DNA breaking activity and mutagenicity of soy sauce: characterization of the active components and identification of 4-hydroxy-5-methyl-3(2H)-furanone.

Soy sauce is a seasoning consumed widely in Southeast Asia. When supercoiled DNA was incubated with soy sauce at pH7.4 and 37 degrees C, extensive breaking of DNA single-strands was caused. It was found that the breaking activity was due to multiple components with different molecular weight and polarity. One of the components with the breaking activity was purified successively by extraction with ethyl acetate, thin-layer chromatography and high performance liquid chromatography, and identified as 4-hydroxy-5-methyl-3(2H)-furanone (HMF), one of the fragrant components in soy sauce. Formation of this component was found due to Maillard reaction of pentoses/amino acids. HMF was readily degraded into the compound with an endiol-ketol structure and reducing activity. Using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in an electron spin resonance-spin trapping technique, generation of hydroxyl radical in an aqueous solution of HMF was confirmed. While DNA breaking by soy sauce was little inhibited by the scavengers of active oxygen radicals, the breaking by HMF was effectively inhibited by superoxide dismutase, catalase, hydroxyl radical scavengers, spin-trapping agents, thiol compounds and metal chelating agents. Hence, DNA breaking activity of HMF was found due to generation of active oxygen radicals. HMF was found mutagenic to Salmonella bacteria without metabolic activation, probably due to generation of active oxygen radicals.