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Title: Biokinetic, toxicology and differential protein expression in molluscs exposed to paralytic shellfish toxins
Other Titles: Ma bei xing bei lei du su dui yi bei lei de sheng wu dai xie, du li xue yi ji ge lei dan bai biao da de ying xiang
痳痺性貝類毒素對貽貝類的生物代謝, 毒理學以及各類蛋白表達的影響
Authors: Choi, Man Chi (蔡敏芝)
Department: Dept. of Biology and Chemistry
Degree: Doctor of Philosophy
Issue Date: 2006
Publisher: City University of Hong Kong
Subjects: Mollusks -- Physiology
Paralytic shellfish poisoning
Notes: CityU Call Number: RA1242.S52 C47 2006
Includes bibliographical references (leaves 176-205)
Thesis (Ph.D.)--City University of Hong Kong, 2006
xxi, 205 leaves : ill., map ; 30 cm.
Type: Thesis
Abstract: Harmful algal blooms (HABs) have drawn great attention from the public due to their increasing frequency, wide geographical distribution, and unpredictable nature. In the past few decades, the increasing demand of protein food resources has triggered the rapid development of shellfish mariculture in China to create a new industry. However, the exponential growth in red tide incidences has caused adverse effects on the mariculture industries. Some HABs are capable of producing highly toxic phycotoxins that can be transferred along the food chain through their vector, filter-feeding bivalves, to higher trophic levels including humans. Hence, the consumption of toxin-laden shellfish has posed a significant public health risk. Among the phycotoxins, paralytic shellfish toxins (PSTs) are the most well-known phycotoxins that are associated with HABs. An understanding of toxin uptake and depuration kinetics, as well as toxin transformation and distribution among different tissue compartments, can be useful in developing predictive models of toxin kinetics that may help to predict toxin concentrations in bivalves in the natural environment and provide useful information for the assessment of the public health risks of PSTs. In addition, as most studies to date have concentrated on the acute effects of PSTs, it is conceivable that PSTs may also exert sublethal effects on shellfish species. A study of the effects of PSTs on organisms at lower levels of biological organization will not only allow us to know more about the toxic mechanisms of PSTs, but might be useful in identifying biomarkers that can serve as early warning systems for environmental monitoring. In this study, the biokinetics, toxicology, and differential protein expression in several commercially important molluscs that are exposed to paralytic shellfish toxins were studied. The study was divided into four parts: (1) field depuration and biotransformation of paralytic shellfish toxins in the scallop Chlamys nobilis and the green-lipped mussel Perna viridis, (2) the trophic transfer of paralytic shellfish toxins from clams (Ruditapes philippinarum) to gastropods (Nassarius festivus), (3) the relationships between tissue concentrations of paralytic shellfish toxins and the antioxidative responses of clams (Ruditapes philippinarum), and (4) differential protein expression in clams (Ruditapes philippinarum) that are exposed to algae Alexandrium tamarense (clone ATCI01) that produce paralytic shellfish toxins (PSTs). For part (1) of this study, the scallop Chlamys nobilis and the mussel Perna viridis were exposed to N-sulfocarbamoyl toxins (C2 toxin), a paralytic shellfish toxin (PST) that is produced by a local toxic strain of dinoflagellate Alexandrium tamarense (ATDP). The bivalves were subsequently depurated in the field and their depuration kinetics, biotransformation, and toxin distribution were quantified. Depuration was characterized by a rapid loss within the first day, followed by a secondary slower loss of toxins. In the fast depuration phase, scallops detoxified PSTs more quickly than the mussels (depuration rate constants for scallops and mussels were 1.16 d-1 and 0.87 d-1, respectively). In contrast, mussels detoxified PSTs more quickly than the scallops in the slow depuration phase, and the calculated depuration rate constants from Day 2 to Day 13 were 0.063 + 0.009 d-1 (mean + S.E.) and 0.040 + 0.019 d-1 (mean + S.E.) for mussels and scallops, respectively. The differences in the appearances of gonyautoxins GTX2 and GTX3 and their decarbamoyl derivatives, dcGTX2, dcGTX3, and GTX5, that are all derivatives of the C2 toxin, indicated active and species-specific biotransformation of the algal toxins in the two bivalves. In both species of bivalves, the non-viscera tissue contained fewer toxins and lower concentrations than the viscera-containing tissue compartment. In scallops, very little toxin was distributed in the adductor muscle. In mussels, most of the PSTs were found in the digestive gland with a significant transport of toxins into the digestive gland from other tissues during the course of depuration. The toxin profiles of scallops and mussels differed from each other and from that of the toxic algae that were used to feed the mussels. A significant fraction of GTX5 was detected in the mussels but not in the scallops. Our study demonstrates species specificity in the depuration kinetics, biotransformation, and tissue distribution of PSTs among different bivalves. For part two of this study, a local strain of the dinoflagellate Alexandrium tamarense (ATCI01), which predominantly produces the C2 toxin, was fed to the clams (Ruditapes philippinarum) under laboratory conditions. Concentrations of paralytic shellfish toxins (PSTs) in the dosed clams were determined by high performance liquid chromatographic (HPLC) analyses, and the clams were homogenized and then fed to the gastropods (Nassarius festivus). In the toxin accumulation phase, which lasted for 42 days, concentrations of PSTs increased in the snails gradually, reaching a maximum of 1.10 nmole g-1 at the end of the exposure period. The toxin content of the homogenized clams (food) was 13.18 nmole g-1, which was about 12-fold higher than the PST content in the snails. Between Days 43 and 82, the snails were fed with non-toxic clams, and this period represented the depuration phase. Accumulation and depuration rates of PSTs in the snails, N. festivus, were determined by fitting the experimental data to a one-compartment model. Two different modeling approaches were used to derive the accumulation and depuration rates. The first approach is to derive both values from the data for the toxin uptake. The second approach is to derive the depuration rate from the depuration data and derive uptake rate, allowing for toxin depuration, from the data for toxin uptake. The first approach yielded more consistent results for the toxin concentration at the end of the uptake period, when compared with the experimental data. The toxin uptake and depuration rates were 1.64 (pmole of toxin into snail per day) per (nmole g-1 of toxin in food) and 0.06 ± 0.02 day-1 (mean ± SE), respectively. The toxin profiles of snails were similar to the clams, but different from the algae. In addition to C toxins (C1 and C2), dcGTX2 and dcGTX3 were also detected in both clams and snails. The β:α epimer ratio gradually decreased during trophic transfer and approached a ratio of 1:3 (26.4 mol%:73.6 mol% at Day 42) in the snails, near the end of the accumulation period. For part (3) of this study, the effects of dinoflagellate cells that contained paralytic shellfish toxins (PSTs) on antioxidant parameters were investigated in the gill and hepatopancreas of clams, Ruditapes philippinarum, under laboratory conditions. The clams were exposed to PST-producing dinoflagellates (Alexandrium tamarense, Clone ATCI01) for 15 days, and different antioxidant parameters – including glutathione-S-transferase (GST); oxyradical scavenger, glutathione (GSH); antioxidant enzymes [superoxide dismutase, (SOD) and glutathione peroxidase (GPx)]; and a measure of the degree of oxidative damage, lipid peroxidation (LPO) – were quantified. The tissue concentrations of PSTs were also determined for individual clams. The findings of the present investigation showed that the C2 toxin was transformed into other PST derivatives, such as C1 and dcGTX2/3 toxins in the clams. Gill GST showed a significant negative correlation with total PST concentrations. Hepatic GPx and gill LPO showed a significant positive correlation with total PST concentrations. Gill GPx, GSH and hepatic GST, GSH, LPO, and SOD showed no responses to an increase in the tissue burden of PSTs. The induction of GPx in hepatopancreas would probably prevent the propagation of lipid peroxidation in this organ, but such an induction was not observed in gill tissues. For the final part of this study, differential protein expression in clams (Ruditapes philipinnarum) that were exposed to PST- producing algae, Alexandrium tamarense (clone ATCI01) was studied using two-dimensional gel electrophoresis. In this study, clams (Ruditapes philipinnarum) were exposed to an environmentally realistic concentration of PST-producing dinoflagellate, Alexandrium tamarense (ATCI01) for 15 days. Proteins from the whole clam tissue were separated using two-dimensional gel electrophoresis (2-DE). Proteome between the treatment and control groups were compared using ImageMaster 2D platinum software. Changes in protein expression due to PST exposure were identified. Based on image analysis, 227 ± 25 (n=3) protein spots were detected in the non-toxic clams, while 211 ± 21 (n=3) were detected in clams that had been exposed to PSTs. Among these protein spots, 30 protein spots were unique to the non-toxic clams, while 27 protein spots were unique to clams that were exposed to PST producing algae. Such changes in protein expression patterns could help to identify possible toxic mechanisms of PSTs and the associated defense mechanisms in the organisms. Moreover, the specific protein spots that were found in response to PST exposure might be potential biomarkers to distinguish PST-contaminated and non-toxic clams. Overall, the above findings could provide useful information for the assessment of public health risks of PSTs, enhance our understanding on the toxic mechanisms of PSTs, and serve as a preliminary study for the identification of useful biomarkers for PSTs.
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