Browning reaction systems as sources of mutagens and antimutagens.

Heated food systems contain hundreds of chemical compounds, some being mutagenic and others being antimutagenic. Studies have indicated that foods exposed to drying, frying, roasting, baking, and broiling conditions possess net mutagenic activity as assessed by the Ames/Salmonella/microsome mutagenicity test and the chromosome aberration assay with Chinese hamster ovary (CHO) cells. With the above-mentioned heat treatment of food, nonenzymic browning reactions are generally proceeding at rapid rates and are involved in the development of mutagens. Caramelization and Maillard reactions are two important pathways in the nonenzymic browning of food and are responsible for the formation of volatile aromatic compounds, intermediate nonvolatile compounds, and brown pigments called melanoidins. Heated sugar-amino acid mixtures possessed mutagenic activities which have been assessed by short-term bioassays. Purified Maillard and caramelization reaction products such as reductones, dicarbonyls, pyrazines, and furan derivatives have exhibited mutagenicity and clastogenicity. The water-insoluble fraction (WIF) of instant coffee and a model-system melanoidin (MSM) have been shown to inhibit the mutagenicity of known carcinogens--aflatoxin B1 (AFB1), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and benzo(a)pyrene (BP)--in aqueous dispersion. WIF and MSM were found to be effective binding agents for the carcinogens.

Induction of mitotic gene conversion by browning reaction products and its modulation by naturally occurring agents.

Mitotic gene conversion in the D7 strain of Saccharomyces cerevisiae was significantly enhanced by exposure to non-enzymatic browning reaction products. These products were formed during the heating of sugar (caramelization reaction) or sugar-amino acid mixtures (Maillard reaction) at temperatures normally used during the cooking of food. Several modulating factors of this convertogenic activity were identified. These factors included two main groups: (1) trace metals which are widely distributed in the environment; and (2) several cellular enzymatic systems. The convertogenic activities of a heated glucose-lysine mixture and a commercial caramel powder were completely suppresses when yeast were concurrently exposed to these products and to either FeIII or CuII. Equimolar concentrations of MnII or sodium selenite had no effect on the convertogenic activity of the products of either model system. Horse-radish peroxidase, beef liver catalase and rat liver S9 preparations each decreased the frequency of gene conversion induced by the caramel powder and the heated glucose-lysine products. This modulating activity of the enzymes was lost if they were heat-inactivated. These studies indicate the presence of a variety of protective mechanisms which can modify genotoxic components in complex food mixtures.

Clastogenic activity of bile acids and organic acid fractions of human feces.

Chloroform extracts of fecal material from 4 subjects on normal mixed western diets were fractionated to obtain an acid fraction and a hexane extract containing neutrals and bases. The acid fraction from at least 2 of the donors induced an elevated frequency of chromosomal aberrations and exchanges in Chinese hamster ovary (CHO) cells. Since acid steroids are expected to be present in the acid fraction, 5 bile acids were assayed for clastogenic activity in CHO cells. Ursodeoxycholic acid induced chromosomal aberrations and exchanges, and this effect was enhanced by the addition of a microsomal S9 mix. However, the enhancement is probably due to physical factors rather than to enzymatic activity.

Antimutagenic activity of browning reaction products.

The Salmonella typhimurium assay was used to determine the antimutagenic effect of products of 2 non-enzymatic browning reactions obtained by heating a lysine-fructose mixture at 121 degrees C for 1 h and by carmelizing D-sucrose at 180 degrees C for 1.5 h. The antimutagenic effect was tested by exposing strain TA1535 in suspension to N-methyl-N' -nitro-N-Nitrosoguanidine (MNNG) in the presence of the browning reaction products. In the case of aflatoxin B1, strain TA98 was used and the browning reaction products were added to the precarcinogen and an S9 mixture. The mutagenic activity of both carcinogens was significantly suppressed by the browning reaction products.

The action of transition metals on the genotoxicity of simple phenols, phenolic acids and cinnamic acids.

Simple phenols (catechol, 4-methyl catechol, resorcinol, phloroglucinol and pyrogallol), phenolic acids (p-hydroxybenzoic acid, protocatechuic acid, vanillic acid, gallic acid, syringic acid and salicylic acid), a phenylacetic acid (3,4-dihydroxyphenylacetic acid) and eugenol were assayed for clastogenic activity in Chinese hamster ovary (CHO) cells with and without the addition of a n S9 mixture, Cu2+ (10-4M) and Mn2+ (10-4M). All dihydroxylated and trihydroxylated phenolics induced chromatid breaks and exchanges. The introduction of a methyl group seems to reduce the clastogenic capacity. The addition of an S9 mixture or the transition metals Cu2+ and Mn2+ enhanced the chromosome-damaging activity in some phenolics and suppressed it in others.

A comparative genotoxicity study of chlorogenic acid (3-0-caffeoylquinic acid).

Chlorogenic acid, a compound which occurs naturally in many food items, was assayed for genotoxic activity in 3 different test systems: reverse mutations in the preincubation test with Salmonella typhimurium, gene conversion with Saccharomyces cerevisiae strain D7, and chromosome aberrations in Chinese hamster ovary (CHO) cells. Chlorogenic acid was directly convertogenic and clastogenic, but lacked a mutagenic capacity in the Salmonella bioassay. The transition metal Mn2+ enhanced the clastogenic and convertogenic activity of chlorogenic acid. In the presence of Mn2+ (10(-4)M), chlorogenic acid increased the frequency of his+ revertants in TA98 and TA100 strains of S. typhimurium. Caffeic acid and, to a lesser degree, quinic acid, which are components of chlorogenic acid, also showed genotoxic activity. The results show the importance of using several assays in combination with transition metals when testing for genotoxicity.

Clastogenicity of furans found in food.

Cultured Chinese hamster ovary (CHO) cells were exposed for 3 h to furan and 6 furan derivatives (furfural, furfuryl alcohol, 5-methyl furfural, 2-methyl furan, 2,5-dimethyl furan and 2-furyl methyl ketone). Each of the 6 furan derivatives induced a relatively high frequency of chromatid breaks and chromatid exchanges in the absence of a liver microsomal activation preparation. The response of the furans to the addition of an S9 mixture differed. The clastogenic activities of 5-methyl furfural, 2-furyl methyl ketone, furfural and furfuryl alcohol were increased, whereas that of 2-methyl furan and 2,5-dimethyl furan were significantly decreased. Furan itself showed a clastogenic activity only in the presence of an S9 mixture.

Clastogenic activity of dried fruits.

The clastogenic activities of several commercially-dried fruits, including black and golden-seedless raisins, medium-sized California prunes, table dates, bananas, California black mission figs and breakfast apricots, were examined using Chinese hamster ovary (CHO) cells as the test organism and chromosome aberrations as the endpoint. Treatment of the CHO cells with water extracts of these dried fruits significantly increased the frequencies of metaphase plates with 1 chromosome break or exchange as well as the average number of chromosome exchanges per metaphase plate. A liver microsomal S9 mixture reduced this clastogenic activity. Dried fruits represent an example of widely consumed food products with strong genotoxic activities.

Clastogenic activity of caramel and caramelized sugars.

Cultured Chinese hamster ovary (CHO) cells were exposed for 3 h to caramelized solutions of the sugars sucrose, glucose, mannose, arabinose, maltose and fructose. Each of these caramelized sugars induced a relatively high frequency of chromosome breaks and exchanges in the treated cells. The non-caramelized sugars did not increase the frequency of chromosome aberrations. A potent clastogenic effect was also observed when a commercially used caramel powder was assayed. Up to 54% of all examined metaphase plates of the treated CHO cells had at least one chromosome break or exchange. This chromosome-damaging action of commercial caramel powder was reduced in the presence of liver microsomal (S9) preparation or FeII and FeIII. The transition metals CuII and MnII neither enhanced nor reduced the clastogenic activity of the caramel powder.

Rapid Method for Detection and Quantitation of Lipid Soils on Food Contact Surfaces: Evaluation of a Model System.

A rapid method for detection and quantitation of lipid-containing food soils on food-contact surfaces has been developed to ascertain whether these surfaces have been properly cleaned. The method is based on transfer of lipid-based soils from a food-contact surface to a polyethylene film and subsequent quantitation of the lipid, at 1750 cm-1, by infrared spectrophotometry. Peak height at 1750 cm-1 is linearly related to the quantity of lipid on the polyethylene surface. Standard curves for peak-height against lipid distribution on the polyethylene film were constructed for stainless steel, glass and three types of plastic cutting board material (high density polyethylene, smooth nylotrol and rough nylotro).

Synthesis, purification and characterization of 7-ketocholesterol and epimeric 7-hydroxycholesterols.

Various methods were assessed for the synthesis of 7-ketocholesterol and epimeric 7-hydroxycholesterols. Upon the oxidation of cholesteryl acetate with t-butyl chromate, the resulting ketosterol acetate crystallized from methanol consisted of about 25% unoxidized cholesteryl acetate. After the sterol acetates were hydrolyzed in an aqueous K(2)CO(3) medium, preparative TLC was used to fractionate the ketone from cholesterol.Of all the reducing agents employed, only LiAlH(4) reduced completely the purified 7-ketocholesterol to 7-hydroxycholesterols without side reaction products and ketone contamination. Yields of 21.3 mg of 7alpha-hydroxycholesterol and 72.6 mg of 7beta-hydroxycholesterol were obtained by preparative TLC of a diol mixture prepared by the LiAlH(4)-reduction of 100 mg of 7-ketocholesterol. To accomplish the preparative TLC separation of diol bands without overlapping, a double development of a chromatoplate with ethyl ether-cyclohexane (90ratio10, v/v) and ethyl ether was essential.Data on the melting point, optical rotation and infrared spectra, as well as TLC and GLC characteristics, were obtained for purified 7-ketocholesterol and epimeric 7-hydroxycholesterols.

Isolation and characterization of cholesterol-5B,6B-oxide from an aerated aqueous dispersion of cholesterol.

An unknown autoxidation product in an aerated cholesterol sol was isolated by preparative thin layer chromatography. This compound was identified as cholesterol-5beta,6beta-oxide by gas liquid chromatography along with infrared and mass spectrometry.