Uptake Of microcystin-LR Research Articles

358 In Vitro Uptake of Harmful Algal Bloom Toxin Microcystin-LR in Human Placental Cells

OBJECTIVES/GOALS: Harmful algal blooms (HABs) are increasing in both frequency and intensity due to climate change. HABs release the toxin microcystin-LR (MC-LR) which enters cells via organic anion-transporting polypeptides (OATPs). In this study, we sought to assess the ability of MC-LR to accumulate in trophoblasts, potentially disrupting placental functions. METHODS/STUDY POPULATION: Intracellular accumulation of MC-LR at exposure concentrations of 0.1, 1, and 10 µM over 6 hrs was evaluated in immortalized JAR placental cytotrophoblasts. Western blotting was used to evaluate protein-bound MC-LR accumulation in JAR cells. The function of OATP transporters in JAR cells was determined by pre-incubating cells with 10µM cyclosporin A, a general OATP inhibitor for 1 hr, and then incubated with 1µM OATP substrate fluorescein for up to 40 min. Fluorescence of fluorescein was measured at Ex/Em: 494nm/515nm by spectrophotometry. RESULTS/ANTICIPATED RESULTS: A concentration-dependent increase of MC-LR bound proteins in JAR cells was observed at 6 hrs with the greatest intracellular accumulation of MC-LR at 10µM. In the transporter experiments, a significant decrease of fluorescein uptake by up to 45% into JAR cells was observed following cyclosporin A inhibition of OATPs. These findings are consistent with the functional expression of OATP transporters in JAR placenta cells. Ongoing studies are evaluating whether the cyclosporin A-mediated inhibition of OATPs also inhibits the uptake of MC-LR. DISCUSSION/SIGNIFICANCE: Although MC-LR is well-known for its hepatotoxic and neurotoxic effects, there is growing interest in examining its potential adverse impacts on female reproductive health, particularly during pregnancy. Active uptake of MC-LR into the placenta could interfere with placental and fetal development.

The presence of polystyrene nanoplastics enhances the MCLR uptake in zebrafish leading to the exacerbation of oxidative liver damage

The accumulation of diminutive plastic waste in the environment, including microplastics and nanoplastics, has threatened the health of multiple species. Nanoplastics can adsorb the pollutants from the immediate environment, and may be used as carriers for pollutants to enter organisms and bring serious ecological risk. To evaluate the toxic effects of microcystin-LR (MCLR) on the liver of adult zebrafish (Danio rerio) in the presence of 70 nm polystyrene nanoplastics (PSNPs), zebrafish were exposed to MCLR alone (0, 0.9, 4.5 and 22.5 μg/L) and a mixture of MCLR + PSNPs (100 μg/L) for three months. The results indicated that groups with combined exposure to MCLR and PSNPs further enhanced the accumulation of MCLR in the liver when compared to groups only exposed to MCLR. Cellular swelling, fat vacuolation, and cytoarchitectonic damage were observed in zebrafish livers after exposure to MCLR, and the presence of PSNPs exacerbated these adverse effects. The results of biochemical tests showed the combined effect of MCLR + PSNPs enhanced MCLR-induced hepatotoxicity, which could be attributed to the altered levels of reactive oxygen species, malondialdehyde and glutathione, and activities of catalase. The expression of genes related to antioxidant responses (p38a, p38b, ERK2, ERK3, Nrf2, HO-1, cat1, sod1, gax, JINK1, and gstr1) was further performed to study the mechanisms of MCLR combined with PSNPs aggravated oxidative stress of zebrafish. The results showed that PSNPs could improve the bioavailability of MCLR in the zebrafish liver by acting as a carrier and accelerate MCLR-induced oxidative stress by regulating the levels of corresponding enzymes and genes.

Bioavailability of microcystin-LR in two different soil types to the legume Alfalfa Medicago sativa L.

The adverse effects of exposure to microcystins in terrestrial crops have been well documented. However, the retention and bioavailability of microcystin-LR, one of the most prevalent cyanotoxins, from soil to plants, is poorly understood. In the present study, the amount of free microcystin-LR from two soil types, a silty sand and clayey loam, with exposure to three toxin concentrations and time was investigated. Using the two soil types, the effects on Medicago sativa (Alfalfa) growth after microcystin-LR exposure via irrigation with spiked water and pre-spiked soil was investigated and the amount of microcystin-LR taken up by the plant quantified. After 3 weeks of growth, the amount of free microcystin-LR remaining in the two soil types with each treatment was quantified. The results indicated that in clayey loam more microcystin-LR is bound to the soil. However, the growth of Alfalfa was only affected in the clayey loam with microcystin/LR exposure via irrigation. Nevertheless, microcystin-LR was detected in Alfalfa grown in both soil types exposed by both irrigation and via pre-spiked soil. Interestingly, more microcystin-LR remained in the silty sand after 3 weeks; yet, more microcystin-LR was taken up by the Alfalfa grown in the silty sand, with a larger concentration in the roots compared to the shoots. The results indicate that the soil type substantially influences the bioavailability and uptake of microcystin-LR and present some insight into the ecological risk posed by microcystin-LR.

Development and evaluation of a sensitive, Diffusive Gradients in Thin-Films (DGT) method for determining microcystin-LR concentrations in freshwater and seawater

A Diffusive Gradients in Thin-Films (DGT) passive sampling technique was developed for microcystin-LR (MC-LR), one of the most common and toxic microcystins. Three types of resins (HP20, SP700, and XAD18) were evaluated for MC-LR uptake kinetics, capacities, and extraction efficiencies and simple procedures were developed for determining MC-LR concentration in binding disc extracts by Adda-ELISA (U.S. EPA Method 546). The XAD18-DGT/Adda-ELISA method had a 7-d deployment time detection limit of ≈0.05 μg/L and capacity of >250 μg/L of MC-LR in water samples which encompass U.S. EPA and WHO advisory concentrations for drinking and recreational waters. The XAD18-DGT/Adda-ELISA method determined time-averaged MC-LR concentrations in waters with wide ranging pH (4.9–8.3) and ionic strength (0.04–0.8 M) under well-stirred and quiescent conditions with 90–101% accuracy. In addition to high sensitivity and accuracy, the method is simple, inexpensive, and applicable for determining MC-LR and related MCs concentrations in waterbodies with wide ranging chemical characteristics and hydrodynamic conditions.

Removal of Microcystin-LR from spiked natural and synthetic waters by anion exchange

Cyanobacterial blooms are becoming a serious challenge across the globe due to changing climate and rainfall patterns as a consequence of human activities. In the present study, the fundamental interactions involved during the removal of Microcystin-LR (MCLR), one of the most commonly occurring cyanobacterial toxins, were investigated by employing strongly basic anion exchange (IX) resins. Several factors including the stoichiometric coefficients, competitive fractions and solute affinities were determined under various concentrations of inorganic ions and natural organic matter. The results indicated that suphates were the most competitive fractions with high affinity (α (affinity coefficient) values ~ 9) followed by nitrates (α ~ 4.7) and NOM fractions (α ~ 4.5, p < 0.05). The Equivalent Background Concentration Mode (EBC), that arises from the Ideal Adsorption Solution Theory (IAST), indicated a competitive fraction of ~2 μeq/L NOM, which approximates to <10% of the initial NOM concentrations, indicating a small fraction of the NOM resulting in the competitive effect. Further, studies with natural surface waters indicated that the MCLR uptake could be modeled using the IAST-EBC model and the IX resin could simultaneously removal of >90% of NOM, inorganic ions and MCLR at resin dosages of 3.6 meq/L or higher.

Characteristics of competitive uptake between Microcystin-LR and natural organic matter (NOM) fractions using strongly basic anion exchange resins

Microcystins are the most commonly occurring cyanotoxins, and have been extensively studied across the globe. In the present study, a strongly basic anion exchange resin was employed to investigate the removal of Microcystin-LR (MCLR), one of the most toxic microcystin variants. Factors influencing the uptake behavior included the MCLR and resin concentrations, resin dosage, and natural organic matter (NOM) characteristics, specifically, the charge density and molecular weight distribution of source water NOM. Equivalent background concentration (EBC) was employed to evaluate the competitive uptake between NOM and MCLR. The experimental data were compared with different mathematical and physical models and pore diffusion was determined as the rate-limiting step. The resin dose/solute concentration ratio played a key role in the MCLR uptake process and MCLR removal was attributed primarily to electrostatic attractions. Charge density and molecular weight distribution of the background NOM fractions played a major role in MCLR removal at lower resin dosages (200 mg/L ∼ 1 mL/L and below), where a competitive uptake was observed due to the limited exchange sites. Further, evidences of pore blockage and site reduction were also observed in the presence of humics and larger molecular weight organic fractions, where a four-fold reduction in the MCLR uptake was observed. Comparable results were obtained for laboratory studies on synthetic laboratory water and surface water under similar conditions. Given their excellent performance and low cost, anion exchange resins are expected to present promising potentials for applications involving the removal of removal of algal toxins and NOM from surface waters.

Tumor-promoting cyanotoxin microcystin-LR does not induce procarcinogenic events in adult human liver stem cells

HL1-hT1 cell line represents adult human liver stem cells (LSCs) immortalized with human telomerase reverse transcriptase. In this study, HL1-hT1 cells were found to express mesenchymal markers (vimentin, CD73, CD90/THY-1 and CD105) and an early hepatic endoderm marker FOXA2, while not expressing hepatic progenitor (HNF4A, LGR5, α-fetoprotein) or differentiated hepatocyte markers (albumin, transthyretin, connexin 32). In response to microcystin-LR (MC-LR), a time- and concentration-dependent formation of MC-positive protein bands in HL1-hT1 cells was observed. Cellular accumulation of MC-LR occurred most likely via mechanisms independent on organic anion transporting polypeptides (OATPs) or multidrug resistance (MDR) proteins, as indicated (a) by a gene expression analysis of 11 human OATP genes and 4 major MDR genes (MDR1/P-glycoprotein, MRP1, MRP2 and BCRP); (b) by non-significant effects of OATP or MDR1 inhibitors on MC-LR uptake. Accumulation of MC-positive protein bands in HL1-hT1 cells was associated neither with alterations of cell viability and growth, dysregulations of ERK1/2 and p38 kinases, reactive oxygen species formation, induction of double-stranded DNA breaks nor modulations of stress-inducible genes (ATF3, HSP5). It suggests that LSCs might have a selective, MDR1-independent, survival advantage and higher tolerance towards MC-induced cytotoxic, genotoxic or cancer-related events than differentiated adult hepatocytes, fetal hepatocyte or malignant liver cell lines. HL1-hT1 cells provide a valuable in vitro tool for studying effects of toxicants and pharmaceuticals on LSCs, whose important role in the development of chronic toxicities and liver diseases is being increasingly recognized.

Simultaneous uptake of NOM and Microcystin-LR by anion exchange resins: Effect of inorganic ions and resin regeneration

This study investigated the efficiency of a strongly basic macroporous anion exchange resin for the co-removal of Microcystin-LR (MCLR) and natural organic matter (NOM) in waters affected by toxic algal blooms. Environmental factors influencing the uptake behavior included MCLR and resin concentrations, NOM and anionic species, specifically nitrate, sulphate and bicarbonate. A860 resin exhibited an excellent adsorption capacity of 3800 μg/g; more than 60% of the MCLR removal was achieved within 10 min with a resin dosage of 200 mg/L (∼1 mL/L). Further, kinetic studies revealed that the overall removal of MCLR is influenced by both external diffusion and intra-particle diffusion. Increasing NOM concentration resulted in a significant reduction of MCLR uptake, especially at lower resin dosages, where a competitive uptake between the charged NOM fractions and MCLR was observed due to limited active sites. In addition, MCLR uptake was significantly reduced in the presence of sulphate and nitrate in the water matrix. Moreover, performance of the resin proved to be stable from one regeneration cycle to another. Approximately 80% of MCLR and 50% of dissolved organic carbon (DOC) were recovered in the regenerated brine. Evidences of resin saturation and site reduction were also observed after 2000 bed volumes (BV) of operation. For all the investigated water matrices, a resin dosage of 1000 mg/L (∼4.5 mL/L) was sufficient to lower MCLR concentration from 100 μg/L to below the World Health Organization guideline of 1 μg/L, while simultaneously providing more than 80% NOM removal.

The organic anion transporting polypeptide 1a5 is a pivotal transporter for the uptake of microcystin-LR by gonadotropin-releasing hormone neurons

Microcystins (MCs) are widely distributed hepatotoxic polypeptides produced by cyanobacteria. Microcystin-LR (MC-LR) has the broadest distribution and strongest toxicity among more than 80 isoforms of hepatotoxic MCs. MC-LR suppresses the expression of gonadotropin-releasing hormone (GnRH) that is critically required for the release of testosterone, resulting in the induction of male reproductive toxicity. However, the specific mechanisms of the uptake of MC-LR by GnRH-secreting neurons still remain unclear. In this study, GT1-7 cells were exposed to MC-LR in order to determine whether the GnRH-secreting neurons were the target of MC-LR that could induce male reproductive toxicity. Our data demonstrated that at least four organic anion transporting polypeptides (Oatp1a4, Oatp1a5, Oatp5a1, Oatp2b1) were expressed in GnRH neurons at the mRNA level, but only Oatp1a5 was expressed at the protein level. Furthermore, we demonstrated that MC-LR could not be transported into Oatp1a5-deficient GT1-7 cells which were protected from cell viability loss induced by MC-LR. These data suggest that Oatp1a5 may play an important role in the toxic effect of MC-LR on GnRH neurons.

Intraperitoneal exposure of whitefish to microcystin-LR induces rapid liver injury followed by regeneration and resilience to subsequent exposures

To date, there has been no systematic approach comprehensively describing the sequence of pathological changes in fish during prolonged exposure to microcystin-LR (MC-LR). Towards this aim, juvenile whitefish individuals received an intraperitoneal injection with pure MC-LR, and the injection was repeated every week to maintain continuous exposure for 28days. During the exposure period, growth and condition of the fish were assessed based on biometric measurements. Additionally, selected biochemical markers were analysed in the fishes' blood, and their livers were carefully examined for morphological, ultrastructural, and molecular changes. The higher dose of MC-LR (100μg·kg−1) caused severe liver injury at the beginning of the exposure period, whereas the lower dose (10μg·kg−1) caused less, probably reversible injury, and its effects began to be observed later in the exposure period. These marked changes were accompanied by substantial MC-LR uptake by the liver. However, starting on the 7th day of exposure, cell debris began to be removed by phagocytes, then by 14th day, proliferation of liver cells had markedly increased, which led to reconstruction of the liver parenchyma at the end of the treatment. Surprisingly, despite weekly-repeated intraperitoneal injections, MC-LR did not accumulate over time of exposure which suggests its limited uptake in the later phase of exposure. In support, mRNA expression of the membrane transport protein oatp1d was decreased at the same time as the regenerative processes were observed. Our study shows that closing of active membrane transport may serve as one defence mechanism against further MC-LR intoxication.

Microcystin-LR induces a wide variety of biochemical changes in the A549 human non-small cell lung cancer cell line: Roles for protein phosphatase 2A and its substrates.

Our previous studies have described the toxic effects of microcystin-LR (MC-LR) in various normal cell lines and human hepatoma SMMC-7721 cells, but the specific effects of MC-LR in other types of cancer cells with respect to protein phosphatase 2A (PP2A) have not been fully elaborated. A549 human lung adenocarcinoma cells have been identified to express organic anion-transporting polypeptides (OATP) involved in cellular uptake of MC-LR, and thus probably make an appropriate in vitro model to assess MC-LR's cytotoxicity. Hence, in our present study, A549 cells were treated with various concentrations of MC-LR for 24 h. The presence of MC-LR in A549 cells was confirmed, and PP2A activity, PP2A substrates, cytoskeleton, apoptosis, and proliferation were subsequently explored. The results showed that 5-10 μM MC-LR inhibited PP2A activity significantly but 0.5-1 μM MC-LR did not change PP2A activity dramatically. The inhibition could result from the hyperphosphorylation of PP2A/C at Tyr307, an elevation in the total PP2A/C expression and the dissociation of α4/PP2A/C complexes. Moreover, MC-LR led to rearrangements of filamentous actin and microtubules, which might be correlated with the hyperphosphorylation of Ezrin, VASP and HSP27 due to PP2A inhibition and mitogen-activated protein kinase (MAPK) activation. However, exposure to MC-LR for 24 h failed to trigger either apoptosis or proliferation, which might be related to PP2A-inhibition-induced hyperphosphorylation of Bcl-2 and Bad and the activation status of Akt. In conclusion, our data indicated that MC-LR induced extensive molecular and cellular alterations in A549 cells through a PP2A-centered pathway, which differed in some respects from our previous study in SMMC-7721 cells. To our knowledge, this is the first report comprehensively demonstrating the effects of MC-LR in A549 cells, and our findings provide insights into the mechanism of MC-LR toxicity in cancer cells. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1065-1078, 2017.

Comparative effects of nodularin and microcystin-LR in zebrafish: 1. Uptake by organic anion transporting polypeptide Oatp1d1 (Slco1d1)

Microcystin-LR (MC-LR) and nodularin are hepatotoxins produced by several cyanobacterial species. Their toxicity is based on active cellular uptake and subsequent inhibition of protein phosphatases PP1/2A, leading to hyperphosphorylation and cell death. To date, uptake of MC-LR and nodularin in fish is poorly understood. Here, we investigated the role of the organic anion transporting polypeptide Oatp1d1 in zebrafish (drOatp1d1, Slco1d1) in cellular uptake in zebrafish. We stably transfected CHO and HEK293 cell lines expressing drOatp1d1. In both transfectants, uptake of MC-LR and nodularin was demonstrated by competitive inhibition of uptake with fluorescent substrate lucifer yellow. Direct uptake of MC-LR was demonstrated by immunostaining, and indirectly by the high cytotoxicity in stable transfectants. By means of a synthesized fluorescent labeled MC-LR derivative, direct uptake was further confirmed in HEK293 cells expressing drOatp1d1. Additionally, uptake and toxicity was investigated in the permanent zebrafish liver cell line ZFL. These cells had only a low relative abundance of drOatp1d1, drOatp2b1 and drOatp1f transcripts, which correlated with the lack of MC-LR induced cytotoxicity and transcriptional changes of genes indicative of endoplasmic reticulum stress, a known effect of this toxin. Our study demonstrates that drOatp1d1 functions as an uptake transporter for both MC-LR and nodularin in zebrafish.

Naringin attenuates the cytotoxicity of hepatotoxin microcystin-LR by the curious mechanisms to OATP1B1- and OATP1B3-expressing cells

Microcystin-LR, which is an inhibitor of serine/threonine protein phosphatase (PP)1 and PP2A, induces liver injury by its selective uptake system into the hepatocyte. It is also thought that microcystin-LR induces reactive oxygen species (ROS). We tried to establish the chemical prevention of microcystin-LR poisoning. We investigated the effect of grapefruit flavanone glycoside naringin on cytotoxicity of microcystin-LR using human hepatocyte uptake transporter OATP1B3-expressing HEK293–OATP1B3 cells. We found cytotoxicity of microcystin-LR was attenuated by naringin in a dose dependent manner. The inhibition magnitude of total cellular serine/threonine protein phosphatase activity induced by microcystin-LR was suppressed by naringin. In addition, uptake of microcystin-LR into HEK293–OATP1B3 cells was inhibited by naringin. Furthermore, microcystin-LR induced phosphorylation of p53 was inhibited by naringin. Regardless of the difference in the exposure pattern of pre-processing and post-processing of naringin, the toxicity of microcystin-LR was comparable. These results suggested that naringin is promising remedy as well as preventive medicine for liver damage with microcystin-LR. In addition, involvement of ROS production after exposure to the sublethal concentrations of microcystin-LR in the onset of cytotoxicity was negligible. Therefore, inhibition of microcystin-LR uptake and the pathway other than ROS production would be involved in the effect of naringin on the attenuation of microcystin-LR toxicity.

Microcystin-LR induces anoikis resistance to the hepatocyte uptake transporter OATP1B3-expressing cell lines.

Microcystin-LR is a cyclic peptide released by several bloom-forming cyanobacteria. Understanding the mechanism of microcystin-LR toxicity is important, because of the both potencies of its acute cytotoxicity and tumor-promoting activity in hepatocytes of animals and humans. Recently, we have reported that the expression of human hepatocyte uptake transporter OATP1B3 was critical for the selective uptake of microcystin-LR into hepatocytes and for induction of its fatal cytotoxicity. In this study, we demonstrated a novel function of microcystin-LR which induced bipotential changes including anoikis resistance and cytoskeleton reorganization to OATP1B3-transfected HEK293 cells (HEK293-OATP1B3). After exposure to microcystin-LR, HEK293-OATP1B3 cells were divided to the floating cells and remaining adherent cells. After collection and reseeding the floating cells into a fresh flask, cells were confluently proliferated (HEK293-OATP1B3-FL) under the microcystin-LR-free condition. Both the proliferated HEK293-OATP1B3-FL and remaining adherent HEK293-OATP1B3-AD cells changed the character with down- and up-regulation of E-cadherin, respectively. Additionally, these cells acquired resistance to microcystin-LR. These results suggest that microcystin-LR could be associated with not only tumor promotion, but also epithelial-mesenchymal transition-mediated cancer metastasis. Furthermore, microcystin-LR might induce the cytoskeleton reorganization be accompanied epithelial-mesenchymal transition.

Inhibitory effect of naringin on microcystin-LR uptake in the freshwater snail Sinotaia histrica

Gastropods are an important food source for aquatic animals, and have been demonstrated to transfer microcystin (MC) to higher trophic levels through the food web. In this study, we performed an oral administration experiment to evaluate whether naringin can inhibit MC-LR uptake in the freshwater snail Sinotaia histrica. We also observed the effect of MC-LR on the organizational pathology of the hepatopancreas in S. histrica. Following exposure to cells of Microcystis ichthyoblabe, S. histrica showed vacuolization and separation of the basal lamina from cells in the hepatopancreas. Initial treatment with 1mM naringin resulted in the prevention of MC-LR uptake rate by approximately 60% over 8days, whereas initial treatment with 10mM naringin suppressed microcystin uptake in 2days, despite an increase in MC-LR levels in the snail from days 5 to 8. With continuous treatment of 10mM naringin, the uptake prevention rate was 100%. Overall, we observed a strong inhibitory effect against MC-LR with naringin treatment. This study provides a potential mechanism to prevent the uptake of microcystin in the aquatic food web, thereby limiting its toxicity in cyanobacterial bloom-polluted areas where the environment can be controlled and may have further applications in the aquaculture of gastropods.

Microcystin-LR induced oxidative stress and ultrastructural alterations in mesophyll cells of submerged macrophyte Vallisneria natans (Lour.) Hara

Microcystins produced by cyanobacteria in the aquatic environment are a potential risk to aquatic plants. In the present study, the uptake of microcystin-LR (MC-LR) and related physiological and biochemical effects on Vallisneria natans (Lour.) Hara were investigated at concentrations of 0.1–25.0 μg L −1. Results showed that O 2 − intensity was significantly induced at 1.0 μg L −1 and reached a maximum level at 5.0 μg L −1. Superoxide dismutase (SOD) and peroxidase (POD) were induced with increasing MC-LR concentrations as an antioxidant response. Catalase (CAT) was significantly induced while GSH/GSSG (reduced/oxidized glutathione) ratio was significantly reduced at 0.1 μg L −1. The induction of glutathione S-transferase (GST) and inhibition of GSH revealed that GSH was involved in the detoxification of MC-LR in plants. Oxidative damage was evidenced by the significant increase of malondialdehyde content at 1.0 μg L −1. A pigment pattern change and a series of significant ultrastructural alterations were also observed due to MC-LR exposure. The lowest non-effect concentration of MC-LR for V. natans at the subcellular and molecular level is around 0.5 μg L −1. These results imply that even at relatively low MC-LR concentrations the aquatic plants may still suffer a negative ecological impact.

Microcystin-LR induced cellular effects in mammalian and fish primary hepatocyte cultures and cell lines: A comparative study

The impact of microcystin-LR, one of the most common cyanobacterial toxins, on liver and gut cells originating from mammals and fish was compared. Upon exposure of human and rainbow trout ( Oncorhynchus mykiss) cell lines up to 2.5 μM microcystin-LR, no alteration in cell viability was observed as assessed with three fluorescent indicators dyes, CFDA-AM, Alamar Blue and neutral red. The lack of sensitivity of the trout cell lines coincided with an absence of detectable mRNA levels of organic anion transporter polypeptide ( OATP), which is implicated in the uptake of microcystin-LR. In contrast to the cell lines, primary rainbow trout and mouse hepatocytes showed damage to subcellular structures, particularly the lysosomes, as indicated by neutral red. This led us to propose a thus far undetected role of lysosomes as targets and mediators of microcystin-LR elicited cellular damage. An inhibitor of OATP, rifampicin, partly protected hepatocytes from this damage. Yet, the sensitivity of rainbow trout hepatocytes rapidly declined in culture, accompanied by decreasing levels of OATP mRNA. The sensitivity of mouse hepatocytes toward microcystin-LR also declined in culture but overall was about 25-fold greater than that of the trout cells. These differences mirror those observed in vivo and suggest the use hepatocytes for deciphering the species differences.

Variation in the response of juvenile and adult gastropods (Lymnaea stagnalis) to cyanobacterial toxin (microcystin-LR)

Owing to the increasing public health problem related to the proliferation of toxin-producing cyanobacteria in aquatic ecosystems, we have investigated the response of the pond snail Lymnaea stagnalis exposed to 33 microg/L microcystin-LR for 6 weeks, through its life traits (survival, growth, fecundity) and locomotion; uptake of microcystin-LR was also quantified in the snail body tissues. To study the potential plasticity of the response related to the development stage, snails were exposed to the toxin as sexually immature and mature. According to our results, microcystin-LR accumulated in snail tissues at a higher level in juveniles (7.96 ng/g fresh weight) versus adults (2.17 ng/g fresh weight). Whatever the age, survival, growth, and locomotion were not affected by the toxin, and fecundity of polluted adults was reduced by half. These results are discussed in terms of negative effects of aqueous microcystin-LR occurrence on the dynamics of natural populations of gastropods.

Metabolism of the cyanobacterial toxin mycrocystin-LR is comparable in different aquatic organisms

The high nutrient load in many aquatic ecosystems leads to increased abundances of blue-green algae (cyanobacteria). Cyanobacteria are known to produce a wide range of toxins with adverse effects on many terrestrial and aquatic organisms. Furthermore, cyanobacterial blooms have been increasingly recognized as animal and human health hazards. In order to withstand chemical stressors, many organisms have developed a detoxication metabolism. It consists of three phases: (1) activation (P450-monooxygenases); (2) conjugation (glutathion S-transferases, and other transferases); and (3) further processing, deposition, or excretion. This study investigated the detoxication metabolism of microcystin-LR which is the most common cyanobacterial toxin. Uptake of microcystin-LR, effects of this toxin on glutathion S-transferase activities, and first detoxication steps were studied in different aquatic organisms. The organisms were choosen from different trophic levels of the aquatic ecosystem but showed a similar startpoint in the detoxication of microcystin-LR.

Uptake and effects of microcystin‐LR on detoxication enzymes of early life stages of the zebra fish (Danio rerio)

The effects of cyanotoxins on fish have been studied mainly in adults, rather than in early life stages which could be more sensitive or, in view of their immobility, more readily affected. The uptake of microcystin-LR by different early life stages of the zebra fish (Danio rerio) was investigated using 14C-labelled microcystin-LR. The effects on the activity of the detoxication enzymes, microsomal and soluble glutathione S-transferases (GST), and glutathione peroxidase (GP-X) were examined. There was a detectable uptake of microcystin from the first day of embryonic development up to 5 day old larvae. On average, an absorption of 0.5 ng microcystin for eggs and eleuthero-embryos was calculated over the entire exposure time. Because of the differences in volume of the eggs and eleuthero-embryos, there was an increase in the microcystin-LR concentration between these stages. In the eggs, approximately 25% of the medium concentration was found, and in eleuthero-embryos an equilibrium between fish and medium was reached. The activity of the detoxication enzymes differed during ontogenesis, but the effects of activation and suppression of these enzymes were similar at all stages. Minor activation of the soluble GST was found and a marked activation of GP-X was evident. The reaction of the microsomal GST was not so obvious. These results showed that there was an uptake of microcystin-LR by early life stages of the zebra fish and that the detoxication system reacted to this toxin, possibly indicating the ability of the organism to metabolize microcystin-LR to a less harmful compound. Chronic toxic effects, such as reduction in growth, in such early life stages when organogenesis is not finished and hence the microcystin-LR affects not one single target organ but the whole organism, might be due to the increased energy demand of these detoxication processes. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 89–95, 1999