Formation of new heterocyclic amine mutagens by heating creatinine, alanine, threonine and glucose.

A mixture of alanine, threonine, creatinine and glucose was heated in diethylene glycol and water (5:1, v/v) for 15 min at 200 degrees C. The mutagens formed were purified by high-performance liquid chromatography using the Ames/Salmonella mutagenic activity to guide the purification. The structures of the purified mutagens were determined using UV absorption, mass and NMR spectrometry. A new mutagenic compound with a mass number of 217 was found and its mass spectrum did not correspond to any known mutagen derived from food. This new compound accounted for 4% of the total mutagenic activity. Other mutagenic compounds were identified as MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline), 4,8-DiMeIQx (2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline), and a new mutagen 4,7,8-TriMeIQx (2-amino-3,4,7,8-tetramethylimidazo[4,5-f]quinoxaline) with a mutagenic activity of 73,000 TA98 revertants per microgram. The percentage of the mutagenic activity attributable to MeIQx, 4,8-DiMeIQx and 4,7,8-TriMeIQx was 10%, 70% and 3%, respectively. The yield of MeIQx, 4,8-DiMeIQx and 4,7,8-TriMeIQx was 10, 36 and 6 nmole/mmole creatinine. The formation of TriMeIQx from natural meat components suggests that this new quinoxaline mutagen may be present in cooked foods.     

Identification of the mutagenic quinoxaline isomers from fried ground beef

Two mutagens isolated from fried-beef patties were compared to a series of synthetic structural isomers of 2-aminodimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-aminotrimethylimidao[4,5-f]quinoxaline (DiMeIQx). Comparison by NMR spectrometry and HPLC coelution showed that one beef mutagen (molecular weight of 213) was identical to the 8-MeIQx isomer not the 7-Me isomer. Another quinoxaline beef mutagen, having 3 methyl groups (molecular weight of 227), had an NMR spectrum different from the 5,8- or 7,8-DiMeIQx isomers, but not clearly distinguishable from the 4,8- or 4,7-DiMeIQx isomers. The HPLC separation of the DiMeIQx isomers and subsequent addition of the beef mutagen showed the beef-derived compound to coelute with the 4,8-DiMeIQx and not with the 4,7-DiMeIQx. The number and position of methyl groups was responsible for a 7-fold range of mutagenic response in the Ames/Salmonella assay. In conclusion, the major quinoxaline mutagens isolated from fried beef were identified as 8-MeIQx and 4,8-DiMeIQx isomers.     

Use of nitrite and hypochlorite treatments in determination of the contributions of IQ-type and non-IQ-type heterocyclic amines to the mutagenicities in crude pyrolyzed materials

The mutagenic heterocyclic amines Glu-P-2, MeA alpha C and Phe-P-1, which possess a 2-aminopyridine structure in their molecule (non-IQ-type mutagens), were found to be inactivated by nitrite treatment under acidic conditions, as observed previously with Trp-P-1, Trp-P-2, Glu-P-1 and A alpha C. In contrast, MeIQx, 4,8- and 7,8-DiMeIQx, which were originally isolated from fried beef or heated model mixtures of creatinine, amino acids and glucose, and which have a 2-aminoimidazole moiety in their molecules (IQ-type mutagens), were very resistant to nitrite treatment like IQ and MeIQ. Both types of mutagenic heterocyclic amines were completely inactivated by treatment with hypochlorite. This differential inactivation of mutagenic heterocyclic amines by nitrite and hypochlorite was used in determination of the contributions of IQ-type and non-IQ-type mutagens to the total mutagenicities of various pyrolyzed materials. The percentage contributions of IQ-type mutagens to the mutagenicities of broiled sardine, fried beef, broiled horse mackerel, cigarette smoke condensate and albumin tar were 88, 75, 48, 6 and 4, respectively.     

Formation of mutagens, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), in heated fish meats

Fish meats were heated under conditions close to those used for cooking and processing. The mutagenic activity of the heated fish meats was estimated toward Salmonella typhimurium TA98 with metabolic activation after extraction with boiling water and adsorption to blue cotton. The numbers of His+ revertant colonies/5 g of the meat heat-dried without charring at 220 degrees C for 15 min were about 3000 for bonito, about 1000 for tunny, less than 500 for mackerel, salmon, swordfish, sardine, horse mackerel and cod, and 0 for cuttlefish. The mutagens in the heat-dried bonito meat were purified by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). They were identified as 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) by comparison with the authentic specimen with respect to Rf values in TLC, retention times in HPLC, ultraviolet absorption spectra and mass spectra. The contents of MeIQx and 4,8-DiMeIQx in the bonito meat were estimated to be 5.2 and 5.4 ng/g, respectively. The major mutagens produced in the bonito, tunny and mackerel meats heated without charring at 100 degrees C for 48 h and at 220 degrees C for 15 min were found to be MeIQx and 4,8-DiMeIQx. It is interesting to note that the bonito and sardine meats grilled with charring for 15 min contained MeIQx and 4,8-DiMeIQx but higher mutagenicity was observed in the fraction that may contain 2-amino-3-methylimidazo[4,5-f] quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and/or 2-aminodipyrido[1,2-a: 3',2'-d]imidazole (Glu-P-2).     

Mutagenic and carcinogenic heterocyclic amines in Chinese cooked foods

Samples of 7 foods commonly eaten in the Northeast of China (i.e. fried and broiled fishes and broiled meat) were tested for mutagenicity on Salmonella typhimurium TA98 with S9 mix. The basic fractions of the samples were mutagenic, inducing 33-2930 revertants/g of cooked food. Fried walleye pollack (a kind of cod fish heated on a stainless steel pan) showed the highest mutagenicity, so attempts were made to isolate mutagens from the basic fraction of this food. The mutagens were purified by treatment with blue cotton and HPLC on a semi-preparative ODS column and analytical cation exchange and ODS columns. 5 mutagens were isolated and identified as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). 1 g of fried fish was estimated to contain 0.16 ng of IQ, 0.03 ng of MeIQ, 6.44 ng of MeIQx, 0.10 ng of 4,8-DiMeIQx and 69.2 ng of PhIP. MeIQx and PhIP accounted for 24% and 4.7%, respectively, of the total mutagenicity. The other 3 heterocyclic amines were each responsible for only 0.3-1.2% of the total mutagenicity.     

Effects of monosaccharides and disaccharides on the formation of food mutagens in model systems

The formation of the mutagenic imidazoquinoxalines (MeIQx, DiMeIQx) was studied using a modification of a previous model system. Creatine or creatinine (0.9 mmole) was heated together with glycine (0.9 mmole) and various sugars (0.45 mmole) dissolved in diethylene glycol and water (3 ml, 5 : 1) for up to 15 min at 180°C. This system produced the same amount of mutagenicity after 10 min at 180°C as a previous one during 2 h of reflux boiling at 12dgC. MeIQx (4 nmole/mmole creatin(in)e was the major mutagen produced together with minor amounts of DiMeIQx, both 4,8- and 7,8-DiMeIQx according to HPLC-MS. A few other mutagenic peaks were also separated on HPLC, but they were not identified. Varying the concentration (0–2.4 mmole) and type of monosaccharides and disaccharides greatly affected the yields of all the mutagenic compounds. Sugar in molar amounts lower than the creatin(in)e concentration increased the yield until an optimum was reached. In higher concentrations the formation of all the mutagens was markedly reduced. The same was found for glucose, fructose, sucrose, and lactose, though the monosaccharides showed the most pronounced inhibitory effects.

The inhibition of the formation of the mutagenic compounds by an excess of sugars is proposed to be an effect of Maillard reaction products, which may block the formation of imidazoquinoxalines by attacking creatine. Support for this mechanism is given by data showing a lower recovery of unreacted creatine with increasing concentration of glucose and also by an inhibitory effect on the formation of these mutagens after adding a typical Maillard reaction product, 5-hydroxymethyl-2-furfural.     

Formation of 2-amino-3,7,8-trimethylimidazo [4,5-f]quinoxaline, a new mutagen, by heating a mixture of creatinine, glucose and glycine

When a mixture of creatinine, glycine and glucose was heated for 2 h at 128 degrees C in diethylene glycol containing 14% water, two mutagens were formed. One of them, responsible for 90% of the mutagenicity, has already been identified as 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). The other mutagen was purified and characterized. The UV absorption, mass and NMR spectra suggested that this mutagen was 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-DiMeIQx). Comparison of the spectral properties of the compound obtained from the heated model mixture with those of synthetic material confirmed this structure. 7,8-DiMeIQx is a newly identified compound which, at a dose of 1 microgram, induced 163 000 and 9900 revertants of Salmonella typhimurium TA98 and TA100, respectively, with S9 mix.     

Occurrence of heterocyclic amines in several home-cooked meat dishes of the Spanish diet

Heterocyclic amines (HAs) were determined in several of the most frequently eaten meat dishes in Spain such as fried beef hamburger, fried pork loin, fried chicken breast, fried pork sausages, griddled chicken breast, griddled lamb steak and griddled beef steak. All of the products tested were household cooked. The HAs were analysed in the selected meat dishes using an analytical method based on solid-phase extraction followed by liquid chromatography coupled to tandem mass spectrometry. DMIP, MeIQx, 4,8-DiMeIQx, Norharman, Harman, PhIP, Trp-P-1, AalphaC and MeAalphaC were the amines most frequently found at concentrations of up to 47 ng g(-1) of cooked meat. Glu-P-2, IQ, MeIQ, Glu-P-1, 7,8-DiMeIQx and Trp-P-2 were only found in a few of the meat dishes and their concentrations were lower than 1 ng g(-1) of cooked meat. The highest amounts of HAs, especially PhIP and DMIP, were formed in fried chicken breast and the lowest were formed in fried beef hamburger and in fried pork sausages. Daily intake of HAs in Spain was estimated at 606 ng of mutagenic HAs per capita and day, DMIP and PhIP being the main contributors.     

Determination of heterocyclic aromatic amines in beef extract, cooked meat and rat urine by liquid chromatography with coulometric electrode array detection

This paper describes a method for the determination of heterocyclic aromatic amines (HAs; DMIP, IQ, MeIQ, MeIQx, 4,8-DiMeIQx, 7,8-DiMeIQx, AalphaC, PhIP) by high-performance liquid chromatography (HPLC) with coulometric electrode array detection. The compounds are separated on reversed phase columns (LiChroCart Superspher 60 RP-select B, 250 mm x 2 mm, 4 microm and LiChrospher 60 RP-select B, 250 mm x 4 mm, 5 microm) using mobile phases consisting of acetonitrile/buffer/distilled water and detected at eight working electrodes at potentials between +190 and +680 mV against modified palladium electrodes. In the context of an EU-interlaboratory exercise, the method was applied to analyse a standardised test solution and--after isolation of the analytes by several clean-up steps--for the analysis of standardised beef extract and grilled meat. Further, the method could be applied for the analysis of HAs in suspensions of bacteria and rat urine without any sample preparation step beyond sample dilution. The data obtained show that HPLC with coulometric electrode array detection gives accurate results.     

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.     

Formation of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in a model system by heating creatinine, glycine and glucose

A mixture of creatinine, glucose and glycine was heated in diethylene glycol containing 14% water for 2 h at 128 degrees C, and the mutagens formed were purified by XAD-2 column chromatography, acid-base partition, Sephadex LH-20 column chromatography, 'blue cotton' treatment and HPLC. Two mutagenic substances were isolated by HPLC. The major mutagen was identified by its UV absorption and mass and NMR spectra as 2-amino-3,8- dimethylimidazo [4,5-f]quinoxaline, which was originally isolated from fried beef. This finding supported the idea that creatinine, amino acids and sugars present in meat are precursors in the formation of the mutagenic imidazoquinoxaline derivative.     

Mutagenicity of the Alternaria metabolites altertoxins I, II, and III

The Ames Salmonella typhimurium assay was used to demonstrate that an extract of the mold Alternaria alternata was mutagenic. The mutagenic extract was fractionated, and the Ames test was used to determine which fractions were mutagenic. Subsequently, altertoxins I and II and a new compound referred to as altertoxin III were isolated by liquid chromatography and shown to be hydroxyperylenequinone compounds by mass spectrometry and infrared, ultraviolet, and proton magnetic resonance spectroscopy. Altertoxins I, II, and III were mutagenic to S. typhimurium TA98, TA100, and TA1537 with and without metabolic activation.     

Mutagenicity of the Alternaria metabolites altertoxins I, II, and III.

The Ames Salmonella typhimurium assay was used to demonstrate that an extract of the mold Alternaria alternata was mutagenic. The mutagenic extract was fractionated, and the Ames test was used to determine which fractions were mutagenic. Subsequently, altertoxins I and II and a new compound referred to as altertoxin III were isolated by liquid chromatography and shown to be hydroxyperylenequinone compounds by mass spectrometry and infrared, ultraviolet, and proton magnetic resonance spectroscopy. Altertoxins I, II, and III were mutagenic to S. typhimurium TA98, TA100, and TA1537 with and without metabolic activation.     

Mechanism(s) involved in meat mutagen formation and inhibition.

The Maillard reaction, which involves Amadori rearrangement as a key step, also results in sugar fragmentation and free radical formation. The imidazoquinoline meat mutagens (2-amino-3-methylimidazo[4,5-f]-quinoline, or IQ, and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, or MeIQ) are formed from a reaction mixture containing alkylpyridine free radicals and creatinine. The imidazoquinoxaline meat mutagens (2-amino-3,4-dimethylimidazo[4,5-f]-quinoxaline, or MeIQx, and 2-amino-3,4,8-trimethylimidazo[4,5-f]-quinoxaline, or 4,8-DiMeIQx) may be produced by reacting a mixture containing dialkylpyrazine free radicals and creatinine. Two different pathways for free radical formation are proposed. One involves bimolecular ring formation from the enaminol form of the glycoaldehyde alkylimine and is followed by oxidative formation of the free radical. The other pathway involves formation of N,N1-dialkylpyrazinium ions from glyoxal monoalkylimine followed by reduction to produce the free radicals. The respective intermediates (glycoaldehyde alkylimine and glyoxal monoalkylamine) are formed by reacting glycoaldehyde and glyoxal with amino compounds. The glycoaldehyde system reacts faster and produces more free radicals than the glyoxal system. The reactions help to explain the formation of imidazoquinoxaline meat mutagens and their predominance in fried fish and why these mutagens are present in larger quantities in fried ground beef than the imidazoquinoline-type meat mutagens. These two pathways may not be the only mechanisms involved in formation of meat mutagens, but other free radical reactions may also contribute to meat mutagenicity and are mentioned briefly.(ABSTRACT TRUNCATED AT 250 WORDS)     

4,8-anhydro-N-acetylneuraminic acid. Isolation from edible bird's nest and structure determination

A new, sialic-acid-derived compound was isolated from the acid hydrolysate of edible bird's nest by ion-exchange chromatography. Combined use of mass spectroscopy and 1H- and 13C-NMR spectroscopy established that it is the 4,8-anhydro derivative of N-acetylneuraminic acid and that in solutions it exists in two tautomeric forms. The formation of the new compound supports and earlier findings that in the glycoprotein of edible bird's nest at least a portion of N-acetylneuraminic acid is acetylated at HO-4.     

The structure and distribution of ceramide aminoethylphosphonates in the oyster (Ostrea gigas).

1. Ceramide aminoethylphosphonate was isolated from the adductor, gills, mantle and viscera of oysters. 2. After drastic acid hydrolysis of the lipid, aminoethylphosphonic acid was the only water-soluble carbon-phosphorous compound detected. 3. The main fatty acids of ceramide aminoethylphosphonates were hexadecanoic acid (77-90%) and 2-hydroxy hexadecanoic acid (13-15%). 4. Hexadeca-4-sphingenine, octadeca-4-sphingenine and octadeca-4,8-sphingadienine were identified as the major long chain base components. However, the ratio of the three bases was characteristic for each tissue; the adductor muscle contains primarily hexadeca-4-sphingenine, and the viscera, octadeca-4,8-sphingadienine. The gills and mantle contain the three bases in approximately equal concentration. 5. The main molecular species in the adductor muscle was hexadecanoyl-hexadeca-4-sphingenyl 2-aminoethylphosphonate, while in the viscera hexade-canoyl-octadeca-4,8-sphingenyl 2-aminoethylphosphonate predominated.     

2,7-Diamino-3,8-dimethylphenazine as the major mutagenic product from the reaction of 2,4-diaminotoluene with hydrogen peroxide

The mutagenicity of 2,4-diaminotoluene (DAT) in Ames's Salmonella/microsome test was remarkably enhanced by treatment with hydrogen peroxide. Therefore, identification of the major mutagenic reaction product of 2,4-DAT with hydrogen peroxide at room temperature has been performed. Red precipitates were produced in a 2-day reaction mixture and were column chromatographed on silica gel. 5 fractions having mutagenic potency were obtained. The red crystalline needles, obtained as the major reaction product, were separated from fraction 2 and were subjected to high resolution mass spectrometry, 1H- and 13C-NMR spectrometry. The structure of the compound was determined to be 2,7-diamino-3,8-dimethylphenazine from physicochemical and chemical evidence. The compound induced 212 revertants/nmole in Salmonella typhimurium TA98 with 25 microliters S9 per plate.     

Diversity of fungal isolates from fungus-growing termite Macrotermes barneyi and characterization of bioactive compound from Xylaria escharoidea

Owing to their potential applications,as well as their structural diversity,the discovery of novel secondary metabolites from insect-associated fungi has been of interest to researchers in recent years.The aim of this study was therefore to estimate the diversity of fungi associated with fungus-growing termites and bioprospecting these for potential secondary metabolites.In total,18 fungal species were isolated and described from the gut and comb of Macrotermes barneyi based on 18S ribosomal DNA gene sequence analysis.Antimicrobial activity assays were carried out on all the known fungi,and nine isolates were recorded as active against pathogenic fungi.Xylaria escharoidea,the best performing isolate,was grown at laboratory scale and 4,8-dihydroxy-3,4-dihydronaphthalen-l(2H)was isolated and characterized.The minimum inhibitory concentration of this isolated compound against tested pathogenic organisms was found to be 6.25 fig.In addition,molecular docking studies have revealed that 4,8-dihydroxy-3,4-dihydronaphthalen-l(2H)is a prominent antibacterial agent with a marked interaction with key residues on protein A(agrAc)that regulates the accessory gene.The findings of this study support the drug discovery of antimicrobial properties in insect-associated fungi,which may lead to novel secondary metabolites.     

Isolation, identification and bacterial mutagenicity of 2-nitro-9-fluorenone from diesel-exhaust particle extracts

Organic extracts of diesel-exhaust particles show direct mutagenic activity in the Salmonella typhimurium bacterial mutagenicity assay. Nitro-aromatic compounds are believed to be responsible for part of the mutagenicity. A previously unidentified polyfunctional nitro-aromatic compound, 2-nitro-9-fluorenone (2N-Fone) was isolated from diesel-exhaust particles using a two-step fractionation scheme consisting of Sephadex LH20 chromatography and silica-gel thin-layer chromatography. Positive identification was by gas chromatography/mass spectroscopy and coelution with an authentic standard. Direct and indirect mutagenicities of 2N-Fone in several bacterial strains were also determined. The results indicated that 2N-Fone produces 60-70 rev/nmole of direct mutagenic activity, and is about 1/5 to 1/10 as mutagenic as 1-nitropyrene.     

Potent antimutagenic activity of white tea in comparison with green tea in the Salmonella assay.

There is growing interest in the potential health benefits of tea, including the antimutagenic properties. Four varieties of white tea, which represent the least processed form of tea, were shown to have marked antimutagenic activity in the Salmonella assay, particularly in the presence of S9. The most active of these teas, Exotica China white tea, was significantly more effective than Premium green tea (Dragonwell special grade) against 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and four other heterocyclic amine mutagens, namely 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (4,8-DiMeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). Mechanism studies were performed using rat liver S9 in assays for methoxyresorufin O-demethylase (MROD), a marker for the enzyme cytochrome P4501A2 that activates heterocyclic amines, as well as Salmonella assays with the direct-acting mutagen 2-hydroxyamino-3-methylimidazo[4,5-f]quinoline (N-hydroxy-IQ). White tea at low concentrations in the assay inhibited MROD activity, and attenuated the mutagenic activity of N-hydroxy-IQ in the absence of S9. Nine of the major constituents found in green tea also were detected in white tea, including high levels of epigallocatechin-3-gallate (EGCG) and several other polyphenols. When these major constituents were mixed to produce "artificial" teas, according to their relative levels in white and green teas, the complete tea exhibited higher antimutagenic potency compared with the corresponding artificial tea. The results suggest that the greater inhibitory potency of white versus green tea in the Salmonella assay might be related to the relative levels of the nine major constituents, perhaps acting synergistically with other (minor) constituents, to inhibit mutagen activation as well as "scavenging" the reactive intermediate(s).