Esther Fultz

Effect of microwave pretreatment on heterocyclic aromatic amine mutagens/carcinogens in fried beef patties

To investigate a method to reduce the amount of mutagenic/carcinogenic heterocyclic aromatic amines formed during frying of ground beef, the mutagenic activity in Salmonella strain TA98 was assessed and the amount of known heterocyclic amines was determined by solid-phase extraction and HPLC. The beef patties received microwave treatment for various times before frying. Microwave pretreatment for 0, 1, 1.5, 2 or 3 min before frying at either 200 degrees C or 250 degrees C for 6 min per side reduced heterocyclic aromatic amine precursors (creatine, creatinine, amino acids, glucose), water, and fat up to 30%, in the patties and resulted in a decrease in mutagenic activity up to 95%. The sum of the four heterocyclic aromatic amines shown to be present--2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4,8-trimethylimidazo-[4,5-f]quinoxaline (DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)--decreased three- to nine-fold compared with control, non-microwaved beef patties fried under identical conditions.

Mutagen formation in a model beef boiling system. III. Purification and identification of three heterocyclic amine mutagens-carcinogens

Abstract An extensively boiled supernatant2 (S2) fraction from beef round steak contains two major Salmonella frameshift mutagens, designated as HPLC peaks A and B. These same mutagens arise, but in different proportions, when S2 is boiled with creatine phosphate (CP), and they are produced in much greater quantities from a boiled mixture of S2 + L‐tryptophan (Trp) + CP + FeSO4 (S2 ∗). A third mutagen, peak C, is also generated in the S2 ∗ mixture. Mutagen peaks A, B, and C were purified to homogeneity and shown by their absorption spectra, mass fragmentation patterns, and other data to be 2‐amino‐3‐methylimidazo[4,5‐f]quinoline (IQ), 3‐amino‐l‐methyl‐5H‐pyrido[4,3‐b]‐indole (Trp‐P‐2), and 3‐amino‐l,4‐dimethyl‐5H‐pyrido[4,3‐b]indole (Trp‐P‐1), respectively. This is the first demonstration that IQ, Trp‐P‐2, and Trp‐P‐1 can form under aqueous conditions at 100°C from reactions between low molecular wt precursors that are present in meat juice. Further simplification of and studies with the S2 fraction of be...

14C AMS quantification of biomolecular interactions using microbore and plate separations

AMS sensitivity arises from the direct counting of radioisotopes without interference from molecular isobars. No chemical or physical information other than a bulk isotope ratio is available from the usual AMS instrument. Chemical or biological significance of the isotope ratio depends on the definition of the sample prior to conversion to material used in the ion source. The authors use AMS to quantify biochemical interactions between labeled xenobiotics and their potential targets of toxicity. These potential target molecules are separated and defined by various types of plate and microbore separations, including thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and gel electrophoresis (GE) in quantifying the binding of {sup 14}C-labeled compounds to specific DNA and protein fragments. They discuss their methods of using these microbore and plate separations of biomolecules while controlling contamination from {sup 14}C in laboratory equipment and give examples.

Mutagen formation in a model beef boiling system. I. Conditions with a soluble beef-derived fraction

Abstract Fried beef and commercial beef extract contain Ames/ Salmonella frameshift mutagens that require microsomal (S‐9) activation. To ascertain which fraction(s) of beef muscle contain(s) the essential precursors, aqueous (1:1) homogenates of lean round steak were centrifuged to give an insoluble residue1 (R1) and a soluble supernatant1 (S1). S1 was then boiled for 30 min and again centrifuged, yielding a residue2 (R2) and a soluble supernatant2 (S2). S2 represents only 5% of the meat dry wt and it contains only 10∗ of the H2O‐soluble protein, but it contributes all of the S‐9 dependent Salmonella TA1538 mutagenic activity in boiled homogenates. Mutagen formation from S2 boiled for 0–30 h at a constant volume increases exponentially with time and displays sharp pH optima at 4.0 and 9.0. By molecular ultrafiltration the pH 4.0 mutagen precursors in S2 have molecular wts < 500. They are also stable to lyophilization. These observations and the disappearance of certain amino acids upon boiling at pH 4.0 ...

Mutagen formation in a model beef boiling system II. Effects of proteolysis and comparison of soluble fractions from several protein sources

Abstract Fried beef and commercial beef extracts contain Ames/Salmonella frameshift mutagens that form at relatively low temperatures (100–200°C). To investigate the types of natural components in beef muscle that give rise to frameshift mutagenic activity, we previously devised a model boiling system and demonstrated that all of the Salmonella TA1538 activity is formed from H2O‐soluble, < 500 molecular wt compounds that are present in round steak supernatant fractions (S1 and S2). S2 is derived from S1, the soluble fraction of homogenized beef, by a brief 30 min boil and centrifugation. We now report that proteolysis of beef S1 with papain, trypsin, or chymotrypsin (± carboxypeptidase A) increases the mutagenic activity of boiled S2 by 1.8–4.5 fold over the baseline range of 90–100 TA1538 revertants/108 bacteria/g dry beef/14 h at pH 4.0. Sequential treatment of beef S1 with chymotrypsin followed by carboxypeptidase A is the most efficient mutagen enhancing digestion (415 revertants/g dry beef or 7.7 rev...

Beef Supernatant-Fraction-Based Studies of Heterocyclic Amine-Mutagen Generation

To characterize the reaction conditions that generate frameshift mutagens in cooked meats, we have concentrated on a supernatant fraction (S2) prepared from (1:1) aqueous homogenates of lean round steak. Soluble compounds <500 MW 1n S2 are the sources of the S-9 dependent Salmonella TA1538 mutagenic activity 1n these homogenates, Irrespective of how they are heated (100°C boiled, 200–300°C aqueous-pressure, or 200–300°C dry), as well as the outer surfaces (crust) of griddle-fried ground beef. Water is an important inhibitory reactant that influences not only the total TA1538 activity, but also the proportions of HPLC-polar, nitrite-resistant 2-amlno-3-methyl-imidazo-type mutagens, as opposed to HPLC-nonpolar, nitrite-sensitive mutagens. Dry-state heating beef S2 favors the former. It yields eight of the heterocylic amine-mutagens that have been identified in the surfaces of 100g beef patties fried at 250–300°C, including 2-amlno-3-methylimldazo [4,5-f]quinoline (IQ), 2-amino-3-8-dimethylimidazo[4,5-f]qulnoxaline (NeIQx), and the predominant mutagen 2-amino-l-methyl-6-phenylimidazo [4,5-b]pyridinea (PhIP). Oven-baking the amounts of L-phenylalanine (Phe) and creatine (Cr) present in 100g (raw beef) equivalents of S2 yields sufficient IQ and PhIP to accomodate the ppb quantities reported for high temperature fried beef patties. Dry-heating with heavy-isotope-labeled forms of Phe and Cr shows precisely how their C and N atoms are incorporated into PhIP. Our findings indicate that phIP and other 2-amino-3-methyl-imidazo-mutagens most likely arise in 250–300°C fried beef patties independent of Maillard reactions.