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S Grivas

Publications and source records attributed to S Grivas.

At least 19 recordsLinked to original sources
PubMed1998

Microsomal metabolism and activation of the environmental carcinogen 2-amino-3-methyl-9H-pyrido[23-b]indole.

2-Amino-3-methyl-9H-pyrido[2,3-b]indole (MeA alpha C) is a mutagenic and carcinogenic heterocyclic amine formed as a pyrolysis product during cooking of food and combustion of tobacco. Hepatic microsomes from PCB-induced rats metabolized MeA alpha C to four products, of which three were non-mutagenic and one was mutagenic without S9 activation. The three non-mutagenic products, which accounted for 83% of the metabolism of MeA alpha C, were characterized by mass spectrometry and NMR spectroscopy as 6-hydroxy-MeA alpha C, 7-hydroxy-MeA alpha C and 3-hydroxy-methyl-A alpha C. The mutagenic metabolite, accounting for 17% of the metabolism of MeA alpha C, was characterized as N2-hydroxy-MeA alpha C by comparison with the HPLC retention time and UV spectrum of N2-hydroxy-MeA alpha C obtained by chemical synthesis. N2-Hydroxy-MeA alpha C was very reactive and a part of it bound covalently to microsomal proteins during incubation and a part was degraded to other products during incubation or chromatography. N2-Hydroxy-MeA alpha C was mutagenic in Salmonella typhimurium TA98 without metabolic activation, resulting in 5070 revertants/microgram, which was > 20 times the specific mutagenic activity of the parent compound.

Animals↗
PubMed1997

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.

Animals↗
PubMed1996

Biotransformation of the cyclopenta-fused polycyclic aromatic hydrocarbon benz[j]aceanthrylene in isolated rat liver cell: identification of nine new metabolites.

The biotransformation of benz[j]aceanthrylene (B[j]A) was studied in suspensions of hepatocytes isolated from Aroclor 1254-treated or untreated rats. Using radiolabeled cofactors and metabolic inhibitors combined with UV, mass and 1H-NMR spectroscopy, we have detected five known metabolites and characterized nine new metabolites: metabolite 1 was tentatively assigned as B[j]A-1,2-dihydrodiol-8-sulfate; metabolite 2, B[j]A-1,2,9,10-tetrahydrotetrol; metabolite 3, B[j]A-1,2-dihydrodiol-10-O-glucuronide; metabolite 4, B[j]A-1-one-8-sulfate; metabolite 5, B[j]A-1,2-dihydrodiol-10-sulfate; metabolite 6, the sulfate conjugate of B[j]A-dihydrodiol-phenol; peak 7 in the chromatogram is a mixture of one glutathione conjugate and two sulfate conjugates of a B[j]A-metabolite; metabolite 8, B[j]A-10-O-glucuronide; metabolite 8', B[j]A-1,2-dihydrodiol; metabolite 9, B[j]A-10-sulfate; metabolite 9', B[j]A-9,10-dihydrodiol and metabolite 10, B[j]A-9,10-dihydro-9-hydroxy-10-sulfate. The metabolites identified support the notion that epoxidation at the cyclopenta region is an important activation step of B[j]A. Furthermore, sulfation appears to play a very important role in the conversion of hydroxylated B[j]A metabolites into more polar excretable products.

Animals↗
PubMed1995

Structure-mutagenicity relationships of four amino-imidazonaphthyridines and imidazoquinolines.

We tested four isomeric imidazonaphthyridines and one imidazoquinoline compound for mutagenic activity in the Ames/Salmonella mutagenicity assay, using strain TA98 and strain YG1024, an analogue of strain TA98 with elevated O-acetyltransferase levels. Their potency was related to calculated electronic parameters. Five compounds with a linear arrangement of 3 rings showed a positive response in strain YG1024. Compound 2 (1-methylimidazo[4,5-b][1,7]naphthyridin-2-amine) is the most mutagenic in both strains, giving specific activities of about 200 and 30 revertants per microgram in strains YG1024 and TA98, respectively. Three of the compounds were weak mutagens, giving a positive dose-response only in strain YG1024, with 3-5 revertants per microgram. A higher response of all five compounds in strain YG1024 as opposed to TA98 indicates that they require O-acetyltransferase activity for their metabolism. Mutagenic potencies in strain YG1024 were positively correlated to the energy of the LUMO (lowest unoccupied molecular orbital) of the nitrenium ion.

Dose-Response Relationship, Drug↗
PubMed1995

Antibodies to the food mutagens, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline: useful for immunoassay and immunoaffinity chromatography of biological samples.

Monoclonal mouse IgG1 and IgG3 antibodies were developed to the food mutagens, 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) and 2-amino-3,4,8-trimethylimidazo[4,4-f] quinoxaline (4,8-DiMeIQx) in order to make specific and sensitive detection and purification systems suitable for biological samples. The antibodies were developed with the strategy that cross-reaction with analogues modified in the N2-position was desirable. Competitive enzyme-linked immunosorbent assays (ELISA) with 50% inhibition by 0.4-6 pmol food mutagen were developed. The epitopes recognized by the antibodies have been characterized by ELISA using 52 synthetic analogues and metabolites of PhIP, 4,8-DiMeIQx, and other food mutagens. One of the anti-PhIP antibodies only recognizes PhIP and those PhIP-analogues which have minor modifications in the N2-amino group, whereas the other, 7B7-1, is less stringent and also recognizes several other modified metabolites, including bulky adducts at the N2-amino group e.g. the major guanine and deoxyguanosine adducts isolated from PhIP-modified DNA. The antibodies to DiMeIQx also recognize the food mutagens 2-amino-3,4-dimethylimidazo[4,5-f]quinoxaline (4-MeIQx), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), and the corresponding quinolines (4-MeIQ and 8-MeIQ). Two of these antibodies only bind analogues with minor modifications in the free amino group, whereas analogues with major modifications in this position, including a deoxyguanosine adduct, react with the third antibody. Urine samples and faecal extracts from 3H-PhIP or 2-14C-DiMeIQx dosed rats were analysed by these ELISA assays, and high correlations between radioactivity and response in the ELISA assays were observed. Urine samples and faecal extracts from 3H-PhIP-dosed rats were purified on an affinity column containing the less stringent anti-PhIP antibody, 7B7-1. The affinity column was found by high performance liquid chromatography (HPLC) analysis to concentrate exclusively labelled material. This affinity column also bound PhIP-related materials from dilute samples of acid hydrolysed PhIP-DNA with high efficiency. Only approximately 40% of the 4,8-DiMeIQx related materials found in dilute acid hydrolysed samples of 4,8-DiMeIQx-DNA was bound by an affinity column containing the less stringent anti-4,8-DiMeIQx antibody, 2C5-1. We conclude that our anti-PhIP and anti-DiMeIQx antibodies can be used to determine the presence of these food mutagens and some of their activated or conjugated metabolites in complex biological samples.

Animals↗
PubMed1995

Synthetic routes to the food carcinogen 2 amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx) and related compounds.

A review of five different routes to the synthesis of the grilled or fried food carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx) and of closely related compounds developed in different laboratories is presented. Interest in the synthesis of these amines began in the late 1970s when the research group led by Professor T. Sugimura (National Cancer Center, Tokyo) detected extremely high mutagenicity in the charred parts of fish and meat that could not be explained only by the presence of polyaromatic hydrocarbons. A number of new mutagenic heterocyclic amines have been detected, isolated and identified since then (de Meester, 1989; Overvik and Gustafsson, 1990; Felton and Knize, 1991; Jägerstad et al., 1991). It is still not entirely clear how these compounds are formed during cooking. For the "IQ-group" of the amines (2-amino-3-methylimidazo-quinoline and -quinoxaline congeners), a formation pathway from Maillard reaction products and creatinine was conceived by Professor K. Olsson (this laboratory) and presented at the 183rd meeting of the American Chemical Society, Las Vegas in 1982 (Jägerstad et al., 1983). However, the amounts of the amines formed during cooking or in model reaction systems are very small. Therefore, efficient and unambiguous synthetic methods yielding the compounds in isomerically pure form are required for reference purposes in analytical work and structure-biological activity studies. For instance, compare the mutagenicity of 4,8- and 5,8-DiMeIQx, and PhIP and its 3-methyl isomer shown on the following page. The pure compounds are also required in large quantities for long-term animal feeding studies. The length of this article does not allow a presentation of the published synthetic methods for all the heterocyclic amines. Therefore, the syntheses of only one of the food mutagens, 8-MeIQx, and some related compounds are presented. This will hopefully demonstrate the sort of problems the organic chemist encounters and some of their possible solutions. For a relatively recent reference list covering the synthesis, isolation, detection, formation and biological activity of these food mutagens and carcinogens the reader is referred to Hatch et al. (1988).

Animals↗
PubMed1994

In vitro formation and degradation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) protein adducts.

Adduct formation between the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and rat serum albumin (RSA) was studied in vitro using hepatic microsomes isolated from polychlorinated biphenyl-induced rats. With 1-methyl-2-nitro-6-phenylimidazo[4,5-b]pyridine (2-nitro-PhIP) as starting material, four main products were formed. Pretreatment of RSA with beta-mercaptoethanol markedly increased the yield of one of them. In this adduct, the C-2 of PhIP was linked to cysteine of RSA at position 34 in a C-S linkage. With N2-acetoxy-PhIP as starting material, unstable conjugates were formed with RSA as well as with glutathione (GSH) and cysteine. The suggested structures of the GSH and cysteine conjugates, GSH-S-N2-PhIP and cysteine-S-N2-PhIP respectively, are based on mass spectra and UV spectra. The degradation of the conjugates of GSH and cysteine as well as of the protein adduct were monitored. They all resulted in the same degradation product, identified as 2-amino-5-hydroxy-1-methyl-6-phenylimidazo[4,5-b]pyridine (5-hydroxy-PhIP).

Animals↗
PubMed1994

Formation of DNA adducts by the food mutagen 2-amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (4,8-DiMeIQx) in vitro and in vivo. Identification of a N2-(2'-deoxyguanosin-8-yl)-4,8-DiMeIQx adduct.

The covalent binding of the mutagenic N2-hydroxy metabolite of the food mutagen 2-amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (4,8-DiMeIQx) to 2'-deoxynucleosides and DNA was investigated in vitro and in vivo. N2-Hydroxy-4,8-DiMeIQx reacted to a small extent spontaneously with 2-deoxyguanosine. However, acetylation of N2-hydroxy-4,8-DiMeIQx with acetic anhydride to form the N2-acetoxy derivative prior to reaction with 2-deoxyguanosine resulted in much higher yield of adduct. N2-Acetoxy-4,8-DiMeIQx did not form adducts with 2'-deoxyadenosine, 2'-deoxycytidine or 2'-deoxythymidine. The adduct formed between the N2-OH metabolite of 4,8-DiMeIQx and 2-deoxyguanosine was analysed by mass spectrometry and NMR spectroscopy and the structure of the adduct was shown to be N2-(2'-deoxyguanosin-8-yl)-4,8-DiMeIQx. N2-Acetoxy-4,8-DiMeIQx reacted with calf thymus DNA and formed a covalently bound 4,8-DiMeIQx residue, which could not be removed by repeated precipitations or solvent extractions. The 4,8-DiMeIQx-DNA was hydrolysed enzymatically with nuclease P1/acid phosphatase and HPLC analysis showed that 70% of the bound mutagen was recovered as N2-(2'-deoxyguanosin-8-yl)-4,8-DiMeIQx. An additional minor adduct accounting for approximately 15% of the bound mutagen showed UV spectral characteristics similar to N2-(2'-deoxyguanosin-8-yl)-4,8-DiMeIQx and is probably an undigested oligomer. 32P-Postlabelling analysis of calf thymus DNA modified with 4,8-DiMeIQx in vitro and liver DNA from rats dosed with 50 mg/kg 4,8-DiMeIQx showed a similar adduct pattern. In both samples N2-(2'-deoxyguanosin-8-yl)-4,8-DiMeIQx accounted for 60-70% of the bound mutagen. Thus, these results show that 4,8-DiMeIQx similar to other heterocyclic amines form adducts with C-8 of guanine both in vitro and in vivo via its N2-OH metabolite.

Acetylation↗
PubMed1994

Microsomal metabolism of the food mutagen 2-amino-3,4,8-trimethyl-3H-imidazo[4,5-f]-quinoxaline to mutagenic metabolites.

Heterocyclic aromatic amines are formed in the crust of meat during ordinary cooking. These aromatic amines are potent bacterial mutagens and also potent rodent carcinogens. 2-Amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (DiMeIQx) is one of the more abundant heterocyclic aromatic amines, accounting for approximately 20% of the mutagenic material found in cooked meat. DiMeIQx is metabolically activated, by hepatic microsomes from PCB treated rats, to two major and three minor metabolites. One major and one minor metabolite were identified as 2-hydroxyamino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline and 3,4,8-trimethyl-2-nitro-3H-imidazo[4,5-f]quinoxaline, respectively, confirmed by comparison of HPLC retention times, and UV and mass spectra of synthetic standards. Both metabolites were mutagenic in Salmonella typhimurium TA98 without metabolic activation. The other major metabolite was identified as 2-amino-8-hydroxymethyl-3,4-dimethyl-3H-imidazo[4,5-f]quinoxaline by mass and NMR spectral analysis. The two remaining minor metabolites were identified as the 2-hydroxyamino- and 2-nitro- derivatives of 2-amino-8-hydroxymethyl-3,4-dimethyl-3H-imidazo]4,5-f]quinoxaline by UV and mass spectral analysis. Both of these metabolites were mutagenic in S. typhimurium TA98 without metabolic activation.

Animals↗
PubMed1993

Mutagenic activity of three synthetic isomers of the food carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in the Ames test.

The mutagenic activities of three isomers of IQ, with the pyridine N-atom at different positions, were measured in the Ames test with Salmonella typhimurium TA98 and enzymatic activation (S9). All test compounds showed lower activities than IQ. Interestingly, the amount of protein in the S9 mix does not seem to influence the mutagenic activity of the '8-IQ' isomer.

Animals↗
PubMed1993

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.

Animals↗
PubMed1992

Reaction of the N2-acetoxy derivative of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) with 2'-deoxyguanosine and DNA. Synthesis and identification of N2-(2'-deoxyguanosin-8-yl)-PhIP.

The direct acting mutagenic N2-hydroxylated metabolite of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) does not react with DNA. Upon acetylation of the N2-hydroxy-PhIP with acetic anhydride two products could be detected. Mass spectrometric analysis showed that both products were monoacetyl derivatives of N2-hydroxy-PhIP. One of the products did not show any reactivity towards DNA and is probably the N-acetyl derivative of N2-hydroxy-PhIP. The other product which is most likely to be N2-acetoxy-PhIP reacted with DNA and 2'-deoxyguanosine but not with 2'-deoxycytidine, 2'-deoxyadenosine or 2'-deoxythymidine. The PhIP-2'-deoxyguanosine adduct was purified and characterized by mass spectral, 1H and [13C]NMR analysis, showing that PhIP like the other cooked food mutagen 2-amino-3-methylimidazo[4,5-f]quinoline, had reacted with C-8 of guanine forming N2-(2'-deoxyguanosin-8-yl)-PhIP. HPLC analysis of enzymatically hydrolyzed calf thymus DNA which had been reacted with N2-acetoxy-PhIP showed one adduct which was chromatographically and spectroscopically identical to N2-(2'-deoxyguanosin-8-yl)-PhIP. HPLC separation followed by liquid scintillation counting of hydrolyzed liver DNA from a rat dosed with [3H]PhIP showed that radioactivity coeluted with the hydrolysis product of the synthetic PhIP-2-deoxyguanosine adduct, indicating that PhIP in vivo also forms an N2-(2'-deoxyguanosin-8-yl)-PhIP adduct.

Animals↗
PubMed1992

Determination of human exposure to the dietary carcinogen 3-amino-1, 4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) from hemoglobin adduct: the relationship to DNA adducts.

A quantitative method for estimation of the exposure of the food-borne carcinogen, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), was developed by the analysis of its hemoglobin binding in rats. This method was then applied to show the presence of Trp-P-1-hemoglobin adducts in human blood. In rat experiments, 0.2 and 0.07% of the administered [14C]Trp-P-1 formed stable covalent adducts with blood hemoglobin and plasma proteins respectively. Subsequent strong acidic treatment (6 N HCl, 110 degrees C, 24 h) of the Trp-P-1-hemoglobin adducts cleaved peptide bonds of globin, and yielded mainly three derivatives of Trp-P-1. One of them (TPHB) represented approximately 50% of the total Trp-P-1-hemoglobin adducts and was suitable for detection through its strong fluorescence. TPHB was used as a surrogate marker of the Trp-P-1-hemoglobin adducts. Linear dose dependency of Trp-P-1 binding to liver DNA and hemoglobin in rats was confirmed by 32P-postlabeling analysis and TPHB assay. The absence of Trp-P-1-DNA adducts and TPHB in nontreated rats was also confirmed. Using TPHB as a tool for human dosimetry of Trp-P-1, human blood samples from four healthy individuals were examined. TPHB was detected in all samples ranging from 0.23 to 4.33 pmol/g hemoglobin. These results suggest human exposure to Trp-P-1, probably from cooked foods or cigarette smoke, and its possible relationship to human carcinogenesis.

Adult↗
PubMed1991

Formation of a glutathione conjugate and a semistable transportable glucuronide conjugate of N2-oxidized species of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in rat liver.

We have previously shown that 2-hydroxamino-1-methyl-6-phenylimidazo[4,5-b]pyridine(2-h ydroxamino-PhIP) is the principal metabolite leading to mutations in Salmonella typhimurium TA98 and DNA damage in mammalian cells. In rat hepatocytes this metabolite can be further conjugated to 2-(N-beta-D-glucuronopyranosyl (hydroxamino)-1-methyl-6-phenylimidazo[4, 5-b]pyridine[N(OH)-gluc-PhIP]. Its rate of formation was increased in hepatocytes from polychlorinated biphenyl (PCB)-pretreated animals. This metabolite is the main metabolite of PhIP in bile and it is hydrolyzed both by human and rat intestinal bacteria. Smaller amounts are excreted into urine. The evidence for the proposed structure is based on 1H- and 13C-NMR, beta-glucuronidase-lability giving 2-hydroxamino-PhIP upon hydrolysis and on the results obtained by using biochemical enzyme inhibitors. N(OH)-gluc-PhIP may be important for genotoxic lesions and tumors of 2-amino-1methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) in extrahepatic tissue. In hepatocytes and bile from PCB-pretreated rats a PhIP-glutathione conjugate, 2-glutathionyl-1-methyl-6-phenylimidazo[4,5-b]pyridine (GSH-PhIP) was also found. The evidence for the proposed structure is based on 1H-NMR and high-resolution mass spectrometry. The metabolite can also be produced by a direct nucleophilic substitution of the nitro group in 2-nitro-PhIP by glutathione (GSH) in vitro. The metabolite did not form from 2-hydroxamino-PhIP and GSH either directly or in the presence of glutathione S-transferase. The formation of GSH-PhIP in rat liver and isolated cells only at a high rate of 2-hydroxamino-PhIP formation (PCB-treated animals) indicates that 2-nitro-PhIP may be formed in the liver during such N-oxidation of PhIP.

Animals↗
PubMed1990

Formation of a nitro derivative of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline by photo-irradiation.

A direct-acting mutagen to Salmonella typhimurium TA98 was found to be formed by exposing 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) in acetone to sunlight for 60 min. The direct-acting mutagen in the irradiated sample was purified by HPLC and identified as 3,4-dimethyl-2-nitroimidazo-[4,5-f]quinoline (NO2-MeIQ). The yield of NO2-MeIQ from MeIQ was estimated to be 0.3%.

Chromatography, High Pressure Liquid↗