POLYCYCLIC AROMATIC HYDROCARBON O-QUINONES MUTATE p53 IN HUMAN LUNG ADENOCARCINOMA (A549) CELLS

Abstract

Human lung adenocarcinoma (A549) cells form reactive and redox active PAH-o-quinones via constitutively expressed aldo-keto reductases. To determine whether these metabolites mutate p53, A549 cells were treated with N-methyl-N-nitroso-N-nitroguanidine (MNNG an alkylating mutagen), anti-BPDE (diol-epoxide) and benzo[a]pyrene-7,8-dione (o-quinone). p53 cDNA was amplified from the treated cells and cotransfected with a gap-repair plasmid designed to express p53 in yeast. When mutant p53 is expressed, it fails to drive the expression of an ADE2 reporter, and the yeast strain yIG397 turns red. The following mutational frequencies were observed versus the solvent controls: 1 mM MNNG (24.2% red colonies p = .02), 1 μ M anti-BPDE (9.9% red colonies p = .02), and 10 μ M BP-7,8-dione (9.3% red colonies p = .03). MNNG gave a high frequency (49%) of the expected C > T (G > A) mutations (p = .25). While anti-BPDE and BP-7,8-dione gave A > G (T > C) transitions (p = .034 – p = .054). These mutations would result from stable N⁶-deoxyadenosine adducts, but not from oxidatively damaged bases.

Keywords:

• aldo-keto reductase
• ortho-quinones
• polycyclic aromatic hydrocarbon
• reactive oxygen species

Acknowledgments

This work was supported by Grants R01 CA39504 and P01 CA92537 awarded to Trevor M. Penning. The authors thank the NCI Chemical Carcinogen Standard Reference Repository for (±)-anti-BPDE.

Notes

*p = .02 and

**p = .03.

^a Under an assumption of randomness, the frequency of mutation in each of the nine groups would be 11.1%. The null hypothesis is that the mutations are randomly distributed among the nine outcomes. For each treatment, the observed distribution is highly significantly different from the random distribution as indicated by the p-value.