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

Biooxidation of Ciguatoxins Leads to Species-Specific Toxin Profiles

Ciguatoxins (CTXs) contaminate fish worldwide and cause the foodborne illness ciguatera. In the Pacific, these toxins are produced by the dinoflagellate Gambierdiscus toxicus, which accumulates in fish through the food chain and undergoes oxidative modification, giving rise to numerous analogs. In this study, we examined the oxidation of CTXs in vitro with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using reference toxins, and found that CTX4A, CTX4B, and CTX3C, which are produced by the alga, are oxidized to the analogs found in fish, namely CTX1B, 52-epi-54-deoxyCTX1B, 54-deoxyCTX1B, 2-hydroxyCTX3C, and 2,3-dihydroxyCTX3C. This oxidation was catalyzed by human CYP3A4, fish liver S9 fractions, and microsomal fractions prepared from representative ciguateric fishes (Lutjanus bohar, L. monostigumus, and Oplegnathus punctatus). In addition, fish liver S9 fractions prepared from non-ciguateric fishes (L. gibbus and L. fulviflamma) in Okinawa also converted CTX4A and CTX4B to CTX1B, 54-deoxyCTX1B, and 52-epi-54-deoxyCTX1B in vitro. This is the first study to demonstrate the enzymatic oxidation of these toxins, and provides insight into the mechanism underlying the development of species-specific toxin profiles and the fate of these toxins in humans and fish

A Protein Phosphatase 2A-Based Assay to Detect Okadaic Acids and Microcystins

Okadaic acids (OAs) are causative agents of diarrhetic shellfish poisoning, produced by the dinoflagellates Dinophysis spp. and Prorocentrum spp. Microcystins (MCs) are cyclic heptapeptide hepatotoxins produced by some cyanobacteria genera, including Microcystis spp. Traditionally, toxicity detection and quantification of these natural toxins were performed using a mouse bioassay (MBA); however, this is no longer widely employed owing to its lack of accuracy, sensitivity, and with regard to animal welfare. Therefore, alternative toxicity analyses have been developed based on MCs’ and OAs’ specific inhibition of protein phosphatase 2A (PP2A), using p-nitrophenylphosphate (p-NPP) as a substrate. The assay is simple, inexpensive, ready for use on site, and can be applied to several samples at once. For OA detection, this assay method is appropriate for widespread application as a substitute for MBA, as evidenced by its alignment with the oral toxicity of MBA. In this review, we summarize the structure and function of PP2A, the inhibitory activities of OAs and MCs against PP2A, and the practical applications of the PP2A assay, with the aim of improving understanding of the PP2A assay as an OAs and MCs detection and quantification method, as well as its suitability for screening before confirmatory chemical analysis

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

Estimation of slip parameters associated with frictional heating during the 1999 Taiwan Chi-Chi earthquake by vitrinite reflectance geothermometry

To estimate the slip parameters and understand the fault lubrication mechanism during the 1999 Taiwan Chi-Chi earthquake, we applied vitrinite reflectance geothermometry to samples retrieved from the Chelungpu fault. We found a marked reflectance anomaly of 1.30% ± 0.21% in the primary slip zone of the earthquake, whereas the reflectances in the surrounding deformed and host rocks were 0.45% to 0.77%. By applying a kinetic model of vitrinite thermal maturation together with a one-dimensional heat and thermal diffusion equation, we determined the shear stress and peak temperature in the slip zone during the earthquake to be 1.00 ± 0.04 MPa and 626°C ± 25°C, respectively. Taking into account the probable overestimation of the temperature owing to a mechanochemically enhanced reaction or flash heating at grain contacts, this temperature should be considered an upper limit. The lower limit was previously constrained to 400°C by studies of fluid-mobile trace-element concentrations and magnetic minerals. Therefore, we inferred that the peak temperature during the Chi-Chi earthquake was 400°C to 626°C, corresponding to an apparent friction coefficient of 0.01 to 0.06. Such low friction and the previous evidence of a high-temperature fluid suggest that thermal pressurization likely contributed to dynamic weakening during the Chi-Chi earthquake.This study was supported by a Japan Ministry of Education, Culture, Sports, Science and Technology Grant-in-Aid for Young Scientists (A) 23684039, 2013

Efficient production of recombinant PP2A at a low temperature using a baculovirus expression system

Protein phosphatase 2A (PP2A) is an enzyme useful for detecting several natural toxins represented by okadaic acid and microcystins. We found that the production of the recombinant human PP2A catalytic subunit (rhPP2Ac) in High Five insect cells could markedly increase when the cells were cultured at 19 °C instead of 27 °C used under conventional conditions. The yield and purity of the enzyme increased four- and three-folds, respectively. The benefit of the altered culturing temperature was observed with the recombinant human protein phosphatase 2B but not 2Cα. The different responses among the enzymes suggest the involvement of an enzyme-specific mechanism that leads to the catalytic subunit overexpression. This is the first report to produce rhPP2Ac at a temperature lower than that used under conventional culture conditions (27 °C) used in the baculovirus expression system with High Five insect cells