An in vitro evaluation of epigallocatechin gallate (eGCG) as a biocompatible inhibitor of ricin toxin

Paul D.R Dyer,Arun K Kotha,Alex S Gollings,Susan A Shorter,Thomas R Shepherd,Marie W Pettit,Bruce D Alexander,Giulia T.M Getti,Samer El-Daher,Les Baillie,Simon C.W Richardson

doi:10.1016/j.bbagen.2016.03.024

Paul D.R Dyer, Arun K Kotha + Show 9 more

Open Access

https://doi.org/10.1016/j.bbagen.2016.03.024

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Abstract

The catechin, epigallocatechin gallate (eGCG), found in green tea, has inhibitory activity against a number of protein toxins and was investigated in relation to its impact upon ricin toxin (RT) in vitro. The IC50 for RT was 0.08±0.004ng/mL whereas the IC50 for RT+100μM eGCG was 3.02±0.572ng/mL, indicating that eGCG mediated a significant (p<0.0001) reduction in ricin toxicity. This experiment was repeated in the human macrophage cell line THP-1 and IC50 values were obtained for RT (0.54±0.024ng/mL) and RT+100μM eGCG (0.68±0.235ng/mL) again using 100μM eGCG and was significant (p=0.0013). The documented reduction in ricin toxicity mediated by eGCG was found to be eGCG concentration dependent, with 80 and 100μg/mL (i.e. 178 and 223μM respectively) of eGCG mediating a significant (p=0.0472 and 0.0232) reduction in ricin toxicity at 20 and 4ng/ml of RT in Vero and THP-1 cells (respectively). When viability was measured in THP-1 cells by propidium iodide exclusion (as opposed to the MTT assays used previously) 10ng/mL and 5ng/mL of RT was used. The addition of 1000μM and 100μM eGCG mediated a significant (p=0.0015 and <0.0001 respectively) reduction in ricin toxicity relative to an identical concentration of ricin with 1μg eGCG. Further, eGCG (100μM) was found to reduce the binding of RT B chain to lactose-conjugated Sepharose as well as significantly (p=0.0039) reduce the uptake of RT B chain in Vero cells. This data suggests that eGCG may provide a starting point to refine biocompatible substances that can reduce the lethality of ricin.

Highlights

There is a clear and pressing need for improved first-line treatment and prophylaxis to combat the ever-increasing threat posed by agents of bioterrorism
The characterization of ricin toxin (RT), RT B chain (RTBC) and RT A chain (RTAC) was performed (Fig. 1; panel a) and with the exception of the re-folded RT preparation, there was no detectable RTBC in the RTAC preparations and no detectable RTAC in the RTBC preparations (Fig. 1; panel a)
Ratio of RTBC to RTAC was obtained. This was an unavoidable consequence of the protocol that was necessary to ensure that the majority of the RT preparation was heterodimeric [25]

Summary

Introduction

There is a clear and pressing need for improved first-line treatment and prophylaxis to combat the ever-increasing threat posed by agents of bioterrorism. Amongst these agents is ricin toxin (RT), a protein produced by the castor bean (Ricinus communis). The availability of RT was underpinned by both the intoxication of Roger Bergendorff in 2008 [4] and the discovery of RT containing letters sent to Bill Frist in 2004, who was, at the time the US senate majority leader [5]. There is no cure for intoxication with medical support being palliative [6]. The UK and US government exploited these observations during the production of “compound W” (RT) prior to the Biological and Toxin Weapons Convention of 1972 and the Chemical Weapons Convention of 1973 [7]

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