domoic acid

Abstract

Domoic acid (DA) is a potent toxin, marine biotoxin, and primarily produced by Pseudo-nitzschia. The DA hapten was coupled with bovine serum albumin (BSA), and ovalbumin (OVA) as carrier proteins. DA-BSA conjugate was used as immunogen and DA-OVA as coating antigen. Cell fusion between spleen cells and sp2/0 myeloma cells developed 1C3 hybridoma clone producing 1C3 monoclonal antibody (mAb). Hybridoma was injected into the mice to produce ascites, and further purified by caprylic acid/ammonium sulfate method. The mAb was of IgG3 subclass, and was specific to DA with high affinity (2.5 × 108 L/mol). Moreover, western blot exhibited significant specificity to the DA antigens. Indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) showed DA working range of 0.006–0.2 ng/mL. The IC50 was 0.03 ng/mL with low limit of detection (LOD) of 0.006 ng/mL. Average DA recovery from spiked shellfish extract was 100.56 ± 2.8% with the coefficient variation of 0.01–0.1%. Hence, mAb producing 1C3 hybridoma was successfully developed and could be used to detect DA in contaminated samples.

2.4. Subclass and Chromosome Count of 1C3 Hybridoma Cell Line

The hybridoma cell line 1C3 against DA was analyzed for subclass using a commercial isotyping kit (IgM, IgG1, IgG2a, IgG2b, IgA, and IgG3). The results of the subclass analysis showed that the mAb producing 1C3 hybridoma cell had the IgG3 subclass (A). The chromosome count analysis by Geimsa staining exhibited that the chromosome number of 1C3 hybridoma cell line was 102 ± 4 (B) among the experiment replicates, and was produced by cell fusion between splenocytes with chromosome number 66 ± 4 and Sp2/0 myeloma cells with chromosome number 39 ± 1, respectively []. Therefore, the results indicated that the 1C3 hybridoma clone was produced by successfully fusing splenocyte and the Sp2/0 cell.

2.8. Preparation of the Standard Curve

The standard curve and recovery test of the 1C3 mAb was analyzed by competitive inhibition ELISA. The standard curve was prepared and the data was analyzed by Microcal OriginPro 9.1 to test the relationship of the concentration of DA with its inhibition. The standard curve was analyzed by the logistic curve and the equation was y = 0.447/(1 + (x/0.0349)0.795), and the correlation coefficient (R2) was about 0.98 (A). The linear equation was y = 0.2507 − 0.0452x with the correlation coefficient (R2) 0.99 (B). In the present study, the half inhibitory concentration (IC50) was 0.03 ng/mL, and the linear range of detection was 0.006–0.2 ng/mL with a lower LOD of 0.006 ng/mL.

2.9. Analysis of Recovery Test and Sample Detection by ic-ELISA Using 1C3 mAb

Recovery test was carried out to assess the quality of the established ic-ELISA, Non-contaminated shellfish extract was diluted properly to minimize the matrix effect and was spiked with DA at various concentrations (0.5, 5, 100, 1000 ng/mL) (). The results demonstrated that the recovery range was from (95.03 ± 5.4)% to (106.18 ± 1.7)% with an average recovery of (100.56 ± 2.8)%, and its coefficient of variation (CV) was ranged from 0.01 to 0.1% with an average CV of 0.03% (). Real shellfish samples (abalone, sea mussel, fresh oyster, giant clam, king scallop, razor clam, fresh water mussel and triangular clam) were randomly purchased from the seafood market for sample analysis. After extraction, the samples were assayed independently in duplicates to check the working of the established ic-ELISA for DA detection in complex matrix. The results showed that four samples from all shellfish samples were DA positive with levels under 30 ng/mL (120 ng/g), and the extracts of razor clam and giant clam showed the highest levels of DA at 6.70 ng/mL (26.80 ng/g) and 2.89 ng/mL (11.56 ng/g), respectively. Moreover, extracts of abalone (0.76 ng/mL) and fresh oyster (0.02 ng/mL) exhibited least detection, demonstrated DA levels lower than 1.0 ng/mL (4 ng/g). Similarly, all other shellfish samples were DA free with no contamination compared with the control in ic-ELISA at OD 450 nm using 1C3 mAb in the study ().

Abstract

The king scallop Pecten maximus retains the amnesic shellfish poisoning toxin, domoic acid (DA), for a long time. Most of the toxin is accumulated in the digestive gland, but this organ contains several cell types whose contribution to the accumulation of the toxin is unknown. Determining the time-course of the depuration by analyzing whole organs is difficult because the inter-individual variability is high. A sampling method, using biopsies of the digestive gland, has been developed. This method allows for repetitive sampling of the same scallop, but the representativeness of the samples obtained in this way needs to be validated. In this work, we found that the distribution of DA in the digestive gland of the scallops is mostly homogeneous. Only the area closest to the gonad, and especially its outer portion, had a lower concentration than the other ones, probably due to a transfer of the toxin to the intestinal loop. Samples obtained by biopsies can therefore be considered to be representative. Most of the toxin was accumulated in large cells (mostly digestive cells), which could be due to differences during the toxin absorption or to the preferential depuration of the toxin from the small cells (mostly secretory).

2.2. Distribution of Domoic Acid between Cellular Types

Domoic acid was preferentially accumulated into large (mostly digestive) cells (A, p = 0.0039 by Wilcoxon paired samples test). The measured concentration of this toxin in small cells (basophilic, mostly secretory) was less than 25% of that in large cells.

3. Discussion

The variability of the toxin concentration among areas of the digestive gland of the scallops is moderate-low. It is lower than the usual inter-individual variation in scallops [,] and other bivalves [,,]. Most sectors of the digestive gland had very similar concentrations, with the only significant exception being the sector adjacent to the gonad. In addition, in this sector the outer region was shown to have a lower concentration than the inner one. The most likely cause for this difference is the migration of the toxin to the intestinal loop located in the gonad. In previous studies [], it has been observed that gonadal growth during depuration produces an increase in DA burden in this organ. The origin of the toxin incorporated by the intestinal loop should be the digestive gland, which is where the DA in the scallops is mainly stored. The mechanism involved, nevertheless, is unknown. A possibility could be the reabsorption of the toxin excreted by the digestive gland by the intestinal cells. It has been shown that those cells can significantly absorb different substances [], but the very low depuration rate of the scallops [,] suggests that the amount of DA that could be reabsorbed is very low. In any case, this mechanism would not be expected to affect some parts of the gonad differently. A second possibility could be the transfer of the toxin from the digestive gland cells to the newly generated intestinal ones. Transfers of DA between organs have been documented but are significant only in exceptional situations, such as post-spawning periods [,,]. This mechanism could or could not have an effect on the distribution of DA in the different areas of the digestive gland. The third possibility is that the intestinal loop cells which contain the toxin derive from cells from the digestive gland. If the new cells of the intestinal loop are generated in the nearby area of the digestive gland, a reduction in the toxin concentration in that area would be expected. Intestinal cells are mostly epithelial and the presence of immature cells, as in the digestive gland, has not been observed []. It is therefore possible that the intestinal cells derive from immature cells in the digestive gland and consequently inherit the toxin they contain. This would explain the lower DA concentration in the sector closest to the gonad and also the greater reduction in the outer region relative to the inner one, because the cells in this region are the closest to the intestinal loop. The confirmation of this hypothesis requires additional studies.

3. Discussion

Domoic acid was preferentially retained in large cells, mostly absorptive (digestive cells) []. The concentration measured in small cells, mostly secretory and immature cells, was substantially lower than in the digestive ones. In terms of toxin burden, the importance of large cells was even greater, because they are found in a larger amount in the digestive gland (more biomass). The difference in concentration opens two possibilities regarding the methods of depuration not taken into account to date: (a) the secretory cells depurate the DA they have accumulated during the intoxication phase (which could be the case if both cell types accumulate the toxin to the same extent); and (b) DA is transferred from absorptive to secretory cells, which excrete it. Unfortunately, no information about the distribution of DA during the intoxication phase exists and the transfer between cellular types has not been documented, so new studies would be required to shed some light on the subject. However, in our opinion, neither of the two ways is very likely. Firstly, it seems unlikely that absorptive and secretory cells would absorb and accumulate DA during the intoxication at the same rate, because of their main functions. Secondly, it is difficult to explain how the toxin could pass the cellular membrane of the absorptive cells to go to the secretory ones but not to be excreted (the depuration rate is very low [,]) or transferred to other tissues [,].

Introduction

The community composition and toxigenicity of the diatom Pseudo-nitzschia in the open South Atlantic Ocean were characterized during the austral spring of 2007 by Guannel et al. (). Multiple morphological types of Pseudo-nitzschia were detected in coastal and in open-ocean waters. The toxin produced by Pseudo-nitzschia, DA, was present in at least 10 species in the South Atlantic (Gayoso, ; Reguera, ). Even though Pseudo-nitzschia sp. blooms occur in different environmental conditions, Marchetti et al. () and Wells et al., ) showed a positive correlation between these blooming events and high levels of Fe(III) and NO3-, and low levels of PO43- and SiO44-. Even more, differences in temperature, salinity, pH, high irradiance, and long-term photoperiod can affect the formation of Pseudo-nitzschia blooms (Lelong et al., ). Woods () suggested a possible photo-oxidative stress regulation on DA's higher production under high irradiance. Cellular stress produced by these conditions may favor the production of toxins during HAB events. The main objective of this opinion article is to briefly report the toxicological implications of the harmful marine phycotoxin DA and its intrinsic properties. Special focus will be made on the reported oxidative stress status in marine algae in relation to the exposure to DA. Moreover, because Fe presence is known to be implicated in oxidative stress generation, and its occurrence seems to be a fundamental factor in the production of DA, the oxidative effects of the biotoxin will be discussed in relation to its capacity to bind Fe.

Discussion

On the other hand, DA production starts in late EXP and ST phase of the Pseudo-nitzschia growth cycle (Lelong et al., ). In the late ST phase, cells are physiologically impaired by injury in the structure of the wall and in the membrane (Pan et al., ). Therefore, DA tends to be released into the culture medium, along with ROS generated by the cells (as shown by Cabrera et al., ,). Because of the short life span of ROS, this increased production could not be very effective in damaging other organisms in the aquatic community. However, the release of DA could allopathically affect (protect or injure) other species of non–toxin-producing photosynthetic microalgae. Using a highly sensitive adsorptive cathodic stripping voltammetric technique, Rue and Bruland () showed that DA forms Fe chelates with a conditional stability constant of KcondFeDA,Fe(III) = 108.7±0.5 M−1. The degree to which the DA acts as a trace metal chelator reveals that it has the potential to modify the availability of these metals in the seawater. Thus, the physiological role of DA for toxigenic Pseudo-nitzschia species may be tied closely to the acquisition of Fe in coastal waters. Among several other postulations, the formation of a Fe–DA complex could be a very attractive hypothesis to explain the increased generation of ROS both in producing and non–phycotoxin-generating species. Chemical aspects of the Fe chelators, such as Fe affinity, Fe selectivity, molecular weight, and lipophilicity, in addition to stability and redox properties of the resultant Fe complex, drastically change the ability of the Fe complex to catalyze radical generation. The presence of a catalytically active Fe–DA complex could favor lipid peroxidation and radical generation in a significative amount, by affecting the composition of the labile Fe content within the cells. This is a novel point of view that should be further explored. The capacity of the antioxidant defense in each species will be the main factor to determine the hormetic (beneficial) or the damaging effect of the triggering of the burst of oxidative species.

Discussion

However, other alternatives could be considered under changing environmental conditions that can modify DA-dependent active species generation in aquatic organisms. Hypoxia is a common condition in aquatic environments, and exposure to hypoxia followed by reoxygenation is often believed to induce oxidative stress and activation of relevant signaling molecules such as the hypoxia-inducible factor 1α. This transcription factor is a potent coordinator of acclimation processes in various stress conditions. Eutrophication and extensive algal blooms caused by anthropogenic activity (input of nutrients, fertilizers, and human waste) deplete the ocean and lake bottoms of O2, i.e., make them hypoxic. In the seas, the large hypoxic areas are also known as hypoxic dead zones, and they are expanding quickly (Conley et al., ). Hypoxic bottoms release phosphorus from the aquatic sediments, which feed the algae even further. Hypoxia has an acute effect on the benthic fauna, and if the hypoxic environments are allowed to expand further, the whole ecosystem will be disturbed (Conley et al., ; Conley, ). Recently, Borowiec and Scott () reported that hypoxia acclimation of killifish leads to adjustments in ROS homeostasis and oxidative status that do not reflect oxidative stress, but may instead be part of the suite of responses used to cope with chronic hypoxia. Thus, in situ studies could show the alterations due to other transitory changes in the environment that may occur during the HABs. Overall, the specific cellular response to the increased oxidative stress triggered by DA will be one of the important factors to allow survival of each organism that contribute to determine how the composition of the community will be affected by an increasing magnitude of the HAB season produced by the climate global change.