Activation of 2,4-diaminotoluene to proximate carcinogens in vitro, and assay of DNA adducts.
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/10(6) 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.
Regina Elena Institute for Cancer Research, Rome, Italy.
G
G
Citro
R
R
Galati
A
A
Verdina
S
S
Marini
R
R
Zito
B
B
Giardina
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001711:Biotransformation; D002273:Carcinogens / Q000378:metabolism / Q000493:pharmacokinetics* / Q000633:toxicity; D004247:DNA / Q000187:drug effects / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D007120:Immunochemistry; D008297:Male; D010655:Phenylenediamines / Q000378:metabolism / Q000493:pharmacokinetics* / Q000633:toxicity; D011066:Poly C / Q000378:metabolism; D011068:Poly G / Q000378:metabolism; D011067:Poly dA-dT / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013056:Spectrophotometry, Ultraviolet
D000911:Antibodies, Monoclonal; D002273:Carcinogens; D010655:Phenylenediamines; D011068:Poly G; C008961:poly G-poly C; D011067:Poly dA-dT; D011066:Poly C; D004247:DNA; C010914:2,4-diaminotoluene