The plant-activation of m-phenylenediamine and its mutagenic, biochemical and molecular characterization

A promutagen is a chemical agent that is not mutagenic in itself but can be metabolically transformed into a mutagen by plant or animal systems. Many aromatic amines are promutagens and plants can activate these agents into stable mutagens. The biochemical and molecular mechanisms involved in bioactivation of promutagens into mutagens have been partially revealed in mammalian systems to understand cancer induction mechanisms. This laboratory demonstrated that simple aromatic amines are activated into potent frameshift mutagens by cultured tobacco cells (TX1). In this study, biochemical, genetic, and molecular approaches were used to define the mechanisms involved in the plant-activation of an arylamine model compound, m-Phenylenediamine. This study showed that bacterial acetyl CoA:N-hydroxyarylamine O-acetyltransferase (OAT) as well as human acetyltransferase (NAT) play a key role in the bioactivation of m-PDA into potent mutagens. The mutagenic potency of the isolated m-PDA metabolites were stable for more than 1 year when stored at 4$\sp\circ$C. Mammalian-activated metabolites of aromatic amines have short half-lives. This suggests that plantactivation mechanisms for arylamines fundamentally differ from that of mammalian activation. S. typhimurium tester strain YG1024 was treated with plant-activated m-PDA metabolites. The genomic DNA was analyzed using the $\sp{32}$P-postlabeling method. DNA adducts were quantified using an ion-pairing reversed-phase HPLC. The data demonstrated that m-PDA metabolites covalently adducted to deoxyribonucleotides in the genome of tester strain YG1024. This formation of plant-activated m-PDA metabolite-DNA adducts may contribute to mutation induction. To define the mutagenic specificity of the plant-activated metabolites of m-PDA, a mutant spectrum analysis was performed. S. typhimurium strain TA98 was treated with the m-PDA metabolites and its genomic DNA was extracted. The target hisD3052 region was amplified via polymerase chain reaction. The DNA preparations were hybridized, probed with chemiluminescence-labeled oligonucleotide probes, and analyzed. The data demonstrated that over 93% of total induced revertants produced a single type of $-$2 frameshift mutation at an alternating CG octamer (D878-885) in the hisD3052 allele. The remaining revertants are believed to have originated as spontaneous mutants. This indicated that plant-activated metabolites of m-PDA were exclusively inducing a single type of frameshift mutation at the target gene.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Microbiology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2011

Author dc:creator

• Seo, Kwang-Young

Contributors dc:contributor

• Plewa, Michael J.

• Biology, Molecular
• Health Sciences, Oncology

Statement dc:rights

• Copyright 1996 Seo, Kwang-Young

Language dc:language

eng

Identifier

9780591199901
AAI9712433
(UMI)AAI9712433

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/19257