Desmutagenic Effect of Pheophytin b against Trp-P-2 in the Salmonella Assay

Abstract

Modifications of the standard Ames test and umu test were employed to study the “desmutagenic” response exerted by pheophytin b against the mutagenicity of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). The mixture from which pheophytin b separated was activated with S9 mix and was assayed to determine the remaining mutagenicity of Trp-P-2. The decrease in the His+ revertant colonies of Salmonella typhimurium TA98 was dependent on the increase in pheophytin b concentrations. Binding of Trp-P-2 to DNA and the subsequent induction of the SOS response were investigated using the umu test employing Salmonella typhimurium NM 2009. Trp-P-2 induced induction of β-galactosidase was suppressed when pheophytin b was included in the test. In order to evaluate the mode of action of pheophytin b in the mixture of Trp-P-2, Trp-P-2 was analyzed using HPLC. Compared with a solution of Trp-P-2 alone, the absorbance peak of Trp-P-2 in the HPLC profile of the mixture of Trp-P-2 and pheophytin b was observed to be decreased. Also a new peak was not shown in the HPLC profile. To investigate the possible effect of pheophytin b on the S9 mix, the HPLC profile of a mixture containing Trp-P-2, S9 mix and pheophytin b was compared with a mixture containing only Trp-P-2 and S9 mix. The absorbance peak of the hydroxylated Trp-P-2 in the reaction mixture containing pheophytin b was observed to be smaller than the peak of the mixture without pheophytin b. From these results, it is suggested that pheophytin b limits the formation of hydroxylated Trp-P-2.