Protein Phosphatase 2A Inhibition Assay Research Articles

Specification of the Okadaic Acid Equivalent for Okadaic Acid, Dinophysistoxin-1, and Dinophysistoxin-2 Based on Protein Phosphatase 2A Inhibition and Cytotoxicity Assays Using Neuro 2A Cell Line

Diarrhetic shellfish poisoning (DSP) is a globally occurring disease threatening public health and trade. The causative toxins, okadaic acid (OA), dinophysistoxin-1 (DTX1), and dinophysistoxin-2 (DTX2) are collectively called OAs, and are quantified using the LC-MS/MS method. The hazardous effect of total OAs is expressed as the sum of OA equivalents defined for respective OAs based on mouse lethality, produced by either intraperitoneal (OAip) or oral administration (OAor). OAs are potent inhibitors of protein phosphatase 2A (PP2A) and are cytotoxic, necessitating expansion of the concept of OA equivalents to all relevant bioactivities. In this study, we determined OA equivalents for respective OA members in PP2A inhibition and cytotoxicity assays. To secure result credibility, we used certified OAs, reference materials, and PP2A produced using genetic engineering. The relative ratio of the OA equivalents determined by PP2A inhibition assays for OA, DTX1, and DTX2 were 1.0:1.6:0.3, while the ratio determined using the cytotoxicity assays indicated 1.0:1.5:0.5. OA equivalents showed a similar tendency in the PP2A inhibition and cytotoxicity assays, and matched better with oral toxicity data than intraperitoneal toxicity in mice. The PP2A inhibition assay, which measures the core activity of the OAs, suggested a higher OA equivalent for DTX1 than that currently used.

Isolation and Characterization of [DLeu1]microcystin-LY from Microcystis aeruginosa CPCC-464.

[D-Leu1]MC-LY (1) ([M + H]+ m/z 1044.5673, Δ 2.0 ppm), a new microcystin, was isolated from Microcystis aeruginosa strain CPCC-464. The compound was characterized by 1H and 13C NMR spectroscopy, liquid chromatography–high resolution tandem mass spectrometry (LC–HRMS/MS) and UV spectroscopy. A calibration reference material was produced after quantitation by 1H NMR spectroscopy and LC with chemiluminescence nitrogen detection. The potency of 1 in a protein phosphatase 2A inhibition assay was essentially the same as for MC-LR (2). Related microcystins, [D-Leu1]MC-LR (3) ([M + H]+ m/z 1037.6041, Δ 1.0 ppm), [D-Leu1]MC-M(O)R (6) ([M + H]+ m/z 1071.5565, Δ 2.0 ppm) and [D-Leu1]MC-MR (7) ([M + H]+ m/z 1055.5617, Δ 2.2 ppm), were also identified in culture extracts, along with traces of [D-Leu1]MC-M(O2)R (8) ([M + H]+ m/z 1087.5510, Δ 1.6 ppm), by a combination of chemical derivatization and LC–HRMS/MS experiments. The relative abundances of 1, 3, 6, 7 and 8 in a freshly extracted culture in the positive ionization mode LC–HRMS were ca. 84, 100, 3.0, 11 and 0.05, respectively. These and other results indicate that [D-Leu1]-containing MCs may be more common in cyanobacterial blooms than is generally appreciated but are easily overlooked with standard targeted LC–MS/MS screening methods.

First Report of Microcystis Strains Producing MC-FR and -WR Toxins in Japan.

Microcystins (MCs) are a group of cyclic heptapeptide hepatotoxins produced by Microcystis and several other genera of cyanobacteria. Many structural variants have been characterized using various methods such as liquid chromatography–mass spectrometry (LC-MS) analysis, enzyme-linked immunosorbent assay (ELISA) and protein phosphatase 2A (PP2A) inhibition assay. The representative MC, MC-LR, and related cyanobacterial toxins strongly inhibit PP2A activity and can therefore be assayed by measuring the extent of PP2A inhibition. However, these methods require reference toxin standards for the quantification and identification of known MCs. To obtain various MC-producing cyanobacterial strains, we surveyed and collected MC-producing cyanobacteria from environmental sources of water in Okinawa, Japan. Using a dual assay (LC-MS analysis and PP2A inhibition assay), we identified and isolated Microcystis strains producing five MC variants (MC-LR, -RR, -LA, -FR and -WR). Approximately 4 mg of MC-WR and -FR toxins were purified from the laboratory culture of the Microcystis isolate NIES-4344. Pure MC-WR and -FR variants were prepared for future use as toxin standards in LC-MS analysis. Phylogenetic analysis based on ftsZ revealed that the NIES-4344 strain belongs to the identified groups in Microcystis aeruginosa. This is the first report of Microcystis strains producing mainly MC-WR and -FR toxins in Japan.

Disposable poly (o-aminophenol)-carbon nanotubes modified screen print electrode-based enzyme sensor for electrochemical detection of marine toxin okadaic acid

Based on the inhibition of okadaic acid (OA) on protein phosphatase 2A (PP2A), we developed disposable poly-o-aminophenol-carbon nanotubes (PoAP-CNTs) modified screen print electrode (SPE)-based electrochemical enzyme sensor for OA detection. Electropolymerized PoAP-CNTs composite film was coated on electrode for the immobilization of PP2A. Owing to the excellent physicochemical characteristic of PoAP-CNTs film, the performance of enzyme sensor was enhanced. Under the optimal conditions, the biosensor provides a larger linear range, and lower detection limit for the analysis of OA compared with colorimetric PP2A inhibition assay and PP2A/SPE. The spiked OA shellfish samples were applied to verify the efficiency of method for OA detection. The results shows acceptable recovery rate and low RSD, demonstrating the applicability of this method for in suit OA assessment in the quality and toxicity monitoring of seafood.

Comparative summer dynamics of surface cyanobacterial communities in two connected lakes from the west of Ireland

The eutrophication of lakes is typically associated with high biomass proliferations of potentially toxic cyanobacteria. At a regional level, the sustainable management of water resources necessitates an approach that recognises the interconnectivity of multiple water systems within river catchments. This study examined the dynamics in summer diversity of planktonic cyanobacterial communities and microcystin toxin concentrations in two inter-connected lakes from the west of Ireland prone to nutrient enrichment. DGGE analysis of 16S rRNA gene amplicons of genotype-I cyanobacteria (typically spherical) showed changes in the communities of both Lough Corrib and Ballyquirke Lough throughout the summer, and identified cyanobacterial genotypes both unique and shared to both lakes. Microcystin concentrations, estimated via the protein phosphatase 2A inhibition assay, were greater in August than in July and June in both lakes. This was concomitant to the increased occurrence of Microcystis as evidenced by DGGE band excision and subsequent sequencing and BLAST analysis. RFLP analysis of PCR amplified mcy-A/E genes clustered together the August samples of both lakes, highlighting a potential change in microcystin producers across the two lakes. Finally, the multiple factor analysis of the combined environmental data set for the two lakes highlighted the expected pattern opposing greater water temperature and chlorophyll concentration against macronutrient concentrations, but also indicated a negative relationship between microcystin concentration and cyanobacterial diversity, possibly underlining allelopathic interactions. Despite some element of connectivity, the dissimilarity in the composition of the cyanobacterial assemblages and the timing of community change in the two lakes likely were a reflexion of niche differences determined by meteorologically-forced variation in physico-chemical parameters in the two water bodies.

Comparison of Microcystis aeruginosa (PCC7820 and PCC7806) growth and intracellular microcystins content determined by liquid chromatography–mass spectrometry, enzyme-linked immunosorbent assay anti-Adda and phosphatase bioassay

Cyanobacteria are able to produce several metabolites that have toxic effects on humans and animals. Among these cyanotoxins, the hepatotoxic microcystins (MC) occur frequently. The intracellular MC content produced by two strains of Microcystis aeruginosa, PCC7806 and PCC7820, and its production kinetics during the culture time were studied in order to elucidate the conditions that favour the growth and proliferation of these toxic strains. Intracellular MC concentrations measured by liquid chromatography (LC) coupled to electrospray ionization mass spectrometer (MS) were compared with those obtained by enzyme-linked immunosorbent assay (ELISA) anti-Adda and protein phosphatase 2A (PP2A) inhibition assays. It has been demonstrated there are discrepancies in the quantification of MC content when comparing ELISA and LC-MS results. However, a good correlation has been obtained between PP2A inhibition assay and LC-MS. Three MC were identified using LC-MS in the PCC7806 strain: MC-LR, demethylated MC-LR and a new variant detected for the first time in this strain, [L-MeSer(7)] MC-LR. In PCC7820, MC-LR, D-Asp(3)-MCLR, Dglu(OCH3)-MCLR, MC-LY, MC-LW and MC-LF were identificated. The major one was MC-LR in both strains, representing 81 and 79% of total MC, respectively. The total MC content in M. aeruginosa PCC7820 was almost three-fold higher than in PCC7806 extracts.

Screening Tests for the Rapid Detection of Diarrhetic Shellfish Toxins in Washington State

The illness of three people due to diarrhetic shellfish poisoning (DSP) following their ingestion of recreationally harvested mussels from Sequim Bay State Park in the summer of 2011, resulted in intensified monitoring for diarrhetic shellfish toxins (DSTs) in Washington State. Rapid testing at remote sites was proposed as a means to provide early warning of DST events in order to protect human health and allow growers to test “pre-harvest” shellfish samples, thereby preventing harvest of toxic product that would later be destroyed or recalled. Tissue homogenates from several shellfish species collected from two sites in Sequim Bay, WA in the summer 2012, as well as other sites throughout Puget Sound, were analyzed using three rapid screening methods: a lateral flow antibody-based test strip (Jellett Rapid Test), an enzyme-linked immunosorbent assay (ELISA) and a protein phosphatase 2A inhibition assay (PP2A). The results were compared to the standard regulatory method of liquid chromatography coupled with tandem mass spectroscopy (LC-MS/MS). The Jellett Rapid Test for DSP gave an unacceptable number of false negatives due to incomplete extraction of DSTs using the manufacturer’s recommended method while the ELISA antibody had low cross-reactivity with dinophysistoxin-1, the major toxin isomer in shellfish from the region. The PP2A test showed the greatest promise as a screening tool for Washington State shellfish harvesters.

PP2A Inhibition Assay Using Recombinant Enzyme for Rapid Detection of Okadaic Acid and Its Analogs in Shellfish

Okadaic acid and its analogs (OAs) responsible for diarrhetic shellfish poisoning (DSP) strongly inhibit protein phosphatase 2A (PP2A) and thus are quantifiable by measuring the extent of the enzyme inhibition. In this study, we evaluated the suitability of the catalytic subunit of recombinant human PP2A (rhPP2Ac) for use in a microplate OA assay. OA, dinophysistoxin-1(DTX1), and hydrolyzate of 7-O-palmitoyl-OA strongly inhibited rhPP2Ac activity with IC50 values of 0.095, 0.104, and 0.135 nM, respectively. The limits of detection and quantitation for OA in the digestive gland of scallops and mussels were 0.0348 μg/g and 0.0611 μg/g respectively, and, when converted to the whole meat basis, are well below the regulation level proposed by EU (0.16 μg/g whole meat). A good correlation with LC-MS data was demonstrated, the correlation coefficient being 0.996 with the regression slope of 1.097.

Detection of okadaic acid and related esters in mussels during diarrhetic shellfish poisoning (DSP) episodes in Greece using the mouse bioassay, the PP2A inhibition assay and HPLC with fluorimetric detection

An approach involving chemical, functional and biological techniques was taken for the detection and quantification of the marine toxin okadaic acid (OA) in mussels from Thermaikos and Saronikos Gulfs, Greece, during DSP episodes that occurred in 2006–2007. Samples were analyzed using the mouse bioassay, high performance liquid chromatography with fluorimetric detection (HPLC-FLD), using l-bromoacetylpyrene (BAP), as a precolumn derivatisation reagent, and the protein phosphatase 2A inhibition assay (PP2AIA) using a commercially available kit. Okadaic acid (OA) and its polar and non-polar esters were detected and quantified by HPLC-FLD, after hydrolysis of the samples during preparation. The detection limit of the HPLC method for OA was 5.86 μg OA/kg, which permits this method to be used for the regulatory control of these toxins in shellfish. Comparison of the results by all three methods revealed excellent consistency.

Potentially toxic epiphytic Prorocentrum (Dinophyceae) species in Greek coastal waters

Five epiphytic Prorocentrum species have been identified after the examination of macrophyte, water column and sediment samples collected from 50 sites along North Aegean coastline (Greece) the period from August 2003 to December 2005. Two of the identified Prorocentrum species, P. borbonicum and P. levis, constitute new records for the Mediterranean Sea. Numerous specimens fitted the general description of P. lima exhibiting, however, great variability in several morphological characters, such as cell shape, length and width, number and shape of valve and marginal pores, making this species identification difficult and raising issues regarding its morphological identity. For this reason, all these identified morphotypes, are referred to as “ P. lima complex”. Representatives of “ P. lima complex” were the most abundant (up to 133,000 cells g −1 fresh weight of macrophyte (fwm) on Cymodocea nodosa) among Prorocentrum species, followed by P. rhathymum (11,000 cells g −1 fwm on C. nodosa). P. borbonicum was detected sporadically, while P. levis, P. rhathymum and P. emarginatum showed maximum cell densities in summer and autumn. “ P. lima complex” representatives exhibited peak abundances at the end of November and/or December, while they were found in relatively high densities during summer and autumn. The temporal distribution of Prorocentrum species, combined with specific trends in substrate preference, may imply possible succession patterns and niche separation between potentially toxic dinoflagellates in epiphytic assemblages. Toxicological properties of Prorocentrum species were examined by means of protein phosphatase 2A inhibition assay and/or Artemia bioassay. Results concerning toxicity of Prorocentrum species constitute new information for the Mediterranean Sea, pointing out their potential role as diarrhetic shellfish poisoning (DSP) toxins producers.

Reduction of trihalomethane formation and detoxification of microcystins in tap water by ozonation

The efficiency of ozonation in comparison to chlorination for removal of microcystins and production of trihalomethanes (THMs) in water was investigated. One hundred and ninety water samples of ozone and chlorine treated water were collected at a water treatment plant between August 2004 and March 2005. The level of THMs, total organic carbon and residual chlorine were determined. Protein phosphatase 2A inhibition assay was used to detect microcystins and the presence of microcystins was confirmed by HPLC. The results show that 91.5% of the THM species in treated water was chloroform and 8.5% was bromodichloromethane. The mean THM level± standard error of mean in chlorinated water (CW) (45.1±3.0 μg/L) was higher than the mean of THM level in ozonated water (OW) (18.6±2.2 μg/L). In addition, no OW sample exceeded the first stage U.S. EPA maximum THM contaminant level for drinking water (80 μg/L) and only 8% of these samples exceeded the second stage level (40 μg/L). On the other hand, 3% of CW samples exceeded 80 μg/L and 68% exceeded the 40 μg/L level. The microcystin level in all water samples was below the WHO guideline value (1 μg/L) for drinking water.

Issues arising when interpreting the results of the protein phosphatase 2A inhibition assay for the monitoring of microcystins

Microcystins, cyclic heptapeptidic hepatotoxins produced by a number of bloom forming freshwater cyanobacteria, are considered to represent a serious risk to public health through drinking and recreational water. A highly sensitive bioassay relying on the specific inhibition of the human protein phosphatase 2A was applied to the quantification of microcystins. A systematic approach based on the rational testing of seven purified mcyst variants as well as characterized environmental samples allowed to point out the limits and experimental bias associated with this assay. All the seven microcystin variants known as microcystins RR, YR, LR, LY, LA, LW and LF strongly inhibited the enzyme with IC 50 ranging between 0.29±0.02 and 0.84±0.07 nM for microcystins LW and YR, respectively. Using the model system of Microcystis aeruginosa PCC7820 axenic cultures and within the 1-year study of a Planktothrix agardhii bloom, the PP2A assay was shown to be strongly correlated to high-performance liquid chromatography (HPLC) coupled to ultra violet diode array detection. However the slope of the linear regression was significantly influenced by the sample composition, as confirmed by HPLC coupled to electrospray ionization mass spectrometry. A model based on pure additivity of mcyst effects was established to describe PP2A inhibition by standard mcyst mixtures, and fully agreed with experimental observations.

A perennial bloom of Planktothrix agardhii (Cyanobacteria) in a shallow eutrophic French lake: limnological and microcystin production studies

A perennial bloom of the hepatotoxic filamentous cyanobacterium Planktothrix agardhii was investigated from January 1999 to February 2000 in a shallow eutrophic lake in the south of Paris (Viry-Châtillon, France). The lake is linked to the Seine River by an artificial channel and used for both recreation and fishing. Little data on cyanobacterial blooms is available in France. The aim of this preliminary study was to characterise the cyanobacterial bloom limnologically as well as for toxicity levels at one site in the lake and another in the channel. A relatively high biomass of P. agardhii was maintained throughout the year with maximum density in winter (7.9 ×10 4 to 4.5×10 6 trichomes/L). The only other significant phytoplankton species in the water bodies was also a cyanobacterium: Limnothrix redekei. The nutrient levels in the lake, although still relatively high (SRP varied in the range between 0.13 and 2 μM but NO 3 - stayed under the detection limit), were generally lower than in the channel which reflected the hydraulic influence of exchanges with the eutrophic Seine River. The concentration of microcystins (hepatotoxic cyclic heptapeptides) was measured in bloom extracts throughout the year using two different techniques: protein phosphatase 2A inhibition assay and high performance liquid chromatography coupled to diode array detection (HPLC-DAD). High microcystin concentrations, up to 5.2 μg/L MCYST-LR eq., were found at the two sampling sites, with good correlation between the two methods. The presence of five different microcystins was confirmed by further analysis of samples by LC-MS. Factors affecting seasonal population bloom dynamics, toxicity and the possible public health significance of these toxic cyanobacterial populations in the lake are discussed.

On the presence of peptide synthetase and polyketide synthase genes in the cyanobacterial genus Nodularia

Nodularin is a hepatotoxin produced by the bloom-forming cyanobacterial species Nodularia spumigena. Putative peptide synthetase and polyketide synthase genes were detected in toxic strains of Nodularia by degenerate PCR. Using specific primer sets, peptide synthetase and polyketide synthase gene homologues were detected in nodularin-producing strains indicating a possible role of peptide synthetase and polyketide synthase enzyme complexes in the biosynthesis of nodularin. Strains of Nodularia isolated from around the world were also analyzed for the production of nodularin by the protein phosphatase 2A inhibition assay. The protein phosphatase inhibition assay and the molecular detection of peptide synthetase and polyketide synthase genes in Nodularia may be useful techniques for the assessment of nodularin-producing cyanobacteria in the environment.

On the presence of peptide synthetase and polyketide synthase genes in the cyanobacterial genus Nodularia.

Nodularin is a hepatotoxin produced by the bloom-forming cyanobacterial species Nodularia spumigena. Putative peptide synthetase and polyketide synthase genes were detected in toxic strains of Nodularia by degenerate PCR. Using specific primer sets, peptide synthetase and polyketide synthase gene homologues were detected in nodularin-producing strains indicating a possible role of peptide synthetase and polyketide synthase enzyme complexes in the biosynthesis of nodularin. Strains of Nodularia isolated from around the world were also analyzed for the production of nodularin by the protein phosphatase 2A inhibition assay. The protein phosphatase inhibition assay and the molecular detection of peptide synthetase and polyketide synthase genes in Nodularia may be useful techniques for the assessment of nodularin-producing cyanobacteria in the environment.

A protein phosphatase 2A inhibition assay for a fast and sensitive assessment of okadaic acid contamination in mussels

The specific inhibitory activity exerted by okadaic acid on protein phosphatase 2A was used to assess the presence of okadaic acid in mussels, using a commercially available protein phosphatase 2A preparation. Under the conditions used, okadaic acid inhibits the enzymatic activity dose-dependently, with an IC 50 = 0.26 ng/ml (0.32 nM). The assay is accurate and reproducible. Okadaic acid was detected in concentrations as low as 0.063 ng/ml in aqueous solutions and 2 ng/g in mussel digestive glands. Thirty naturally contaminated mussel samples were submitted to the protein phosphatase 2A inhibition assay as well as to an ELISA assay and to a MTT cytotoxicity assay, with similar results. The proposed assay is sensitive, rapid and does not require expensive equipment. These characteristics make it a good candidate for employment in the routine assessment of okadaic acid shellfish contamination.