iTRAQ-Based Quantitative Proteomic Analysis Reveals Toxicity Mechanisms in Chlamys farreri Exposed to Okadaic Acid

Okadaic acid (OA), produced by dinoflagellates during harmful algal blooms, is a principal diarrhetic shellfish poisoning toxin. This toxin poses a potential threat to bivalves with economic values. To better understand the toxicity mechanism of OA to bivalves, in this study, oxidative stress biomarkers (superoxide dismutase, SOD; catalase, CAT; glutathione S-transferase, GST; malondialdehyde, MDA) and the expression of detoxification genes (heat shock protein 70, HSP70; heat shock protein 90, HSP90; cytochrome P450, CYP450) were assessed in the gills of scallops Chlamys farreri after 24 h, 48 h and 96 h exposure to OA. In addition, the digestive glands of scallops exposed to OA for 96 h were dissected for an iTRAQ based quantitative proteomic analysis. The results of OA exposure experiments showed that OA induces oxidative stress and significant enhancement of the expression of detoxification genes in scallops. The proteomics analysis revealed that 159 proteins altered remarkably in OA-treated scallops, and these proteins were involved in phagosomes, regulation of actin cytoskeleton, adherens junction, tight junction, and focal adhesion. Amino acid biosynthesis, carbon metabolism, pentose phosphate pathway, fructose and mannose metabolism in the digestive glands were also significantly impacted. Our data shed new insights on the molecular responses and toxicity mechanisms of C. farreri to OA.

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.

CRMP2 Is Involved in Regulation of Mitochondrial Morphology and Motility in Neurons

Regulation of mitochondrial morphology and motility is critical for neurons, but the exact mechanisms are unclear. Here, we demonstrate that these mechanisms may involve collapsin response mediator protein 2 (CRMP2). CRMP2 is attached to neuronal mitochondria and binds to dynamin-related protein 1 (Drp1), Miro 2, and Kinesin 1 light chain (KLC1). Treating neurons with okadaic acid (OA), an inhibitor of phosphatases PP1 and PP2A, resulted in increased CRMP2 phosphorylation at Thr509/514, Ser522, and Thr555, and augmented Drp1 phosphorylation at Ser616. The CRMP2-binding small molecule (S)-lacosamide ((S)-LCM) prevented an OA-induced increase in CRMP2 phosphorylation at Thr509/514 and Ser522 but not at Thr555, and also failed to alleviate Drp1 phosphorylation. The increased CRMP2 phosphorylation correlated with decreased CRMP2 binding to Drp1, Miro 2, and KLC1. (S)-LCM rescued CRMP2 binding to Drp1 and Miro 2 but not to KLC1. In parallel with CRMP2 hyperphosphorylation, OA increased mitochondrial fission and suppressed mitochondrial traffic. (S)-LCM prevented OA-induced alterations in mitochondrial morphology and motility. Deletion of CRMP2 with a small interfering RNA (siRNA) resulted in increased mitochondrial fission and diminished mitochondrial traffic. Overall, our data suggest that the CRMP2 expression level and phosphorylation state are involved in regulating mitochondrial morphology and motility in neurons.