Differing activation pathways for 2-acetylaminofluorene to a mutagen in vitro

Area of Science:

• Biochemistry
• Toxicology
• Genetics

Background:

• 2-acetylaminofluorene (AAF) is a known mutagen.
• The Ames test is a standard assay for detecting mutagenicity.
• Liver post-mitochondrial supernatant (S9) fractions are used to mimic metabolic activation in mutagenicity testing.

Purpose of the Study:

• To investigate the mutagenicity of AAF in Salmonella typhimurium TA 1538.
• To characterize the role of rat and cotton rat liver sub-fractions (microsomes and S100) in AAF activation.
• To identify key cofactors and inhibitors involved in the metabolic activation of AAF.

Main Methods:

• Ames test using Salmonella typhimurium TA 1538.
• Sub-fractionation of rat and cotton rat liver S9 into microsomal (M) and 100,000 X g supernatant (S100) fractions.
• Investigation of cofactor (NADPH, NADH) and inhibitor (7,8-benzoflavone, paraoxon) effects on AAF activation.

Main Results:

• Rat liver S9-mediated AAF activation was primarily associated with the S100 fraction.
• Cotton rat liver S9-mediated AAF activation was predominantly linked to the S9 fraction, exceeding the sum of its components.
• Microsomal fractions were the dominant determinant of S9 properties in both species, with NADPH as the principal cofactor, largely replaceable by NADH.
• 7,8-Benzoflavone inhibited activation in both M and S100 fractions, while paraoxon affected cotton rat M activation but not rat S100 activation.

Conclusions:

• Species-specific differences exist in the metabolic activation of AAF.
• Microsomal fractions play a crucial role in AAF activation across species.
• NADPH is the primary cofactor, but NADH can substitute, and specific inhibitors highlight distinct activation pathways.