Short communication
A novel microplate-based assay for screening radioprotectors and its validation based on DNA and membrane system

https://doi.org/10.1016/j.mrgentox.2012.09.004Get rights and content

Abstract

Ionizing radiation leads to damage at various cellular and sub-cellular levels and can be prevented by radioprotectors. There are many in vitro and in vivo but rather expensive assays for screening of radioprotectors from natural and synthetic sources. We have developed a cell free radioprotector screening assay which involves bleaching of crocin pigment, isolated from saffron by radiolytic products of water. Any molecules/compounds which can inhibit the bleaching of the crocin will act as a radioprotector. The developed assay was further validated by the existing in vitro assays. Different radioprotectors have different level for inhibition of bleaching of crocin. The trends of radioprotection offered by crocin bleaching assay, plasmid relaxation and lipid peroxidation are TMG > FA > VA > Amifos > Trox, TMG > VA > FA > Amifos > Trox, and TMG > FA > Trox > VA > Amifos, respectively. We are getting different trends for different assays. This is because different drugs have different mechanisms of radioprotection in different assay systems. In conclusion, the crocin bleaching assay developed here is a simple, fast and economical screening assay and it will have great value in radioprotection programme for screening many potential compounds for radioprotection.

Introduction

With the advent of the nuclear age there came the realization that people would require protection against the toxic effects of radiation that could occur from nuclear accidents, medical exposures, and nuclear war, with the latter risk being the primary driving force for research and development of radioprotectors during the cold war [1], [2]. In recent years there is an increased awareness regarding ones health and hence advances in various areas of human health are of immense importance. Radiation has been considered an enigma to the general public and the use of radiation for therapeutic and other uses has always been associated with skepticism. To combat these situations there is a need to screen as many potential compounds and products for their radioprotective property. Many methods have been developed for measuring radioprotective properties of natural and synthetic compounds under in vitro and in vivo conditions. These methods includes plasmid relaxation assay [3], [4], estimation of lipid peroxidation [5], single cell gel electrophoresis (comet assay) [3], [6], micronuclei assay [7], etc. In recent years due to the rise of a nuclear terrorism threat, there has been a new interest focused on the development of novel effective and non-toxic radioprotectors for potential use in homeland defense as well as in selected medical applications. Since the assays mentioned above are expensive and time consuming, there is need for faster assays so that we can screen many natural and synthetic compounds for radioprotection.

This paper reports the development of a microplate-based assay for estimating the radioprotective efficacy, to screen compounds in minimum amount of time. This assay is based on the bleaching of crocin pigment isolated from saffron. Crocin (C44H64O24, MW 976.96) is the most prominent chemical in saffron and constitutes 2% (w/w) of total saffron (Scheme 1) (Ref. CID 5281233). It is a rare carotenoid found in nature which can easily dissolve in the water [8]. Crocin is bleached by reducing and oxidizing radicals but not by superoxide (O2) and methyl (CH3radical dot) radicals [9]. Crocin is derived from dry stigma of Crocus sativus L. (saffron), and this pigment is responsible for the red color of saffron. The traditional ‘crocin assay’ for antioxidant activity involves bleaching of crocin by radical attack induced by thermal decomposition of azo-initiators, like 2,2′-azobis (2-amidopropane) dihydrochloride (AAPH). In our assay we have used gamma-radiation as a source of radical initiator, after radiolysis of water. The compounds which are able to inhibit the bleaching of crocin against gamma-radiation will have radioprotective property. Further studies were carried out to validate this assay using plasmid relaxation and membrane protection assays. These assays corroborated well with our crocin bleaching assay.

Section snippets

Chemicals

Amifostine (Amifos), ferulic acid (FA), vanillin (VA) and troxerutin (Trox) were obtained from Sigma Chemicals Inc. (St. Louis, MO, USA). Plasmid pBR322 DNA was purchased from Bangalore Genei, Bangalore, India. α-Tocopherol monoglucoside (TMG) was a kind gift from Prof. V.T. Kagiya, Japan. All other chemicals used were of analytical grade procured locally.

Gamma-radiation

A 60Co Gamma Cell 220 (AECL, Otawa, Canada) with a dose rate 5 Gy/min was used for irradiation.

Isolation of crocin from saffron

Crocin was isolated from Saffron by

Results

Fig. 1 showed that exposure of crocin solution to gamma-radiation leads to the dose dependent bleaching of its color and the color was stable up to 24 h post-radiation exposure. In this assay troxerutin, FA, VA and TMG exhibit radioprotective property and level of bleached pigment are stable up to 24 h (Fig. 2). The order of radioprotection by crocin bleaching assay is TMG > FA > VA > Amifos > Trox. Fig. 3, Fig. 4 depicted that the presence of 0.5 mM amifostine, ferulic acid, troxerutin, vanillin and TMG

Discussion

Due to the increased use of ionizing radiation in various aspects of human life, there is a need to develop an effective and non-toxic radioprotector. Though a large number of compounds have been shown to be promising as radioprotectors in laboratory studies, few could pass the transition from bench to bedside [11], [12], [13]. In fact, no radioprotective agent is now available, either alone or in combination to meet all the requisites of an ideal radioprotector [14]. Amifostine is the only one

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgements

The authors thank Mr. P.M. Gonsalves, D. Kathole and Ms. Pritam Patil for their help during experiments.

References (17)

There are more references available in the full text version of this article.

Cited by (0)

View full text