Studies on the mechanisms of haloacentronitrile-induced genotoxicity IV: In vitro interaction of haloacetonitriles with DNA

Abstract

Haloacetonitriles, which are water chlorination by-products, are mutagens, carcinogens and teratogens. In vitro, the reaction of the haloacetonitriles bromoacetonitrile (BAN), chloroacetonitrile (CAN), dichloroacetonitrile (DCAN) and trichloroacetonitrile (TCAN) with calf thymus DNA produced fluorescent DNA derivatives. The reactivity of haloacetonitriles towards DNA was in the order of BAN > CAN > DCAN > TCAN. The emission fluorescence spectra of the reaction product(s) of various haloacetonitriles with DNA has a peak at 404 nm at fixed excitation wavelength (300 nm). The fluorescence intensity of the reaction product(s) was pH dependent with an optimum intensity at pH 7.4. The DNA interaction was dependent on haloacetonitrile concentration. Higher affinity of haloacetonitriles towards single-stranded DNA (SS-CT-DNA) than towards double-stranded DNA (DS-CT-DNA) was observed. To characterize the type of fluorescent product(s) formed, samples of the reaction product of SS-CT-DNA with BAN were hydrolysed (a) enzymatically by micrococcal nuclease and spleen phosphodiesterase to nucleotides and nucleotide derivative(s) or (b) chemically by formic acid to nucleobases and nucleobase adducts. The hydrolysates were analysed by reversed phase HPLC. A major fluorescent peak was detected in the enzymatic hydrolysate that was not present in unreacted DNA. In the acid hydrolysate, one fluorescent peak was detected that was not present in unreacted DNA. Authentic 7-(cyanomethyl)guanine was synthesized by the reaction of CAN with 2′-deoxyguanosine and the product was purified and characterized spectroscopically. The product, 7-(cyanomethyl)guanine, was found to be chromatographically and spectroscopically identical to the fluorescence product that was obtained following haloacetonitrile-DNA interaction. This study shows that haloacetonitriles, in vitro, are capable of alkylating DNA at the guanine moiety to form a 7-(cyanomethyl)guanine adduct.