When inhaled, chromate is a well-known mutagen and human carcinogen [1]. Unlike cationic Cr(III) complexes, anionic chromate readily enters and is distributed in cells via anion transporters, performing phosphate and sulfate influx. In the cellular milieu, chromate is reduced from Cr(VI) to Cr(III) by biological reductants such as ascorbate and non-protein thiols (glutathione and cysteine). The newly formed Cr(III) preferentially forms six-coordinate complexes containing oxygen- and nitrogen-based ligands, including DNA [1,2].

Cr(III) has been proposed to bind to DNA to form binary (DNA–Cr(H2O)5) and ternary (DNA–Cr–small molecule) adducts as well as interstrand DNA crosslinks (ISC) [2,3]. While much has been inferred about these species from biologically oriented experiments, very few spectroscopic or magnetic studies have directly probed the structures of these Cr(III) adducts. In part, this lack is due to the difficulty of obtaining spectroscopic data on a d3 metal ion at biologically relevant concentrations. For example, Cr(III) complexes generally lack intense charge transfer bands in their electronic spectra, while the spin 3/2 metal centers generate NMR features that are extremely broad or even unobservable [4]. Recently, this laboratory has utilized its experience probing the structure of Cr(III) protein and peptide complexes using spectroscopic and magnetic techniques to characterize the structure of Cr(III)–DNA adducts. These studies have shown that Cr(III) at low concentrations binds to the N-7 atom of guanine bases in DNA as the [Cr(H2O)5]3+ forming binary adducts [5]. Additional stabilization of these adducts arises through the hydrogen bonding of aqua ligands to sites in the DNA major groove. In contrast, the addition of Cr(III) and small molecules or Cr(III) chelate complexes to DNA does not result in the formation of ternary adducts [6]. These studies have been performed with synthetic oligonucleotides, where the composition and structure of the resulting double-stranded DNA can be carefully controlled and scrutinized [4,5]. These studies are providing structures and spectroscopic and magnetic properties of adducts of Cr(III) with DNA that will allow for the identification and quantification of such adducts in in vitro and in vivo studies of the mutagenicity and carcinogenicity of Cr(VI) species. Understanding the significance of the formation of Cr(III)-DNA adducts is crucial to determining the major pathway(s) leading to mutations and ultimately cancer from Cr(VI) exposure, particularly as both ternary Cr(III)-DNA-small molecule or protein and Cr(III)-based interstrand crosslinks have been proposed as the primary mutagenic and carcinogenic lesion [3].

While numerous studies have examined the formation of ISC formed from the introduction of chromate and a reducing agent to DNA in vivo or in vitro [[7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19]], little, if anything, is firmly known about the composition of the crosslinks. The presence of chromate-induced ISC is generally deduced by renaturing agarose gel electrophoresis or similar techniques [7,14]; these experiments measure the amount of DNA that remains double-stranded under denaturing alkaline conditions. The addition of Cr(III) to DNA has also been proposed to generate interstrand crosslinks [8,11,[20], [21], [22]]. These crosslinks were manifested in DNA polymerase arrest one base upstream of guanine residues in the template strand [22].

Starting from the observed selectivity of Cr(III) in binary complexes to bind at the guanine N-7 position, it is rapidly apparent that the metal center cannot easily bridge to Lewis basic sites on the complementary DNA strand. Two possibilities exist to overcome this geometric difficulty: either the structure of the DNA is radically distorted to bring a second coordinating atom near the Cr(III) center or the metal complex is lengthened to span the distance to another coordinating atom. The former effect is well-known in the case of platinum intra- and interstrand crosslinks of DNA [23]. However, it has previously been shown that Cr(III) does not form similar intrastrand crosslinks [5]. The observation that aquated Cr(III) complexes are subject to hydrolysis to form hydroxo- and oxo-bridged binuclear species and larger oligomers suggests a route to accomplish the latter task. Notably, the presence of oligonuclear Cr(III) complexes in chromate-induced interstrand crosslinks has been proposed based on the exponential dose dependence of the yield of chromate-induced ISC [12]. Three combinations of sequences can place a pair of guanines at neighboring positions in a DNA sequence: 5′-GG, 5’-GC, and 5’-CG. The first combination would form an intrastrand crosslink while the last two would form ISC. Previous work has demonstrated that 5′-GG sites form simple binary complexes with Cr(III); the metal interacts with one or the other guanine [5]. Herein we report experiments to examine the potential formation of Cr(III)-based ISC by addition of CrCl3•6H2O to oligonucleotide duplex DNAs containing 5’-CG sites. Thus, the self-complementary oligonucleotide, 5′-AT ACG TATA CGT AT-3′ (CG-14), was designed to form the DNA duplex (CG-14)2. Upon hybridization, the DNA contains two symmetry-equivalent 5’-CG sites for Cr(III) binding, simplifying its spectroscopic characterization.