Mouse FM3A cells in culture were treated with a reactive metabolite of 3‐amino‐1‐methyl‐5 H ‐pyrido[4,3‐ b ]indole (Trp‐P‐2), 3‐hydroxyamino‐1‐methyl ‐ 5 H ‐ pyrido[4,3 ‐ b ]indole (Trp ‐ P ‐ 2‐ (NHOH)). When the treated cells, which were judged as viable on the basis of trypan‐blue exclusion, were subjected to nitroblue tetrazolium staining, formazan was formed inside the cells, a fact suggesting the intracellular presence of superoxide. No formazan formation was detected on treatment of the cells with Trp‐P‐2. Single‐strand breaks in the cellular DNA took place during this treatment with Trp‐P‐2(NHOH). Since Trp‐P‐2(NHOH) in solution generates superoxide anion accompanying its oxidative degradation, we conclude that the Trp‐P‐2(NHOH) treatment produces intracellular active oxygens that can damage DNA.
We have studied the inhibitory effect of chlorophyllin‐chitosan (Chl‐Chi) complex, an insoluble form of chlorophyllin, on the DNA adduct formation and mutagenesis by a heterocyclic food mutagen‐carcinogen, 3‐amino‐l‐methyl‐5H‐pyrido[4,3‐b]indole (Trp‐P‐2), in mice carrying the E. coli rpsL gene as a mutagenesis reporter. Upon administration of a diet containing 0.002% or 0.01% Trp‐P‐2, DNA adducts were formed in various tissues in a dose‐dependent manner, with the maximum level observed in the liver. Addition of 3% Chl‐Chi to the diet reduced the Trp‐P‐2 adduct by up to 90%. The rpsL mutant frequencies increased significantly in both the liver and spleen upon administration of a 0.01% Trp‐P‐2 diet. Addition of Chl‐Chi to the diet decreased these induced mutant frequencies to the background level. No harmful effect of Chl‐Chi was detected during these experiments. The results show that Chl‐Chi may be a candidate chemopreventive agent against the genotoxic action of Trp‐P‐2, and possibly also other aromatic carcinogens in the diet.
The Klenow fragment-mediated in vitro DNA elongation was inhibited by the presence of a class of modified cytosines in the template DNA, i.e., the N4-amino(and -methoxy)-5,6-dihydrocytosine-6-sulfonate residues. We have studied the mechanism of the blockage, using as templates bisulfite-hydrazine (and -methoxyamine)- modified single strand phage-M13mp2 DNA and synthetic oligonucleotides. Both N4-amino-5,6-dihydrocytosine-6-sulfonate and N4-methoxy-5,6-dihydrocytosine-6-sulfonate residues blocked the elongation at one nucleotide before these sites. In this blockage, the idling of polymerase at the lesion site due to its 3'-5' exonuclease action appears not to play a major role, because Sequenase that lacks the 3'-5' exonuclease activity still could not readthrough these sites. It seems possible that conformational distortion of the template near these sites is responsible for the blockage, because on conversion of this 5,6-dihydropyrimidine-6-sulfonate structure into a planar pyrimidine, a complete restoration of polymerase-readthrough resulted. In the presence of RecA and SSB proteins, the Klenow fragment was able to partially readthrough these sites. Since there was no decrease in the 3'-5' exonuclease activity during this readthrough, it seems that the binding of these proteins relaxes the distortion in the modified template to allow the polymerase to readthrough the lesion site. These sites on phage DNA can be lethal but also are capable of inducing C-to-T transitions. This observation suggests that these sites can be read by E. coli DNA polymerases in vivo with accompanying errors.
