Abstract
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.
Highlights
Okadaic acid (OA), dinophysistoxin-1 (DTX1), and dinophysistoxin-2 (DTX2) (Figure 1), collectively called OA-group toxins (OAs) due to their structural similarities, are a group of lipophilic natural marine toxins produced by the dinoflagellates Dinophysis spp. and Prorocentrum spp. [1,2,3,4]
AFsigsuhroew2n, itnheFiOguArse 2in, hthibeiOteAdstihnehribPiPte2dAthaectriPvPit2yAinacativity in a dose-dependendtempaenndeernattmcoanncneenrtraatticoonnscreanntgraintigofnrsomran0.g0i0n1gnfMrotmo 400.0n0M1 .nTMhetIoC4500vnaMlu.esThe IC50 deduced fromdtheedsuecceudrfvreosmwtehrees0e.1cu4,rv0.e0s9w, aenrde 0.4145,n0M.09(,0a.1n1d, 00..0475, nanMd(00..3161,n0g./07m, Lan) dfo0r.36 ng/m OA, DTX1, andODAT, XD2T,Xre1s,paencdtiDveTlyX.2T, hreesrpefeocrteiv, ethlye. rTehlaetrievfeorae,titohsefroerlathtievseertahtrieoes fOoAr tshweseerethree OA calculated to beca1l.c0u:1la.5te6d:0.t3o1b1e(T1a.0b:l1e.516):.0T.3h1e1fi(Tguabreles 1ca).nTbheerfoiguunrdeesdcaton 1b.e0:r1o.6u:n0.d3e. d to 1.0:1.6:0.3
DTX2 were cytotoxic to neuro-2a cells in a dose-dependent manner at concentr ranging from 1 to 300 nM for OA and DTX1, and 1 to 200 nM for DTX2
Summary
Okadaic acid (OA), dinophysistoxin-1 (DTX1), and dinophysistoxin-2 (DTX2) (Figure 1), collectively called OA-group toxins (OAs) due to their structural similarities, are a group of lipophilic natural marine toxins produced by the dinoflagellates Dinophysis spp. and Prorocentrum spp. [1,2,3,4]. Sample Preparation for PP2A Inhibition Assay from CRM-DSP-Mus-c and CRM-Zero-Mus PP2A Inhibition Assay rPP2A was synthesized in insect cells (High Five; Invitrogen, Carlsbad, CA, USA)
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