Howard F. Mower

Fecal mutagenicity arising from ingestion of fried ground beef in the human

Fried ground beef has been shown to contain mutagens, and the major mutagenic component has been identified as 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). Mutagens in feces of 3 adult volunteers were fractionated by treatment of the feces with blue cotton followed by chromatography on a carboxymethyl cellulose column. The chromatographic fraction, corresponding to MeIQx in terms of the position of elution, was examined for mutagenicity in S. typhimurium TA98 with metabolic activation. When meals containing no heated meat were eaten, this fraction of feces showed little or no mutagenicity. On eating fried ground beef, the feces excreted in the next two days showed greatly increased mutagenicity in this fraction. By eating no-meat meal subsequent to the meat meal, the mutagenicity resumed the original low level on the fourth day after the meat meal. The components in the mutagenic fraction were probably metabolites of the mutagens present in cooked meat, since analysis by high pressure liquid chromatography of the mutagenic fraction showed that the active components in the feces were different from the mutagens in cooked meat.

The use of rec− bacteria for testing of carcinogenic substances

A series of rec-Escherichia coli strains were tested for their sensitivity to four known carcinogenic compounds by examination of a zone of inhibited bacterial growth around a central well containing the test chemical. The mutants recA-, recB-, recC-, and recA- recB- recC- were all more sensitive to the mutagens than the parental strain AB1157. The recB- recC- strain was examined with a larger series of compounds and was found to respond to many of the substances in a similar way as the Salmonella typhimurium strains of Ames but with some notable exceptions. Nitrosamines, with rat liver microsomal activation, could be detected at lower levels and a group of aromatic amino compounds failed to react with these rec-E. coli. An unusual feature of these rec-mutants is their sensitivity to mixtures of nitrosamines and 2-acetyl amino-fluorene in the absence of microsomal activation.

The mutagenic propeeties of hexachloroacetone in short-term bacterial mutagen assay systems

Hexachloroacetone, CCl3--CO--CCl3, reverts the Ames strains TA98 and TA100 but not the non-plasmid strains TA1537, TA1535 and TA1538. In the absence of solvent, the number of revertant colonies is 5 times the spontaneous reversion rate for TA100 and 10 times the spontaneous reversion rate for TA98 with 26 mg hexachloroacetone per plate. This effect is seen in the absence of rat liver microsomes. In dimethylsulfoxide (DMSO) solution a more complicated pattern is seen. In DMSO solution cooled between 18 and 20 degrees C, The maximum nuber of revertants is similar to that found in the absence of DMSO, but only 1.75 mg hexachloroacetone per plate is needed. When DMSO solution of hexachloroacetone is warmed above 20 degrees C, a yellow color develops and the solution becomes more toxic to the test bacteria. The maximum number of revertants is then produced at about 0.5 mg hexachloroacetone per plate. Hexachloroacetone is found to be active, without microsomal activation, in the E. coli WP-2 and E. coli rec-BC test systems. Hexachloroacetone readily reacts with water in DMSO solutions to form the non-mutagenic hexachloroacetone hydrate.

Free radical involvement in long wavelength UV light activation of nitrosamines to mutagens

Nitrosamines are carcinogenic and mutagenic only after metabolic activation via endoplasmic reticulum bound mixed function oxidase enzyme systems. Rencently a new photochemical process has been discovered by which nitrosamines are converted into unknown mutagenic compounds by irradiation with long wavelength UV light (> 335 nm) in the presence of phosphate ion at neutral pH. The mutagenic activity is detected by Ames Salmonella Typhimurium strain TA100 in the absence of rat liver microsomes. We have shown that mutagen production with nitrosomorpholine is inhibited in the presence of light by various spin trapping agents (N-t-butyl-phenylnitrone, etc.). Concurrent with this inhibition a stable free radical signal has been detected whose kinetics of formation is similar to the time course of mutagen formation during irradiation in the absence of spin trap. The free radical signal is formed only when phosphate or similar ions are present in the reaction mixture. Monomethylphosphate and dimethylphosphate can substitute for phosphate ion but with small ESR signals and mutagen formation. Trimethylphosphate gives a weak, time independent ESR signal and does not cause mutagen formation. The ESR splitting constants (aN and aH) for signals generated with each of the different phosphate species show differences which suggest that these ions may be components of some intermediate free radical species that is involved in stable mutagen formation. Arsenate ion inhibits mutagen formation in the presence of phosphate but is able in the absence of phosphate to form a ESR signal similar to that observed with phosphate ion.

Identification of DNA adducts at specific locations by sequencing techniques

1. A simple procedure for the accurate identification of the positions of adduct formation when viral M13mp18 single stranded DNA is treated with chloroacetaldehyde (CAA) is presented. 2. The normal dideoxy sequencing reaction protocol was employed except no dideoxy nucleotides were used to cause chain termination. 3. Instead, the CAA adducted bases of the template DNA act as points of polymerase fall off causing the appearance of bands of DNA sequencing gel autoradiographs marking locations where adduct formation may have occurred.

CHARACTERIZATION OF THE REACTION OF CHLOROACETALDEHYDE WITH SINGLE AND DOUBLE STRANDED DNA

Abstract Chloroacetaldehyde reacts with viral M13mp18 single and double stranded DNA to form the highly fluorescent adducts, etheno-AMP and etheno-CMP. Absorbance and fluorescence spectroscopy can be used to monitor and characterize these reactions. Both single and double stranded DNA showed increases in the absorbance following reaction with the aldehyde. The fluorescence also increased in these two groups and continued to rise with increasing time of incubation until a point of saturation was reached. The fluorescence of the double stranded moiety was considerably enhanced following reaction with the aldehyde while that of the single stranded population was not, making this method appropriate for the separation of small quantities of the two populations of DNA.

Detection of small quantities of double stranded DNA by enhanced fluorescence of substituted aldehyde modified polydeoxynucleotide-ethidium bromide complexes

A method for the detection of small amounts of double stranded DNA by a simple incubation procedure involving the reaction of the nucleotide bases in DNA with chloroacetaldehyde is described. Following incubation, the presence of DNA may be visualized by the orange fluorescence emitted when ethidium bromide (EtBr) is added. Alternatively, the aldehyde/DNA mixture may be separated by agarose gel electrophoresis and individual double stranded DNA visualized by exposing the gel to ethidium bromide and observing the orange ultraviolet light induced fluorescence of the aldehyde modified double stranded ethidium bromide complex (Waring, M.J. (1965) J. Mol. Biol. 13, 269-282). At least a 20-fold increase in the detection of double stranded DNA is possible by this procedure. An important aspect of this method is that the visualization of single stranded DNA is not enhanced.