Spiros Grivas

Formation of heterocyclic amines using model systems.

Initially, modeling was used to identify the mutagenic heterocyclic amines and their precursors. Major precursors have been shown to be single amino acids or amino acids together with creatine or creatinine. There is also evidence that Maillard reactions are involved since heating sugar and amino acids together with creatine or creatinine has been shown to produce several of the mutagenic heterocyclic amines, especially the aminoimidazoazaarenes (AIA compounds), e.g., IQ, MeIQ, MeIQx, DiMeIQx and PhIP. Due to a low yield in the model systems, the mechanisms behind the formation of the mutagenic heterocyclic amines are still unclear and need further substantiation. The fact that some AIA compounds are also produced in the absence of sugar casts some doubts on an obligatory participation of the Maillard reaction; alternative routes might exist. Further work using isotopically labeled precursors needs to be done and so far such work has only been performed for PhiP. The formation of mutagenic heterocyclic amines is dependent on time, temperature, pH, concentration of the precursors, type of amino acid, and the presence of certain divalent ions. Water may have an impact both as a temperature regulator and as a solvent medium for the reactants.

Creatin(in)e and Maillard reaction products as precursors of mutagenic compounds: Effects of various amino acids

Abstract The participation of creatin(in)e and Maillard reaction products in developing mutagenic activity was studied in model systems. Glucose and an amino acid were boiled under reflux for 2 h at 130°C together with creatine or creatinine dissolved in water-diethylene glycol (1:6). Threonine produced the highest mutagenic activity, 1068 revertants per μmol amino acid, towards TA98 after S9 activation, followed by glycine (410 rev/μmol) and lysine (246 rev/μmol). Proline, glutamic acid and the sulfur-containing amino acids produced less than 40 rev/μmol. Protein-bound amino acids produced no detectable mutagenic activity. When added to the reaction mixtures, the pure Maillard reaction products increased the mutagenic activity significantly. All precursors used occur in free form in meat. Work is now in progress to identify the mutagenic compounds produced in the model systems and to establish whether they also occur in fried meat.

Formation of a new mutagenic DiMeIQx compound in a model system by heating creatinine, alanine and fructose

A mixture of creatinine, D-fructose and DL-alanine was heated in diethylene glycol containing 14% water for 2 h at ca. 128 degrees C. The mutagens formed were extracted with 1-butanol, and purified by cation-exchange column chromatography, C18 reversed-phase Sep-Pak treatment and reversed-phase HPLC. According to its UV absorption, mass and 1H NMR spectra, one isolated fraction was tentatively assigned the chemical name, 3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxalin-2-amine (4,8-DiMeIQx). This finding is in agreement with the suggestion that sugars, amino acids and creatinine present in meat may be the precursors of the mutagenic imidazoquinolin- and imidazoquinoxalin-2-amines (IQ compounds).

Isolation and identification of the food mutagens IQ and MeIQx from a heated model system of creatinine, glycine and fructose

Abstract A mixture of creatinine, d -fructose and glycine was heated in diethylene glycol containing 14% water for 2h at ca . 128°C. The reaction mixture was extracted with 1-butanol and the extract subjected to cation exchange column chromatography, C 18 reversed-phase Sep-Pak treatment and reversed-phase HPLC. The first of the two HPLC fractions isolated was identified as the known beef mutagen, MeIQx. In the second HPLC fraction, MeIQx and the also known potent fish and beef mutagen, IQ, were co-eluted. HPLC, mass and 1 H NMR spectrometry were used for their identification. These results reinforce the hypothesis that creatinine, sugars and amino acids might be the precursors of these extremely potent mutagens.

Analysis of mutagenic imidazo[4,5-f]quinolines and -quinoxalines (IQ compounds): comparison of electrochemical and ultraviolet detection

Two synthetic imidazoquinolin-2-amines (IQ and MeIQ) and two imidazoquinoxalin-2-amines (MeIQx and 4,8-DiMeIQx), all known potent mutagens, have been separated by reversed-phase HPLC and detected by two methods - UV detection and electrochemical (EC) detection. The limits of detection were found to be 2.5 pmoles for UV detection and 0.5-1.5 pmoles for electrochemical detection.

One-Step Synthesis of 2-Amino-1-methylimidazo[4,5-b]quinoline

Abstract The novel title compound, a linear isomer of the food mutagen IQ, has been prepared in 67 % yield by Friedlander synthesis from creatinine and 2-aminobenzaldehyde.

Binding of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to protein- and low molecular weight thiols and its role in ring hydroxylation.

The N-oxidized species of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) have been shown to react with thiols. We have previously characterized a glutathione conjugate of PhIP linked via the C2 of PhIP with apparent loss of the amino group, in rat hepatocytes and PhIP exposed rats. This metabolite was possibly formed from 1-methyl-2-nitro-6-phenylimidazo[4,5-b]pyridine (nitro-PhIP). Upon reacting nitro-PhIP with rat albumin, both in the presence and absence of a reducing system, four products were observed after enzymic proteolysis. One of them was markedly increased after 2-mercaptoethanol treatment of the protein. This adduct was linked to a cysteine-S via C2 of PhIP. Using N2-acetoxy-PhIP as a starting material, an unstable protein adduct was observed which degraded to 50% of the original concentration (t 1/2) after 3 days. This is compatible with the finding that serum PhIP adducts decline rapidly in PhIP exposed rats. Unstable adducts were also formed following the reaction of N2-acetoxy-PhIP with glutathione or cysteine. Based on mass spectroscopy and UV spectra analysis, the suggested structures were RS(-S-)-(H)N2-PhIP. In all cases a degradation product identified as 5-hydroxy-PhIP was formed as characterized by mass spectrometry and NMR spectroscopy. 5-hydroxy-PhIP and its glucuronyl derivative were also observed in rat hepatocytes incubated in vitro with PhIP. In bile of PhIP-exposed rats, only the glucuronyl derivative was observed. Depletion of glutathione reduced the amount excreted in bile and experiments with microsomes indicate that hydroxylation directly at the 5 position is not mediated by cytochrome P-450 mono-oxygenase system. This indicates that 5-hydroxy-PhIP may be formed from N-acetoxy-PhIP via binding to thiols also in cells.

Mutagenic activity of the methyl and phenyl derivatives of the food mutagen 2-amino-3-methylimidazo[4,5-f]quinoxaline (IQx) in the Ames test

The mutagenic activity of 15 different mono-, di-, tri-, and tetramethyl derivatives of the food mutagen IQx (2-amino-3-methylimidazo[4,5-f]quinoxaline), one diphenyl derivative of IQx and two phenyl derivatives of 5-MeIQx (2-amino-3,5-dimethylimidazo[4,5-f]quinoxaline) were studied in the Ames test with Salmonella typhimurium TA98 and enzymatic activation (S9). The number and positioning of the methyl groups strongly affected the mutagenic activity. The phenylated compounds showed weak mutagenic potency. It seems that both resonance stabilization of the nitrenium ion and steric effects are important in determining mutagenic potency.

The synthesis and mutagenicity of the 3-ethyl analogues of the potent mutagens IQ, MeIQ, MeIQx and its 3,7-dimethyl isomer

The title compounds were synthesized and tested for mutagenicity on Salmonella typhimurium TA98 in the presence of S9 mix. All test compounds showed lower activity than their respective 3-methyl analogues (IQ compounds). The replacement of the 3-methyl by an ethyl group should not alter the chemistry of these compounds; hence, their lower mutagenic activity could merely be of steric origin.

Formation of 4,8-DiMeIQx from the model system fructose, alanine and creatinine Comparison with the isomeric 5,8-DiMeIQx

In a previous paper, the main mutagenic compound isolated from the model reaction system D-fructose, DL-alanine and creatinine was tentatively identified as 4,8-DiMeIQx. Its mutagenic activity and spectral characteristics have now been compared with those of the isomer 5,8-DiMeIQx. The comparison clearly demonstrates that the isolated compound was indeed 4,8-DiMeIQx. This finding is in agreement with the hypothesis that sugars, amino acids and creatinine present in meat may be the precursors of the mutagenic imidazoquinolin- and imidazoquinoxalin-2-amines (IQ compounds).