Journal of Hazardous Materials 166 (2009) 1403–1409 Contents lists available at ScienceDirect Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat Toxicity of free and various aminocarboxylic ligands sequestered copper(II) ions to Escherichia coli Subburaj Selvaraj a , K. Chabita Saha b,1 , Anindita Chakraborty a , Sudhindra N. Bhattacharyya a , Abhijit Saha a,∗ a UGC-DAE Consortium for Scientific Research, Kolkata Centre, III/LB-8 Bidhannagar, Kolkata 700 098, India b Chemical Sciences Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700 064, India article info Article history: Received 26 March 2008 Received in revised form 10 December 2008 Accepted 10 December 2008 Available online 24 December 2008 Keywords: Toxicity Metal complexes Cell inactivation E. coli Copper ions abstract Cytotoxicity of free Cu(II) ions and its complexes of EDTA, NTA or IDA in Escherichia coli (AB 4401) wild type cells were assessed by cell inactivation assay. In order to understand the toxic effects of these additives, membrane status by AFM vis-à-vis K + ion efflux were followed in the absence and in the presence of Cu(II) ions or its complexes. This was coupled with the determinations of cellular copper concentrations by atomic absorption spectrometry. The observed results show that free copper ions are more cytotoxic and cause considerable plasma membrane damage compared to that of Cu–EDTA, Cu–NTA and Cu–IDA. Determination of cellular copper reveals that Cu 2+ and Cu–NTA are able to enter inside the cells while Cu–EDTA and Cu–IDA fail to do that. This may be attributed to the electron affinity of free Cu 2+ ions and Cu–NTA, which help in binding with histidine present in copper transport proteins. In addition to cytotoxicity, genotoxicity of free copper and its complexes were also assessed on E. coli isogenic DNA repair proficient and repair-deficient strains. In contrast to free Cu 2+ ions, Cu–NTA does not cause any significant cytotoxicity but render greater genotoxicity. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Copper ion plays important biochemical roles in living organ- isms. It acts as co-factor for a variety of enzymes that are required for essential biochemical process such as Cytochrome-c oxidase, Cu, Zn superoxide dismutase, lysyl oxidase and dopamine-beta- hydroxylase [1]. Though the concentrations of copper ions vary in the source (i.e., soil, water or food), living organisms show remarkable homeostasis at intracellular level [2]. Interestingly, it has been established that like many other metals, copper also man- ifests a biphasic property, depending upon its concentration level. Although copper ion has been found to be essential for living organ- isms, it can as well show toxic effects in many systems when in excess [1,3]. Although the mechanism of cytotoxicity of copper is not yet fully understood, its toxic manifestations may either be mediated through changes in the plasma membrane permeability [4,5] or Abbreviations: EDTA, ethylene diamine tetra-acetate; NTA, nitrilotriacetate; IDA, iminodiacetate; AFM, atomic force microscopy. ∗ Corresponding author at: UGC-DAE Consortium for Scientific Research, Kolkata Centre, Sector-III, Block-LB Plot-8, Bidhannagar, Kolkata 700 098, India. Tel.: +91 33 2335 1866; fax: +91 33 2335 7008. E-mail address: abhijit@alpha.iuc.res.in (A. Saha). 1 Present address: School of Biotechnology, West Bengal University of Technology, BF142 Bidhannagar, Kolkata 700 064, India. efflux of intra-cellular K + during the entry of Cu 2+ ions [4,6]. It can also participate in Fenton-like reactions generating reactive hydroxyl radicals, which can cause cellular damage imparted via oxidative stress [7]. Toxicity of copper is mostly due to free ion activity [8]. How- ever it has been well documented that when metal ions are bound to organic ligands, their toxicity, assimilation and accumulation in organisms are changed to a great extent [9–12]. Recently, copper complexes with different structural features had been shown to bind with double-helical DNA and to promote double-strand DNA damage upon reductant/H 2 O 2 activation [13]. In order to explore the role of aminocarboxylic groups in modifying the toxic prop- erties of Cu 2+ ions, attempts have been made to look into the characteristics of complexes. For this purpose, three complexes of Cu(II) involving ligands of similar chemical nature, namely, EDTA, IDA and NTA have been chosen. Multidentate aminocarboxylic synthetic chelating ligands such as EDTA, NTA form stable, water- soluble complexes with a wide range of metals and radionuclides [14,15]. EDTA forms a hexidentate complex with two nitrogens and four carboxylate oxygens. NTA forms a tetradentate complex using one nitrogen and three carboxylate oxygens. IDA is expected to be a tridentate ligand and can form complexes with metals with one nitrogen and two carboxylate oxygens (Fig. 1). Our earlier in vitro studies showed that Cu-complexes in ques- tion retain the radiosensitizing property of Cu 2+ ions [16–18]. However, in vivo experiments showed that only Cu–NTA could 0304-3894/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jhazmat.2008.12.060