxins daily excreted in the pellets was only 2.26% of the daily amount of toxins assimilated by the copepods. However, the detoxification rate of PSP toxins by the copepods was 0.586 d -1 . Therefore, toxins were either transformed and excreted as other compounds in faecal pellets and/or were eliminated through excretion in dissolved form. A model showed that the copepods accumulated PSP toxins through dietary incorporation, but excreted them after several days. Copepods accumulate toxins up to a threshold without any negative effect on fecundity, but above this threshold, they require a higher amount of food to achieve the same egg production rate.

The purine degradation pathway: Possible role in paralytic shellfish toxin metabolism in the cyanobacterium Planktothrix sp. FP1

The paralytic shellfish toxins (PSTs) are potent neurotoxic alkaloids and their major biological effect is due to the blockage of voltage-gated sodium channels in excitable cells. They have been recognised as an important health risk for humans, animals, and ecosystems worldwide. The metabolic pathways that lead to the production and the degradation of these toxic metabolites are still unknown. In this study, we investigated the possible link between PST accumulation and the activation of the metabolism that leads to purine degradation in the filamentous freshwater cyanobacterium Planktothrix sp. FP1. The purine catabolic pathway is related to the nitrogen microcycle in water environments, in which cyanobacteria use traces of purines and ureides as a nitrogen source for growth. Thus, the activity of allantoicase, a key inducible enzyme of this metabolism, was used as tool for assaying the activation of the purine degradation pathway. The enzyme and the pathway were induced by allantoic acid, the direct substrate of allantoicase, as well as by adenine and, to a lower degree, by urea, one of the main products of purine catabolism. Crude cell extract of Escherichia coli was also employed and showed the best induction of allantoicase activity. In culture, Planktothrix sp. FP1 showed a differential accumulation of PST in consequence of the induction with different substrates. The cyanobacterial culture induced with allantoic acid accumulated 61.7% more toxins in comparison with the control. On the other hand, the cultures induced with adenine, urea, and the E. coli extract showed low PST accumulation, respectively, 1%, 38%, and 5% of the total toxins content detected in the noninduced culture. A degradation pathway for the PSTs can be hypothesised: as suggested for purine alkaloids in higher plants, saxitoxin (STX) and derivatives may also be converted into xanthine, urea, and further to CO 2 and NH 4 + or recycled in the primary metabolism through the purine degradation pathway.

Reduction of Patulin during Apple Juice Clarification

Patulin is a mycotoxin produced by a number of molds involved in fruit spoilage. This compound is carcinogenic and teratogenic. Various methods are currently used to reduce the levels of patulin in apple juice, namely, charcoal treatment, chemical preservation (sulfur dioxide), gamma irradiation, fermentation, and trimming of fungus-infected apples. Many of these processes are expensive and time-consuming. Therefore, there is a need to find a convenient and economical process to control patulin levels. This study was undertaken to evaluate the effectiveness of several clarification processes for the reduction of patulin. Clarification was carried out on a laboratory scale. Apple pulp was spiked with patulin, pressed, and clarified using four different processes, namely, fining with bentonite, enzyme (pectinase)treatment, paper filtration, and centrifugation. Patulin was recovered from the clarified juice by liquid-liquid extraction, and solid-phase chromatography was used for sample cleanup prior to analysis by high-performance liquid chromatography (HPLC). The minimum detectable limit using HPLC was 20 μg/liter. Pressing followed by centrifugation resulted in an average toxin reduction of 89%. Total toxin reduction using filtration, enzyme treatment, and fining were 70, 73, and 77%, respectively. Patulin reduction was due to the binding of the toxin to solid substrates that was verified by analyzing the clarified juice as well as the filter cake, pellet, and sediment. The combined concentrations correlated to the spiked concentration. These results reveal that clarification was successful in the reduction of patulin levels in apple juice. However, clarification resulted in high levels of patulin in the pressed pulp after filtration and centrifugation, and this could be harmful if they are used as animal feeds.

The effect of mussel size, temperature, seston volume, food quality and volume-specific toxin concentration on the uptake rate of PSP toxins by mussels (Mytilus galloprovincialis Lmk).

The accumulation of paralytic shellfish poisoning (PSP) toxins by bivalves is a serious threat to public health all over the world. However, very little is known about the uptake kinetics of these toxins and the environmental factors that may modify this process. We have studied the effect of mussel size, temperature, seston volume, food quality, and volume-specific toxin concentration (VOSTOC), on the uptake rate of paralytic shellfish poisoning (PSP) toxins by mussels (Mytilus galloprovincialis), by means of a second order factorial experiment. Over a 3-day period, the mussels were fed artificial diets containing Alexandrium minutum AL1V (a PSP toxin producer), Tetraselmis suecica, Ensiculifera sp1 and silt, to the levels required by each treatment. Mussel size, seston volume and VOSTOC were found to be statistically significant when the total toxin accumulated per weight of wet tissue was considered. Mussel size affected the uptake negatively and latter two positively. The interactions, mussel size-VOSTOC and mussel size-food quality were also significant. The response was not linear as shown by the significance of the quadratic term of mussel size. Notwithstanding, when the PSP toxins accumulation per mussel was analysed, only one factor, the VOSTOC and the interactions, food quality-mussel size and food quality-seston volume, were found to be significant. VOSTOC was the most important factor in the accumulation of toxins, in our opinion, probably due to toxin assimilation being mainly regulated by the probability of contact between the toxins and the cellular walls of the digestive system. The size of the bivalve is also especially important because toxin concentration is usually calculated per weight of bivalve tissue and because the weight-specific ingestion increases with mussel size. The food quality, which was directly related to the assimilation of organic matter, had an inverse effect on toxin assimilation. In our opinion, this is probably due to the effect of inorganic particles in enhancing the disruption of Alexandrium cells. Temperature had no effect on the uptake rate except for the accumulation of the gonyautoxin GTX1.

Uptake and fate of diarrhetic shellfish poisoning toxins from the dinoflagellate Prorocentrum lima in the bay scallop Argopecten irradians

Bivalve molluscs can acquire diarrhetic shellfish poisoning (DSP) toxins via ingestion of toxigenic dinoflagellates. The dynamics and fate of DSP toxins were investigated in the bay scallop Argopecten irradians exposed to cells of the epibenthic dinoflagellate Prorocentrum lima, a known producer of DSP toxins, in controlled laboratory microcosms. Toxin parameters determined were uptake and detoxification rates, and anatomical compartmentalization. Toxins in tissue and algal extracts were analyzed by liquid chromatography-mass spectrometry (LC-MS). No mortalities occurred and feeding inhibition was not observed for juvenile and adult bay scallops during the 2 wk exposure to P. lima cells. Clearance rates were similar for scallops exposed to equivalent biovolume cell concentrations of toxigenic P. lima and the non-toxic diatom Thalassiosira weissflogii; however, absorption efficiency of organic matter was significantly lower with a diet of P. lima than T. weissflogii. Although DSP toxin concentrations in viscera of bay scallop exceeded commonly accepted regulatory levels (0.2 μg g -1 whole tissue) within 24 h of exposure to P. lima, after 2 wk of exposure total DSP toxin retained in scallop tissues was <1 % of the total toxin ingested over the same period. Most of the total toxin body burden was in the viscera (76%); however, in adult scallops a significant portion was associated with gonadal tissue (12%). Toxin levels were relatively low in gill, mantle and adductor tissue (<12% of total toxin body burden). During the depuration period, rapid release of DSP toxins from scallops indicated that toxins were poorly bound to all tissues, with the exception of the viscera. Detoxification of viscera consisted of a rapid loss of weakly bound toxin components within the initial 3 d of depuration, followed by a much slower release of the remaining toxin at a rate of 8.4 % d -1 .

Etiopathogenesis of nonalcoholic steatohepatitis.

The definable causes of nonalcoholic steatohepatitis (NASH) include jejunoileal bypass surgery (JIB), other causes of rapid and profound weight loss in obese subjects, total parenteral nutrition, drugs, industrial toxins, copper toxicity, and disorders characterized by extreme insulin resistance. However, the etiopathogenesis in most cases of NASH appears multifactorial. Obesity, type 2 diabetes, and hypertriglyceridemia are often associated with hepatic steatosis, and although this does not invariably lead to NASH, the fatty liver is vulnerable to hepatocellular injury initiated by reactive oxygen species (ROS). It is critical to understand not only the triggers for hepatitis (injury and inflammation) in NASH but also how this is perpetuated as chronic liver disease. The present focus is on whether the biochemical processes that generate oxidative stress lead to hepatocyte injury and secondary recruitment of inflammation or whether inflammation is the primary mediator of liver cell injury. Insulin resistance is a reproducible pathogenic factor in NASH. It favors accumulation of free fatty acids in the liver and predisposes to oxidative stress by stimulating microsomal lipid peroxidases and by the direct effects of high insulin levels in decreasing mitochondrial beta-oxidation. CYP2E1 is normally suppressed by insulin but is invariably increased in the livers of patients with NASH. In rodent dietary models of steatohepatitis, CYP2E1 is the catalyst of microsomal lipid peroxidation, while in Cyp 2e1 nullizygous mice, CYP4A proteins are induced and function as alternative microsomal lipid peroxidases. Other studies implicate activation of peroxisome proliferator-activated receptor-alpha (PPAR alpha) as leading to NASH; PPAR alpha is a transcription factor that governs both microsomal (via CYP4A) and peroxisomal (beta-oxidation) pathways of lipid oxidation and ultimately production of ROS. Increased lipid peroxidation is a crucial difference between the livers of rodents with experimental NASH and those of ob/ob genetically obese mice that have uncomplicated steatosis. Administration of endotoxin, through the release of tumor necrosis factor-alpha (TNF-alpha), provokes liver inflammation with hepatocyte injury in the steatotic liver. This may be particularly relevant in JIB and has been suggested as a pathogenic mechanism in primary NASH. It has been proposed that inheriting one or more copies of the hemochromatosis gene, C282Y, promotes fibrotic progression in NASH because of increased hepatic iron deposition, but recent studies have failed to confirm this. The relationship between the severity of hepatitis in NASH and progression to cirrhosis implies that products of the inflammatory infiltrate play a role in fibrogenesis. In summary, NASH can be regarded as the hepatic consequence of the metabolic syndrome (or syndrome X). Attention should now shift from steatosis, a generally benign process that is less evident in the advanced stages of cirrhosis, to the mechanisms for hepatocellular injury, inflammation, and hepatic fibrosis. In particular, the genetic, molecular, and cellular factors that ordain and moderate fibrosis in the context of steatohepatitis will be of greatest relevance to effective therapy and clinical outcome.

INTRA‐CELLULAR AND EXTRA‐CELLULAR TOXINS IN THE RED TIDE DINOFLAGELLATE, GYMNODINIUM BREVE

The marine dinoflagellate, Gymnodinium breve (Davis), produces several neurotoxins that cause neurotoxic shellfish poisoning (nsp), massive fish kills and respiratory irritation in marine mammals and humans. The common method for discerning toxic levels of G. breve for public health advisories is enumeration of live cells in a given water mass. In this study, laboratory cultures, as well as natural blooms, were added to a stirred ultra‐filtration cell concentrator to separate viable cells containing intra‐cell toxins from ambient water containing extra‐cell toxins. Methods were validated using various mixtures of lysed and whole G. breve laboratory culture. Extractions and recovery of brevetoxins were done using a C‐18 bonded‐phase glass fiber extraction disc eluted with methanol. Total PbTx toxin concentrations were quantified by HPLC/UV using a C‐18 column and an 85:15 methanol:water (1 ml min−1) isocratic elution at 215 nm. This method of separation and extraction was subsequently applied to water samples collected during natural blooms along two different areas of the Florida Gulf coast. The results indicated that early stages of G. breve blooms contained primarily intra‐cell toxins with extra‐cell toxins increasing as the bloom progressed, even though very few viable G. breve cells were present. This suggests that enumeration of cells alone may be insufficient and additional toxin quantitation is necessary.

A comparison of clinical, histopathological and cell-cycle markers in rats receiving the fungal toxins fumonisin B 1 or fumonisin B 2 by intraperitoneal injection

Fumonisins B 1 and B 2 (FB 1 and FB 2) are fungal secondary metabolites produced by members of the genus Fusarium. Although FB 1 is usually detected in greater quantities, FB 2 frequently co-occurs in contaminated feeds and foods and contributes to the total toxin load. In the present study, the comparative toxicity of FB 1 and FB 2 was examined in male Sprague–Dawley rats administered toxin (0.75 mg/kg body weight) or vehicle control intraperitoneally (ip) for 2, 4 or 6 consecutive days. Clinical changes, including elevated serum cholesterol, alanine aminotransferase (ALT), creatinine and protein, were slightly more pronounced in FB 1-treated rats. The most consistent hematological change was an increase in vacuolated bone marrow cells, which was more pronounced in FB 1-treated rats. Histopathological changes were similar in FB 1- and FB 2-treated rats and included single cell necrosis in kidneys and liver, cytoplasmic vacuolation in adrenal cortex and lymphocytolysis in thymus. In the liver mRNA expression for the cyclin kinase inhibitor p21 gene was significantly increased in FB 1- and FB 2-treated rats, compared to controls. Expression of mRNA for the cyclin D1 gene was significantly depressed in FB 2-treated rats. Hepatic cyclin E mRNA was elevated in response to FB 1 and FB 2 compared to controls. In FB 2-treated animals this corresponded with decreased liver p27 mRNA expression. Hepatic proliferating cell nuclear antigen (PCNA) transcription was elevated in FB 1- but not FB 2- treated rats. Changes in liver microsomal protein levels of p27, cyclin E and PCNA were similar to changes in gene expression. In contrast, cyclin D1 protein levels were elevated in rats treated with FB 1 and, to a lesser extent, FB 2. The data indicate that FB 1 and FB 2 can alter the expression of genes associated with the cell cycle, and indicate a need for a further understanding of the mechanistic basis of FB 1 and FB 2 toxicity.

Toxicity and ribosomal DNA of the dinoflagellate Dinophysis sacculus (Dinophyta)

High-density blooms of Dinophysis sacculus Stein, a dinoflagellate that produces diarrhetic shellfish toxins, are reported from a brackish lagoon in Sicily, Italy. The diarrhetic shellfish poisoning (DSP) toxicity of plankton assemblages dominated by this species (≥ 97%) was assessed by high-performance liquid chromatography. Spring outbreaks of D. sacculus were associated with the production of okadaic acid and, to a lesser extent, dinophysistoxin-1 (7–24% of the total DSP toxins), as confirmed under controlled conditions, using no nutrient enrichment. Addition of culture medium to the Dinophysis assemblages favoured the simultaneous growth of other taxa, not allowing comparative analyses of toxicity under enriched treatments. The nucleotide sequences of internal transcribed spacers 1 and 2 and 5.8S rDNA were determined from populations of D. sacculus maintained under controlled conditions. Comparison with sequences obtained in our laboratory from other dinoflagellate genera such as Alexandrium and with those reported in the genomic databases suggests that the ITS–5.8S region could be used as a molecular marker for detection of D. sacculus. This could be particularly useful when toxic populations occur at very low densities or switch to a quiescent phase, since they are then easily overlooked by conventional microscopical analyses.

Saxitoxins (PSP toxins) in Danish lakes

The occurrence of neurotoxic cyanobacteria and saxitoxins in Danish lakes was investigated from 104 phytoplankton samples representing 96 localities. Mouse bioassays demonstrated neurotoxicity in 11 localities (13 samples) of which eight (10 samples) contained saxitoxins and the remaining three anatoxin-a(s). The common cyanobacterial neurotoxin anatoxin-a was not detected. The toxin profiles of the samples containing saxitoxins were characterised using standards of saxitoxin (STX), decarbamoyl STX (dcSTX), neoSTX and gonyautoxins 1–5 (GTX1–5). Presence of C-toxins was not investigated. The lakes fell into two groups; in seven lakes STX was the dominant toxin and in four lakes GTX4 was dominant. In the first group, STX comprised 46–100% of the total toxin content; in the latter, GTX4 made up 40–75%. The total toxin content varied between 5.9 and 224.1 μg STX equivalents g DW −1 with median at 34.1 μg STX equivalents g DW −1. Two lakes contained more than 100 μg STX equivalents g DW −1 (182.5 and 224.1 μg, respectively) with STX constituting more than 95% of the total toxin content. Nine of the neurotoxic samples also contained microcystins. Most of the samples were totally dominated by cyanobacteria but unambiguous identification of toxin-producing species was not possible. Anabaena lemmermannii P. Richter was very common in the neurotoxic lakes, and it was distinctly dominant in the two lakes with the highest total contents of saxitoxins. The high frequency of neurotoxic lakes with saxitoxins suggests that these toxins are more common in fresh waters than previously assumed.

Short-term and long-term effects of the toxic dinoflagellate Alexandrium minutum on the copepod Acartia clausi

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 203:161-169 (2000) - doi:10.3354/meps203161 Short-term and long-term effects of the toxic dinoflagellate Alexandrium minutum on the copepod Acartia clausi M. Frangópulos1,*, C. Guisande1, I. Maneiro1, I. Riveiro1, J. Franco2 1Facultad de Ciencias del Mar, Universidad de Vigo, Apartado 874, 36200 Vigo, Spain 2Instituto de Investigaciones Marinas (CSIC), Eduardo Cabello 6, 36208 Vigo, Spain *E-mail: mfr@uvigo.es ABSTRACT: Several experiments were performed to determine the effects of cell toxin concentration, composition and toxicity of Alexandrium minutum on ingestion rate, egg production, hatching success and naupliar fitness of the copepod Acartia clausi. A combination of A. minutum and non-toxic algae (Prorocentrum micans, Tetraselmis suecica and Isochrysis galbana) was used as food. Copepods ingested a higher amount of A. minutum cells as the concentration of these toxic dinoflagellates increased, and also in response to decreasing total food concentration available for the copepods. A positive relationship was obtained between A. minutum cells ingested by copepods and total toxin concentration per copepod. Hatching success and naupliar production were lower when copepods ingested a higher amount of toxic dinoflagellates. This negative effect could has been due to the accumulated toxins in the egg and copepod tissues, and was higher when A. minutum had a higher cell concentration of GTX1. Finally, the results obtained from nauplii incubated with T. suecica and I. galbana showed that nauplii hatched from females fed non-toxic food (T. suecica and I. galbana) reached copepodite stage earlier than those nauplii hatched from females fed with a combined of toxic (A. minutum) and non-toxic (T. suecica and I. galbana) food. KEY WORDS: Dinoflagellate · Copepod · Toxins · PSP · Reproduction Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 203. Online publication date: September 18, 2000 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2000 Inter-Research.

Influence of feeding increasing levels of leucaena leaf meal on the performance of milch goats and metabolism of mimosine and 3-hydroxy-4 (1H) pyridone

Four crossbred (Alpine × Beetal) milch goats (24 months; 30 kg live weight) in early lactation received during 31 days increasing levels of Leucaena leucocephala leaf meal (LLM): 25, 50, 75 and 100% of dietary DM during first, second, third and fourth week onwards, respectively. Remaining dietary DM was supplied through green berseem and a concentrate mixture which constituted the whole diet after the 31 days feeding on LLM. Mimosine and 3-hydroxy-4 (1H) pyridone (3,4 DHP) contents of the LLM were 1.88 and 0.78% respectively. Daily average mimosine, 3,4 DHP and total toxin intake was 196, 82 and 278; 339, 141 and 480; 553, 230 and 783; and 648, 269 and 971 g kg −1 live weight (W) during first, second, third and from fourth week onwards, respectively. DM intake on sole feeding of LLM was 65.9 g kg −1 W 0.75. No clinical toxicity symptoms were noted. Milk yield started declining from Day 27 onwards and reached the lowest level on Day 33 though LLM was discontinued from Day 31 onwards. Milk yield started improving from Day 37 and reached a peak by Day 49. The milk samples were free of mimosine and DHP, possibly due to some kind of barrier between blood and udder. Mimosine was not detectable in blood serum samples. The 3,4 DHP concentration in the blood serum, however, ranged from 97 on Day 8 to 402 μg ml −1 on Day 29 and disappeared completely by Day 36 when animals were on conventional diet. During metabolism trial faecal excretion of mimosine and 3,4 DHP was 4.43 and 17.9% of total toxin intake, respectively. Urinary excretion of mimosine was 1.59% while that of 3,4 DHP and 2,3 DHP through faeces and urine was 52.17% of total toxin consumed. DCP and TDN values of LLM in goats were 16.03 and 61.58%, respectively. Goats were in positive N balance. The T 3 and T 4 levels declined but DM intake was not affected. The SGOT and SGPT values were in the normal range. The study indicated that LLM could form a substantial portion of milch goat ration and milk of such goats being free of mimosine and DHP should be fit for human consumption.

Microcystin production of Microcystis viridis (cyanobacteria) under different culture conditions

SUMMARYToxic cyanobacterium Microcystis viridis was isolated from the eutrophic Dianchi Lake of south‐western China. Three microcystins were detected from this strain by high‐pressure liquid chromatography. Among them, [Dha7] microcystin‐RR is the major component and represents about 70–80% of the total toxins of this strain. We examined its microcystin production pattern in relation to culture conditions such as light intensity, nutrition, temperature, pH and growth phase. Of all the factors examined, light intensity and pH influenced the toxin production much more than other factors. At 25°C, a high production of [Dha7] microcystin‐RR was obtained at light intensity 15 μE s‐1 m2, and both low and high pH (pH 7.0 and pH 9.2) enhanced the yield of microcystin‐RR. No strong relationships were found between total toxin and temperature ranging from 15°C. The highest production of total toxins was measured in the mid‐exponential growth phase and lowest production towards the declining growth phase. Remarkable changes in toxin production and morphology emerged coincidentally following the transfer of nitrogen‐deficient cells into fresh MA medium during the time course. These findings may shed light on the correlation between toxin production and morphogenesis of this strain.

Domoic Acid Levels of Naturally Contaminated Scallops as Affected by Canning

ABSTRACTChanges in domoic acid levels of toxic scallops (Pecten maximus) were monitored during the standard canning processes of pickled scallops and scallops in brine after 60 and 180 days of frozen storage. The concentration of domoic acid in whole scallops decreased by 43% after 180 days of frozen storage, the decrease being mostly in the hepatopancreas. No significant destruction of domoic acid was observed during the canning process but the transfer of toxin to packing media exceeded 30% of total toxin content in the canned product.

P1E115 - Gabaergic-benzodiazepine system is involved in the crotoxin-induced behavioral alterations

We report the comparative proteomic and antivenomic characterization of the venoms of subspecies cascavella and collilineatus of the Brazilian tropical rattlesnake Crotalus durissus. The venom proteomes of C. d. collilineatus and C. d. cascavella comprise proteins in the range of 4–115 kDa belonging to 9 and 8 toxin families, respectively. Collilineatus and cascavella venoms contain 20–25 main toxins belonging to the following protein families: disintegrin, PLA2, serine proteinase, cysteine-rich secretory protein (CRISP), vascular endothelial growth factor-like (VEGF), l-amino acid oxidase, C-type lectin-like, and snake venom metalloproteinase (SVMP). As judged by reverse-phase HPLC and mass spectrometry, cascavella and collilineatus share about 90% of their venom proteome. However, the relative occurrence of the toxin families departs among the two C. durissus subspecies venoms. The most notable difference is the presence of the myotoxin crotamine in some C. d. collilineatus specimens (averaging 20.8% of the total proteins of pooled venom), which is absent in the venom of C. d. cascavella. On the other hand, the neurotoxic PLA2 crotoxin represents the most abundant protein in both C. durissus venoms, comprising 67.4% of the toxin proteome in C. d. collilineatus and 72.5% in C. d. cascavella. Myotoxic PLA2s are also present in the two venoms albeit in different relative concentrations (18.1% in C. d. cascavella vs. 4.6% in C. d. collilineatus). The venom composition accounts for the clinical manifestations caused by C. durissus envenomations: systemic neurotoxicity and myalgic symptoms and coagulation disturbances, frequently accompanied by myoglobinuria and acute renal failure. The overall compositions of C. d. subspecies cascavella and collilineatus venoms closely resemble that of C. d. terrificus, supporting the view that these taxa can be considered geographical variations of the same species. Pooled venom from adult C.d. cascavella and neonate C.d. terrificus lack crotamine, whereas this skeletal muscle cell membrane depolarizing inducing myotoxin accounts for ∼ 20% of the total toxins of venom pooled from C.d. collilineatus and C.d. terrificus from Southern Brazil. The possible relevance of the observed venom variability among the tropical rattlesnake subspecies was assessed by antivenomics using anti-crotalic antivenoms produced at Instituto Butantan and Instituto Vital Brazil. The results revealed that both antivenoms exhibit impaired immunoreactivity towards crotamine and display restricted (∼ 60%) recognition of PLA2 molecules (crotoxin and D49-myotoxins) from C. d. cascavella and C. d. terrificus venoms. This poor reactivity of the antivenoms may be due to a combination of factors: on the one hand, an inappropriate choice of the mixture of venoms for immunization and, on the other hand, the documented low immunogenicity of PLA2 molecules. C. durissus causes most of the lethal snakebite accidents in Brazil. The implication of the geographic variation of venom composition for the treatment of bites by different C. durissus subspecies populations is discussed.

Activités métaboliques et toxicité du dinoflagellé Alexandrium tamarense en culture à dialyse exposée à un milieu estuarien

Metabolic activities and toxicity of the dinoflagellate Alexandrium tamarense were studied for a dialysis culture exposed to unenriched natural seawater and a batch culture grown in an enriched medium. The following observations were made on the dialysis culture compared with the batch culture: (i) Depending on the season period, the cell densities per litre were similar (≈ 10 × 106 – 14 × 106) or greater (66 × 106). (ii) During growth and stationary phases, cellular consumption of the nutrients (NO3− + NO2−) was much greater (5–50 times), (iii) During growth, the composition of the intracellular (IFAA) and extracellular dissolved free amino acid (EFAA) was characterized by the presence of ORN and LYS in the dialysis culture, which represent a substantial fraction of the IFAA pool of the dinoflagellate; a significant depletion of the IFAA was observed, but the recovery of Gln, Glu, and Arg cell concentration levels at the stationary phase was more important. (iv) During the growth phase, the maximum excretion rate of amino acids (fmol∙cell−1∙d−1) was 263 (in decreasing order of importance: Glu, Gin, Ser, Asp, Leu, Ala, G+T, and Orn) compared with 14,5. (v) During stationary phase, the reduction in excretion activity was twofold with Gln, Glu, Leu, and Orn while 9,2 fmol∙cell−1∙d−1 were observed in batch culture, (vi) Total toxin content was about 4 to 5 times higher (means: 95 and 75 fmol∙cell−1 during growth and stationary phases respectively), with greater concentrations in more potent derivates like saxitoxin and neosaxitoxin. This higher cell toxicity may be attributed to the particular growth conditions of the dialysis culture, which more closely approximate the natural environment, through diffusion processes involving a constant elimination of low molecular weight autoinhibitory metabolites and a more efficient nutrient uptake.Key words: Alexandrium tamarense, dinoflagellate, dialysis culture, phycotoxins, nitrogen metabolism.

Persistence and degradation of cyanobacterial paralytic shellfish poisons (PSPs) in freshwaters

Paralytic shellfish poisons (PSPs) extracted from the cyanobacterium Anabaena circinalis persisted for over 90 days when incubated in freshwater. The primary transformation reaction was desulfocarbamoylation of the predominant, low toxicity C-toxins, to the more potent decarbamoylgonyautoxins (dc-GTXs). This transformation caused an initial increase in sample toxicity, in spite of an overall decrease in total toxin concentration (on a molar basis) with time. Incubation of PSPs in irrigation drainage water resulted in a six-fold increase in toxicity after 10 days, with the solution remaining more toxic than the original solution for over 60 days. The dc-GTXs thus formed, and the gonyautoxins (GTXs) present in the original sample, degraded slowly for the duration of the 90 day incubations. Similar results were recorded when PSPs were incubated with sterile deionised water, and with sterile deionised, protein precipitated water, but the build-up of dc-GTXs proceeded at a slower rate than with the drainage water. The other reaction noted to be significant was the slow epimerisation of the less stable β-epimers (C2, GTX3) to α-epimers (C1, GTX2). The degradation of C-toxins, gonyautoxins (GTXs) and dc-GTXs could be modelled by simple first-order loss kinetics. When PSPs were incubated at 90°C, the same loss processes and reaction order was observed. The failure of protein precipitation and high temperature to alter the reaction processes, indicates that desulfocarbamoylation of C-toxins, and degradation off GTXs and dc-GTXs is chemically, and not enzymatically, mediated. Upon the collapse of PSP producing A. circinalis blooms, and the subsequent release of toxins into the water column, contaminated water may become more toxic in the short term before the eventual degradation of PSPs to non-toxic products. Unless significant dilution occurs, PSP contaminated waters may contain toxins for more than 3 months.