STUDIES ON THE MECHANISMS OF HALOACENTRONITRILE-INDUCED GENOTOXICITY .4. IN-VITRO INTERACTION OF HALOACETONITRILES WITH DNA

Haloacetonitriles, which are water chlorination by-products, are mutag ens, carcinogens and teratogens. In vitro, the reaction of the haloace tonitriles bromoacetonitrile (BAN), chloroacetonitrile (CAN), dichloro acetonitrile (DCAN) and trichloroacetonitrile (TCAN) with calf thymus DNA produced fluorescent DNA derivatives. The reactivity of haloaceton itriles towards DNA was in the order of BAN > CAN > DCAN > TCAN. The e mission fluorescence spectra of the reaction product(s) of various hal oacetonitriles with DNA has a peak at 404 nm at fixed excitation wavel ength (300 nm). The fluorescence intensity of the reaction product(s) was pH dependent with an optimum intensity at pH 7.4. The DNA interact ion was dependent on haloacetonitrile concentration. Higher affinity o f haloacetonitriles towards single-stranded DNA (SS-CT-DNA) than towar ds double-stranded DNA (DS-CT-DNA) was observed. To characterize the t ype 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 d erivative(s) or (b) chemically by formic acid to nucleobases and nucle obase adducts. The hydrolysates were analysed by reversed phase HPLC. A major fluorescent peak was detected in the enzymatic hydrolysate tha t was not present in unreacted DNA. In the acid hydrolysate, one fluor escent peak was detected that was not present in unreacted DNA. Authen tic 7-(cyanomethyl)guanine was synthesized by the reaction of CAN with 2'-deoxyguanosine and the product was purified and characterized spec troscopically. The product, 7-(cyanomethyl)guanine, was found to be ch romatographically and spectroscopically identical to the fluorescence product that was obtained following haloacetonitrile-DNA interaction. This study shows that haloacetonitriles, in vitro, are capable of alky lating DNA at the guanine moiety to form a 7-(cyanomethyl)guanine addu ct.