Abstract
4-Nitroquinoline-1-oxide (4NQO) is a UV-mimic carcinogen but does not have activity of DNA-binding or mutagenicity. 4NQO is converted into 4-hydroxyaminoquinoline-N-oxide (4HAQO) by seryl-tRNA synthetase. 4HAQO binds to bases, producing adducts in DNA. It has been suggested that 4NQO has other activation mechanisms, which produce reactive oxygen species (ROS), increasing oxidative stress in cells and damaging lipids, proteins and DNA. But whether this mechanism is involved in mutagenesis is not yet known. mutM and mutY are involved in the base excision repair that is concerned with repair of the DNA damage produced by ROS. The uvrA gene is involved in the nucleotide excision repair and seems to be able to remove DNA adducts.
In this study, we examined the mutation spectrum of E. coli treated with 4NQO in the growth phase. The lactose reversion assay measuring Lac+ revertant colonies can independently detect each of the six possible base-pair substitution mutations. The results indicate that 4NQO induces specific mutations: G:C → A:T, G:C → T:A, and A:T → C:G. We further investigated which 4NQO mutagenesis depended on: the activity as a DNA adduct or generating ROS using a rifampicin reversion assay of mutM, mutY, mutM mutY, and uvrA mutant strains. The rifampicin reversion assay can analyze nonspecific mutation frequency. The results suggested that mutM and uvrA deficient mutants are sensitive to 4NQO, but there mutation frequencies are not increased by 4NQO.
We are currently further examining the survival and mutation frequency of a mutM mutY uvrA triple mutant and katG mutants (deficient for hydrogen peroxidase) treated with 4NQO in order to further clarify the relationship between ROS produced by 4NQO and mutagenesis. In addition, we hope to reveal which has a stronger effect; 4NQO activity as a DNA adduct or as an agent generating ROS.
