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Salmonella typhimurium strain TA100 differentiates several classes of carcinogens and mutagens by base substitution
W H Koch1, E N Henrikson, E Kupchella
1Molecular Biology Branch, Food and Drug Administration, Washington, DC 20204.
Abstract:
The mutational specificity of N-methylnitrosourea (MNU), nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), sodium azide (NaN3), 4-nitroquinoline oxide (4NQO), benzo[a]pyrene (BP), nitrofurantoin (NF), aflatoxin B1 (AFB1), adriamycin (ADM) and UVA-activated angelicin in Salmonella typhimurium strain TA100 has been examined using allele-specific oligonucleotide hybridization and DNA sequence analyses. These ten mutagens produced five unique classes of reversion spectra, distinct from spontaneous, or the previously characterized 5-azacytidine, ultraviolet light (UV), 8-methoxypsoralen plus UVA (PUVA) and 60Co-induced mutation spectra. For example, 90% of MNU and MNNG-induced mutations in strain TA100 revertants were G:C-->A:T transitions with the majority (82%) occurring in the first position of the CCC codon. In contrast, NaN3 preferentially induced G:C-->A:T transitions at the second codon position (78%). Although MMS, NQO, BP, NF, ADM and AFB1 induced primarily G:C-->T:A transversions (73-86%), these mutagens fall into two classes based on site preference: NF and AFB1 yielded almost exclusively position two transversions (69-78%) whereas ADM, NQO, BP and MMS exhibited a two-fold preference for site 2 over site 1 (on average 52% versus 22%). Angelicin photomutagenesis resulted in the recovery of G:C-->A:T and G:C-->T:A mutations at both codon positions in roughly equal proportions (approximately 20-25% each). Approximately 1% of the mutagen-induced revertants occurred via extragenic tRNA suppressor mutations, while 1% were multiple (usually tandem double) base substitutions. Ultraviolet mutagenesis experiments demonstrated that tandem base substitutions are promoted by pKM101-encoded mucAB gene products. A comparison of the mutagenic specificity derived for several carcinogens in hisG46 with the responses of several eukaryotic gene targets (e.g. HPRT, aprt, supF) revealed a high concordance between these targets. Thus, the Salmonella hisG46 locus provides a rapid, simple system for determining base substitution specificity and for studying mechanisms of mutagenesis.
Insights
This study details the mutational specificity of ten chemicals and UVA-activated angelicin in Salmonella typhimurium. The findings reveal unique mutation patterns, aiding in understanding mutagenesis and carcinogen mechanisms.
Area of Science:
- Molecular Biology
- Genetics
- Toxicology
Background:
- Understanding the specific DNA alterations caused by mutagens is crucial for assessing their carcinogenic potential.
- The Salmonella typhimurium hisG46 locus is a well-established system for studying mutagenesis.
- Previous studies have characterized mutation spectra for various agents, but a comprehensive analysis of several common mutagens was needed.
Purpose of the Study:
- To determine the mutational specificity of ten mutagens, including N-methylnitrosourea (MNU) and benzo[a]pyrene (BP), in Salmonella typhimurium strain TA100.
- To compare the observed mutation spectra with those induced by other agents and known carcinogens.
- To evaluate the utility of the Salmonella hisG46 locus as a model for studying base substitution specificity and mutagenesis mechanisms.
Main Methods:
- Utilized allele-specific oligonucleotide hybridization and DNA sequence analyses to examine mutations in Salmonella typhimurium strain TA100.
- Tested ten mutagens: N-methylnitrosourea (MNU), nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), sodium azide (NaN3), 4-nitroquinoline oxide (4NQO), benzo[a]pyrene (BP), nitrofurantoin (NF), aflatoxin B1 (AFB1), adriamycin (ADM), and UVA-activated angelicin.
- Compared resulting mutation spectra to spontaneous mutations and those induced by ultraviolet light (UV) and other agents.
Main Results:
- Ten mutagens generated five distinct classes of reversion spectra, differing from spontaneous and previously characterized mutations.
- MNU and MNNG predominantly caused G:C-->A:T transitions, while NaN3 induced G:C-->A:T transitions at a different codon position.
- MMS, 4NQO, BP, NF, ADM, and AFB1 primarily induced G:C-->T:A transversions, with variations in site preference.
Conclusions:
- The Salmonella hisG46 locus effectively distinguishes the mutational specificity of various chemical mutagens and UV radiation.
- A high concordance was observed between the mutagenic specificity in Salmonella and eukaryotic gene targets, suggesting its utility for carcinogen assessment.
- The study provides a rapid and simple system for determining base substitution specificity and elucidating mutagenesis mechanisms.