Intracellular zinc trafficking and metallothionein gene activation in HUVEC treated with Crotalus atrox venom

Abstract

Hemorrhagic snake venom, such as Crotalus atrox, destroys local tissue and disrupts hemostasis. In this study, we examined zinc trafficking in human umbilical vein endothelial cells (HUVEC) stimulated with C. atrox snake venom. We utilized MTS cytotoxicity assays to monitor the cytotoxic range of C. atrox venom. HUVEC monolayers stimulated with 10 μg/ml C. atrox venom for 3 hours displayed cellular retraction, which coincided with 53.0±6.5 percent viability. In contrast, venom concentrations of 100 μg/ml produced a complete disruption of cellular adherence and viability decreased to 36.6±1.0. Venom induce cellular retraction involves cytoskeletal rearrangements. Therefore, we used the zinc probe FluorZin‐3AM to compare intracellular free zinc in HUVEC stimulated with 10 μg/ml C. atrox venom or 1 μg/ml cytochalasin D for 1 hour. Fluorescent intensity analysis returned values of 287.1±57.7 for C. atrox and 261.7±33.3 for cytochalasin D, demonstrating an increase in labile zinc compared to non‐stimulated controls. Labile intracellular zinc or other metals can induce the gene activation of intracellular metal binding proteins known as metallothioneins. Thus, we measured the gene and protein expression of metallothionein 1X (MT1X) and metallothionein 2A (MT2A) in HUVEC stimulated with C. atrox venom. MT2A gene expression increase by 4.4±0.41 fold compared to 96.1±3.0 for MT1X. This data suggest cell death from C. atrox venom involves intracellular zinc trafficking, and metallothionein gene activation.Support or Funding InformationCETL, OVPR Grant