A Comparative Study of Calf Thymus DNA Binding to Cr(III) and Cr(VI) Ions

Abstract

Chromium(VI) salts are well known to be mutagens and carcinogens and to easily cross the cell membranes. Because they are powerful oxidizing agents, Cr(VI) reacts with intracellular materials to reduce to trivalent form, which binds DNA. This study was designed to investigate the interaction of calf thymus DNA with Cr(VI) and Cr(III) in aqueous solution at pH 6.5-7.5, using Cr(VI)/DNA(P) molar ratios (r) of 1:20 to 2:1 and Cr(III)/ DNA(P) molar ratios (r) of 1:80 to 1:2. UV-visible and Fourier transform infrared (FTIR) difference spectro-scopic methods were used to determine the metal ion-binding sites, binding constants, and the effect of cation complexation on DNA secondary structure. Spectro-scopic results showed no interaction of Cr(VI) with DNA at low anion concentrations (r 1:20 to 1:1), whereas some perturbations of DNA bases and backbone phosphate were observed at very high Cr(VI) contents (r > 1) with overall binding constant of K 508 M 1. Cr(III) chelates DNA via guanine N-7 and the nearest PO 2 group with overall binding constant of K 3.15 10 3 M 1. Evidence for cation chelate formation comes from major shiftings and intensity variations of the guanine band at 1717 and the phosphate asymmetric stretching vibration at 1222 cm 1. At low Cr(III) concentration (r 1:40), the number of Cr(III) ions bound to DNA were 6-7 cat-ions/500 base pairs, and this increased to 30-35 cations/ 500 base pairs at high metal ion content (r 1:4). DNA condensation occurred at high cation concentration (r 1:10). No major alteration of DNA conformation was observed , and the biopolymer remained in the B family structure upon chromium complexation. Chromium(VI) salts are well known to be mutagens and carcinogens and to easily invade the insides of cells (1). Cr(VI) produced DNA cross-links in rat tissues in vivo (2) and in cultured cells in vitro (3, 4). Although Cr(VI) damaged nuclear DNA in whole cells, no reaction of Cr(VI) with isolated DNA occurred in vitro at physiological pH in the absence of a me-tabolizing system (5). The Cr(VI) that is taken up is considered to be reduced by glutathione, cysteine, or ascorbic acid into Cr(III) (6), and the resulting cation reacts with DNA to form Cr(III)-DNA adducts. Because Cr(III) is a final form of chromium within the cell, the interaction of Cr(III) with DNA may play crucial role in the carcinogenetic action of Cr(VI) salts. The conversion of B form into Z form in the purine-pyrimi-dine sequence of DNA has been considered to be a factor in the transcriptional activity of genes (7). Cr(III) is found to interact with the poly(dG-dC) at low concentration and change B form to Z form in the presence of ethanol (8). However, Cr(III) at high concentration causes DNA condensation, inhibiting the alteration of B to Z structure (8). Moreover, the study on the effect of Cr(III) on DNA replication with single-stranded DNA template and micromolar concentration of Cr(III) revealed that Cr(III) bound in a dose-dependent manner to the template DNA and prevents DNA replication (9). However, if the un-bound chromium was removed from the system by gel filtration , the rate of DNA replication by polymerase I (Klenow fragment) on the chromium-bound template increased more than 6-fold relative to control. This increase was paralleled by as much as a 4-fold increase in processivity and a 2-fold decrease in replication fidelity. When the concentration of Cr(III) increased further, DNA-DNA cross-links occurred to inhibit the polymerase activity. Trivalent chromium can bind purified DNA and form lesions capable of obstructing DNA replication in vitro (10, 11). It has also been observed that intact Novikoff ascites hepatoma cells exposed to potassium chromate formed cross-linking of nuclear proteins to DNA (12). Recently, Cr(III) was shown to cause mutational spectrum in shuttle vector systems replicated in human cells (13). Thus, the interaction of Cr(III) with DNA may be responsible for carcinogenic activity of chromium. There are many agents that are specific for guanine alkyla-tion in the O-6, N-7, or C-8 position. Several of these are highly active carcinogens, such as N-acetoxy-N-2-acetylaminoflu-orene, which alkylates in the C-8 position, and nitrosoamines, nitrogen mustards, nitrourea, and aflatoxin, which alkylate on the N-7 position (14). The action of certain carcinogens, e.g. modification of guanine by N-7 methylation or by alkylation at C-8 with N-acetoxy-N-2-acetylaminofluorene facilitated the B to Z transition of poly(dG-dC) (15-17). On the contrary, modification of poly(dG-dC) with the antitumore drug cis-diamine-dicholoplatinum (II) (a cross-linking agent) prevented the B to Z conversion (18, 19). It was found that Cr(III) preferentially binds guanine-containing DNAs (5, 20). The results of a study on the DNA replication system showed that most of Cr(III) binding to the single-stranded template DNA is electrostatic because 40% or more of bound cation could be displaced by high salt wash, whereas only 20% or less chromium is chelatable by EDTA (9). In the present study, we have investigated the complexation of Cr(III) and Cr(VI) with calf thymus DNA using UV-visible and FTIR 1 difference spectroscopy at pH 6.5-7.5 with Cr(III)/