Mutagen formation in a model beef supernatant fraction. IV. Properties of the system

To identify the precursors and elucidate the reaction conditions that yield heterocyclic amine mutagens in cooked meat products and fish, we have used a supernatant 2 (S2) fraction prepared from H2O-homogenized lean round steak. Compounds (MW less than 500) in S2 are the sources of the microsomal-dependent, Salmonella TA 1538 mutagenic activity in open boiled (aqueous), 200 degrees C pressure-heated (aqueous), or 200 to 300 degrees C oven-baked (freeze-dried) homogenates. Combined incorporation-HPLC experiments show that they are also the precursors for frameshift mutagen formation in the outer surfaces of 200 degrees C griddle-fried ground beef. Maximal stimulations of boiled S2 mutagenic activity are given by 10 mM Trp, 2.5 mM creatine phosphate (CP), and synergistically by 10 mM Trp + 2.5 mM CP + 1.0 mM FeSO4 (a mixture abbreviated as S2*). Boiling S2 for 30 hr at the acidic optimum pH of 4.0----600 TA 1538 revertants (no additions) and 1,400 revertants (+CP), while S2*----24,000 revertants/10(8) bacteria/g of dry beef. By the criteria of HPLC, paper electrophoresis, and resistance of the active HPLC fractions to acid-nitrite inactivation, boiled S2 contains 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and a minor amount of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ). Boiling S2 with CP doubles the IQ, halves the Trp-P-2, produced traces of MeIQ, and generates an unknown nitrite-resistant mutagen. Boiled S2* contains these same four mutagens, but both the IQ and Trp-P-2 are increased and large amounts of Trp-P-1 also are generated. The identities of IQ, Trp-P-2, and Trp-P-1 were verified by purification and by light-absorption and mass spectra. Their increments in stimulated S2 indicate that Trp (or its degradation products) and CP (or its degradation products) are the beef juice precursors for the indole ring in Trp-P type mutagens and the NH2-imidazole ring in IQ-type mutagens, respectively. Aqueous (pressure) heating or oven-baking S2 for 2 hr at 200 degrees C greatly elevates its TA 1538 activity----45,000 revertants/10(8) bacteria/g of dry beef; dry heating at 300 degrees C----approximately 180,000 revertants/g of dry beef. Along with the increases in total TA 1538 activity at 200 to 300 degrees C, the number of mutagens formed from the less than 500 MW S2 precursors also multiplies.(ABSTRACT TRUNCATED AT 400 WORDS)