Activation of 2,4-diaminotoluene to proximate carcinogens in vitro, and assay of DNA adducts

Abstract

1. 2,4-Diaminotoluene, which yields adducts with DNA in vivo, has been studied for its ability to form adducts in vitro. Metabolic activation with rat liver post-mitochondrial supernatant gave 300 adducts/10⁶ nucleotides in calf thymus single-stranded DNA, under defined experimental conditions.

2. 2,4-Diaminotoluene-modified DNA and deoxyhomopolymers showed characteristic u.v. absorption spectra, exhibiting hyperchromic effects at 235 and 220 nm, and hypochromic effect at 260 nm. The difference spectra between diamine-modified and untreated DNA, or deoxyhomopolymer, were very similar to the spectrum of 2,4-diaminotoluene alone.

3. 2,4-Diaminotoluene-modified DNA was assayed by ELISA with specific monoclonal antibodies directed against diamine-DNA adducts. Reactions with poly-d(A) or poly-d(A-T) gave no spectral modification, and immunochemical analysis showed that the diamine did not bind to these polynucleotides. On the other hand, in the case of poly-d(G) or poly-d(C-G), strong immunoreactions were observed, demonstrating that the guanine base is involved in the binding of the diamine to DNA.

4. Monoclonal antibodies directed against different diamine-DNA adducts have shown that 80% of the in vitro metabolic activation involves the para amino group of the aromatic diamine.