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An ethanol extract of Piper auritum leaves (PAEE) inhibits the mutagenic effect of three food-borne aromatic amines (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP); 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx); 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx)) in the TA98 Salmonella typhimurium strain. Preincubation with MeIQx demonstrated in mutagenesis experiments that inhibition of Cytochrome P450 (CYP), as well as direct interaction between component(s) of the plant extract with mutagens, might account for the antimutagenic observed effect. Gas chromatography/mass spectrometry analysis revealed that safrole (50.7%), α-copaene (7.7%), caryophyllene (7.2%), β-pinene (4.2%), γ-terpinene (4.1%), and pentadecane (4.1%) as the main components (PAEE). Piper extract and safrole were able to inhibit the rat liver microsomal CYP1A1 activity that participates in the amines metabolism, leading to the formation of the ultimate mutagenic/ molecules. According to this, safrole and PAEE-inhibited MeIQx mutagenicity but not that of the direct mutagen 2-nitrofluorene. No mutagenicity of plant extract or safrole was detected. This study shows that PAEE and its main component safrole are associated with the inhibition of heterocyclic amines activation due in part to the inhibition of CYP1A subfamily activity., (© The Author(s) 2024. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.)

Bacterial nitroreductases (NRs) catalyse the oxygen-insensitive reduction of several nitro-substituted compounds and quinones. SnrA and cnr NRs have been previously identified in Salmonella enterica serovar Typhimurium; they reduce several environmental nitro compounds that display mutagenic activity in the Ames test. Although some of their biochemical properties have been reported, the substrate specificity of each protein over mutagenic nitro compounds is unknown; even more, the possible relationship between their capacity to activate nitro compounds into mutagens and the redox properties of putative substrates has been poorly investigated. We have purified SnrA and cnr and investigated their capacity to activate several mutagens in the Ames test as well as their kinetic parameters K(m) and V(max). Our results show that SnrA and cnr are able to activate 2,7-dinitrofluorene with the same efficiency and a similar mutagenic potency in the YG7132 tester strain; 1-nitropyrene and 1,3-dinitropyrene were efficiently activated by cnr, whereas 1,8-dinitropyrene, 1,6-dinitropyrene and 2-nitrofluorene were scarcely activated by either NR. The mutagenic potency of nitro compounds obtained in the presence of either enzyme correlates with their redox potential reported in the literature. On the other hand, a good correlation was obtained between the catalytic efficiency (V(max)/K(m)) of the purified cnr with the redox potential of eight molecules including nitro-substituted compounds and quinones. No correlation between redox potential and catalytic efficiency by SnrA was observed, suggesting that factors other than redox potential such as the structure of the compounds are involved in the catalytic efficiency of SnrA.

A characterization of nitrocompounds activation by cell-free extracts (CFE) of wild-type (AB(+)), SnrA deficient (B(+)), Cnr deficient (A(+)) and SnrA/Cnr deficient (AB(-)) Salmonella typhimurium strains has been done. The Ames mutagenicity test (S. typhimurium his(+) reversion assay) was used, as well as nitroreductase (NR) activity determinations where the decrease in absorbance generated by nitrofurantoin (NFN) reduction and NADP(H) oxidation in the presence of NFN, nitrofurazone (NFZ), metronidazole (MTZ) and 4-nitroquinoline-1-oxide (4NQO) were followed. Different aromatic and heterocyclic compounds were tested for mutagenic activation: 2-nitrofluorene (2-NF); 2,7-dinitrofluorene (2,7-DNF); 1-nitropyrene (1-NP), 1,3-dinitropyrene (1,3-DNP); 1,6-dinitropyrene (1,6-DNP); and 1,8-dinitropyrene (1,8-DNP). Differential mutagenicity was found with individual cell free extracts, being higher when the wild type or Cnr containing extract was used; nevertheless, depending on the nitrocompound, activation was found when either NR, SnrA or Cnr, were present. In addition, all nitrocompounds were more mutagenic after metabolic activation by CFE of NR proficient strains, although AB(-) extract still showed activation capacity. On the other hand, NR activity was predominantly catalyzed by wild type CFE followed by A(+), B(+) and AB(-) extracts in that order. We can conclude that results from the Ames test indicate that Cnr is the major NR, while NFN and NFZ reductions were predominantly catalyzed by SnrA. The characterization of the residual NR activity detected by the mutagenicity assay and the biochemical determinations in the AB(-) CFE needs further investigation.