Search PubMed⌕ Search

Biomedical subjects

J S Felton

Publications and source records attributed to J S Felton.

At least 19 recordsLinked to original sources
PubMed1992

Chromatographic methods for the analysis of heterocyclic amine food mutagens/carcinogens.

A series of potent heterocyclic amines that are mutagenic and carcinogenic have been discovered that are formed in some heated foods, most notably, meats derived from muscle. Determining the heterocyclic amine content in foods and food products is required for toxicological research, industry quality control, and possibly in the future, regulatory control. The contents of food needs to be determined using reliable analytical techniques. Since heterocyclic amines are present in foods at ng/g levels, a variety of liquid-liquid or solid-phase purification techniques are required, followed by gas or high-performance liquid chromatography. Peak detection has been successful using UV, fluorescence, and mass spectrometric detection, and biological activity using the Ames/Salmonella test. The low levels present require that chromatographic efficiency, and both detector sensitivity and selectivity be optimized. The cartridge solid-phase extraction and high-performance liquid chromatography method have been used to measure the known food-derived heterocyclic amines for several types of food, and to the authors knowledge, this is the only method undergoing intralaboratory comparison and validation. Our analysis of the literature shows that chromatographic analysis of the heterocyclic amines by high-performance liquid chromatography or gas chromatography (with derivatization) is satisfactory for heterocyclic amine analysis in foods although the methods are just now being optimized for routine use. The biggest improvements in speed and accuracy will probably come from improved extraction methods as analysis of complex food samples for heterocyclic amines will always be a challenge.

Amines↗
PubMed1992

Chemical analysis, prevention, and low-level dosimetry of heterocyclic amines from cooked food.

Potent mutagenic and carcinogenic heterocyclic amines are produced from heated food derived from muscle. These compounds are present at part-per-billion levels and consist primarily of the amino-imidazoazaarene class of chemicals. Additional mutagens present in the meat are not as clearly characterized. Commercial fried-beef patties (hamburgers) have low levels of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), 0.1-0.68 ng/g meat for MeIQx and slightly lower for 4,8-DiMeIQx. The formation of these heterocyclic amines can be reduced by microwave pretreatment of meat, with the resulting liquid being poured off before frying. The Ames/Salmonella mutagenic activity was reduced to 5-10% of that of non-microwave-treated samples. MeIQx and DiMeIQx concentrations were reduced to 12% and 50% of levels in the non-microwave-treated samples, respectively. MeIQx adducts, as measured by accelerator mass spectrometry, were found to be linear with doses from 5 mg/kg to 500 ng/kg. Linear DNA binding at low doses is important for assuming linear risk estimation from the high animal-feeding doses causing cancer to the low human-dietary exposures. Extrapolating from the rodent TD50 dose to humans gives a maximum credible risk from consumption of heterocyclic amines of approximately 1/1000 exposed individuals.

DNA↗
PubMed1992

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.

Alanine↗
PubMed1992

Quantitative correlation of mutagenic and carcinogenic potencies for heterocyclic amines from cooked foods and additional aromatic amines.

Aromatic amines have long been recognized as animal and human carcinogens. Recently heterocyclic aromatic amines (thermic amines) have been found in small amounts in cooked foods, primarily meats, and have proven to be potent mutagens and rodent carcinogens. Availability of quantitative databases for mutagenic potency in Salmonella and for carcinogenic potency in rodents has made possible a study of ten heterocyclic thermic amines and 24 aromatic amines. Potencies on mutagenic and carcinogenic scales were significantly correlated. By multiple linear regression analysis and multivariate analysis of variance, two descriptive structural factors were found to modulate the two modes of biological response. These factors were number of rings and methyl substitution at carbon atoms. The quantitative correlation between mutagenic and carcinogenic potencies and the modulating structural factors suggest a significant similarity of molecular mechanisms and support the utility of the short-term bacterial assay in evaluating hazard levels.

Amines↗
PubMed1992

Metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in mice.

The metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP), a heterocyclic amine carcinogen detected in cooked meats, was investigated in mice. In 3-methylcholanthrene-induced mice administered 0.1, 1.0 and 10 mg/kg [14C]PhIP (i.p.), urinary and fecal excretion over 24 h accounted for 16% and 42-56% of the dose respectively. Urinary excretion of unchanged parent compound accounted for only 0.5-0.8% of the administered dose. At all doses, the major urinary metabolite was identified as 4'-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate and this metabolite comprised approximately 5% of the dose. Uninduced mice excreted greater than 13% of a 10 mg/kg dose as the sulfate conjugate. Urinary excretion of both 2-amino-1-methyl-6-(4'-hydroxy)-phenylimidazo[4,5-b]pyridine (4'-hydroxy-PhIP) and a glucuronide conjugate of 2-hydroxyamino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (N-hydroxy-PhIP) was also higher (4-fold) in uninduced versus induced mice. The decreased urinary excretion of P450-derived metabolites via induction contrasted with increased metabolite formation by hepatic microsomal preparations. 4'-Hydroxy-PhIP and N-hydroxy-PhIP were produced in amounts nearly 7- and 3-fold higher respectively by induced versus uninduced microsomal incubations at 50 microM [3H]PhIP. At concentrations less than 10 microM, PhIP was almost exclusively converted by the induced preparations to an unidentified metabolite that was not retained by the C18 column. This metabolite, which also was formed in incubations with either 4'-hydroxy-PhIP or N-hydroxy-PhIP, was produced by microsomes from uninduced animals at a much slower rate. Covalent binding to microsomal protein in incubations with [3H]PhIP was concentration-dependent and 2- to 4-fold higher in induced than uninduced preparations. Covalent binding in liver and kidney of induced mice administered [14C]PhIP was dose dependent. At 10 mg/kg PhIP, adducts were produced at 1.7-fold higher levels in livers of induced versus uninduced mice, but renal binding was higher in uninduced animals. These studies indicate the importance of cytochrome P450 and other xenobiotic enzymes in the metabolism, disposition and activation of PhIP.

Animals↗
PubMed1991

Introduction of cytochrome P450IA2 metabolic capability into cell lines genetically matched for DNA repair proficiency/deficiency.

We introduced into the CHO cell line the cDNA of the mouse cytochrome P3450 (P450IA2) gene, which oxidizes aromatic amines. A cDNA clone of P3450 was transfected into mutant UV5 cells, which is defective in nucleotide excision repair. Expression of the P3450 cDNA was measured using 9000 x g supernatant (S9) fractions from CHO cells to evaluate Salmonella TA1538 mutagenicity with the mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). The P3450-expressing clone UV5P3 was reverted to repair proficiency using ethyl methanesulfonate to obtain the UV-resistant clone 5P3R2, which maintained the same level of P3450 protein activity as UV5P3. These genetically similar cell lines were compared for toxicity and mutation induction at the aprt locus. With 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (the most prevalent mutagen found in fried beef) the differential sensitivity due to repair deficiency/proficiency was approximately 40-fold, and with IQ there were smaller, but significant, differences in sensitivity. These genotoxic effects occurred at doses that were approximately 10 times lower than those that previously gave similar effects in S9-mediated assays. Thus, these cell lines should be valuable for genotoxicity analysis as well as important for assessing DNA repair when evaluating compounds that undergo metabolic activation.

Animals↗
PubMed1991

Quantitative structure-activity relationships of heterocyclic amine mutagens formed during the cooking of food.

The major protein-rich foods, particularly muscle meats, contain part-per-billion quantities of potent mutagens formed by frying or broiling to a well-done state. Related mutagens are formed by pyrolysis of amino acids or proteins and in heated model systems. The thermic mutagens so far identified are heterocyclic aromatic amines of aminoimidazo-azaarene (AIA) and aminocarboline classes. The chemicals require activation by enzymes to form metabolites reactive with nucleic acids. These thermic mutagens, and numerous synthetic congeners, exhibit an enormous range of potency as frameshift mutagens in the Ames/Salmonella assay. However, structural variations are nominal within the two classes. Structural parameters that appeared relevant to determining potency were selected for 38 AIAs and 23 amino-carbolines. For the AIA class these were: the number of fused rings, the number of heteroatoms in Rings 2 and 3, methyl substitution on imidazo ring nitrogen atoms, and methyl substitution on ring carbon atoms. For the amino-carboline class the structural parameters were: the position of the pyridine-type nitrogen atom in Ring 1, the substitution position of the exocyclic amino group on Ring 1, and methyl substitution on ring carbon atoms. These structural parameters may influence mutagenic potency in the following ways. 1) Electronic or steric effects may determine the reactivity and stability of the ultimate mutagenic metabolite. Optimal balance of reactivity and lifetime of this transient intermediate may be required for access to and reaction with nuclear DNA to cause mutations. 2) Substitution on the rings may block detoxication reactions. The structural parameters identified should prove useful in predicting the mutagenicity of untested compounds of these types.

Amines↗
PubMed1991

Expression of mouse cytochrome P450IA1 cDNA in repair-deficient and repair-proficient CHO cells.

Recombinant DNA techniques have been used to develop Chinese hamster ovary cell lines for studying chemically induced genotoxicity. These cell lines express a specific cytochrome P450 isozyme responsible for the metabolism of polycyclic aromatic hydrocarbons and exhibit defined differences in DNA repair capacity. A bacterial gene (neo) conferring resistance to gentamicin was inserted into the pcD expression vector containing the mouse cytochrome P1450 (P450IA1) cDNA to facilitate the selection of transformed cells. This plasmid was introduced into the nucleotide-excision-repair-deficient UV5 cell line by electroporation. Transformed clonal isolates expressing the P1450 cDNA were identified by differential cytotoxicity assays using benzo[a]pyrene (B[a]P). One such clone, termed UV5P1, was mutagenized with ethyl methanesulfonate and selected for resistance to killing by UV radiation to derive a repair-competent clone that expresses similar P1450 activity to that of the parental cell line. Two repair-competent clones were selected and called 5P1R1 and 5P1R3. The resulting cell lines (UV5P1, 5P1R1, and 5P1R3) expressed arylhydrocarbon hydroxylase activity. UV5P1 and 5P1R3 were compared in terms of cytotoxicity and mutagenicity after exposure to B[a]P. Induced mutations were measured at the adenine phosphoribosyltransferase (aprt) and hypoxanthine guanine phosphoribosyltransferase (hprt) loci. Repair-deficient UV5P1 cells were killed by B[a]P at concentrations below 0.1 microM, while the repair-proficient 5P1R3 cells showed no toxicity up to 60 microM. Mutation induction at both loci was also much more efficient in UV5P1 cells. These new cell lines provide a more sensitive system that can be used in a battery of assays to evaluate the genotoxicity of chemicals requiring P450IA1 metabolic activation and to assess the role of DNA repair in modulating the biological effects of DNA damage.

Animals↗
PubMed1991

Metabolic activation and cytogenetic effects of 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) in Chinese hamster ovary cells expressing murine cytochrome P450 IA2.

We have utilized Chinese hamster ovary cell lines which stably express a murine cytochrome P450IA2 (P(3)450) cDNA to characterize more fully the mechanisms of genotoxicity of heterocyclic amines derived from cooked meats. To verify that these cell lines were capable of converting promutagens into active metabolites, we studied the microsomal metabolism and cytogenetic effects of 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pridine (PhIP). Microsomal preparations derived from excision repair-deficient Chinese hamster ovary cells expressing the mouse cytochrome P(3)450 cDNA (UV5P3) converted PhIP to the genotoxic N-hydroxy-PhIP metabolite. Cytotoxic activity in UV5P3 was observed at concentrations of PhIP as low as 1 microM. Cytotoxicity of PhIP was an order of magnitude lower in a matched repair-proficient cell line (5P3R2) expressing the P(3)450 cDNA. PhIP produced a concentration-dependent increase in sister chromatid exchange (SCE) in UV5P3. N-Hydroxy-PhIP, at concentrations as low as 0.1 microM, produced an increase in SCE in both UV5P3 and in UV5 cells which lack the P(3)450 cDNA. Incubation of PhIP with UV5P3 cells increased the frequency of micronuclei (MN) in cytokinesis-blocked cells. Chromatid gaps, but not aberrations also were induced by treatment with PhIP. N-Hydroxy-PhIP produced increases in MN and chromatid gaps in both UV5 and UV5P3 cell lines; chromosomal aberrations were induced in UV5P3 cells. These results suggested that UV5P3 cells metabolize sufficient quantities of PhIP to produce cytogenetic damage and further indicated that N-hydroxylation of PhIP was requisite for mammalian genotoxicity.

Animals↗
PubMed1990

Role of sulfation and acetylation in the activation of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine to intermediates which bind DNA.

Mutagenic activity associated with amino-imidazoazaarene food-derived mutagens such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) appears to be dependent upon N-hydroxylation, though additional metabolic pathways may be involved in the production of the ultimate reactive intermediate which covalently binds DNA. We have evaluated the ability of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP) to bind DNA in vitro and have determined which secondary metabolic pathways are involved in the production of electrophilic intermediates. Incubation of DNA with 10 microM N-hydroxy-PhIP alone or with mouse-liver cytosol did not result in detectable adduct formation. Addition of 3'-phosphoadenosine 5'-phosphosulfate or acetyl coenzyme A to cytosolic incubations containing N-hydroxy-PhIP resulted in DNA adducts which could be detected by 32P-postlabeling at levels of 594 and 30 fmoles/micrograms DNA, respectively. Addition of 3'-phosphoadenosine 5'-phosphosulfate and to a lesser extent acetyl coenzyme A to cytosolic incubations also increased the rate of degradation of the unstable N-hydroxy-PhIP intermediate. These data suggest that both sulfation- and acetylation-dependent metabolic pathways may be important in the mammalian genotoxic actions of PhIP.

Acetyl Coenzyme A↗
PubMed1990

Accelerator mass spectrometry in biomedical dosimetry: relationship between low-level exposure and covalent binding of heterocyclic amine carcinogens to DNA.

Accelerator mass spectrometry (AMS) is used to determine the amount of carcinogen covalently bound to mouse liver DNA (DNA adduct) following very low-level exposure to a 14C-labeled carcinogen. AMS is a highly sensitive method for counting long-lived but rare cosmogenic isotopes. While AMS is a tool of importance in the earth sciences, it has not been applied in biomedical research. The ability of AMS to assay rare isotope concentrations (10Be, 14C, 26Al, 41Ca, and 129I) in microgram amounts suggests that extension to the biomedical sciences is a natural and potentially powerful application of the technology. In this study, the relationship between exposure to low levels of 2-amino-3,8-dimethyl[2-14C]imidazo[4,5-f]quinoxaline and formation of DNA adducts is examined to establish the dynamic range of the technique and the potential sensitivity for biological measurements, as well as to evaluate the relationship between DNA adducts and low-dose carcinogen exposure. Instrument reproducibility in this study is 2%; sensitivity is 1 adduct per 10(11) nucleotides. Formation of adducts is linearly dependent on dose down to an exposure of 500 ng per kg of body weight. With the present measurements, we demonstrate at least 1 order of magnitude improvement over the best adduct detection sensitivity reported to date and 3-5 orders of magnitude improvement over other methods used for adduct measurement. An additional improvement of 2 orders of magnitude in sensitivity is suggested by preliminary experiments to develop bacterial hosts depleted in radiocarbon. Expanded applications involving human subjects, including clinical applications, are now expected because of the great detection sensitivity and small sample size requirements of AMS.

Animals↗
PubMed1990

Role of metabolism on the DNA binding of MeIQx in mice and bacteria.

We report the effects of several inducers of P450 metabolizing enzymes on DNA adduct formation by 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in C57BL/6 mice. We also examined the role of N:O-acetylation and the nitrenium ion in the genotoxicity of MeIQx, since these have been implicated in the activation of other aminoimidazoazaarenes (AIA) to DNA reactive species. Mice were given phenobarbital (PB), Aroclor 1254, beta-naphthoflavone (BNF) or corn oil, i.p., followed 3-5 days later with oral administration of MeIQx. Induction of Aroclor and BNF produced DNA with 8-fold more adducts than either the corn oil-alone or PB-treated animals. Both corn oil-alone and PB-treated animals were similar. Four major adducts were found in all cases with no differences among inducers as judged by co-chromatography. Azido-MeIQx induced calf-thymus-DNA adducts produced identical adduct profiles to those seen for the mouse DNA. Similar adduct profiles were obtained from Salmonella TA98, and the nitroreductase deficient strains (TA98NR and TA98/1,8-DNP6) exposed to MeIQx in the presence of Aroclor-induced-mouse-liver S9. Adduct frequencies in TA98/1,8-DNP6 were significantly lower than in TA98 and TA98NR. The data described in this report demonstrate that induction quantitatively increases adduct numbers but does not affect the types of DNA damage. These data also suggest that the same DNA reactive intermediates are formed in vivo as in vitro and support the hypothesis that the metabolism of MeIQx involves the P450I family of isozymes, N:O-acetyltransferases and possibly a nitrenium ion. The application of radioanalytic scanners for quantitation of 32P-postlabelling adduct maps is described.

Animals↗
PubMed1990

Proposed structures for an amino-dimethylimidazofuropyridine mutagen in cooked meats.

New analytical data allow the structure of a potent mutagen previously reported present in cooked meats to be proposed. The mutagen was isolated from a fried mixture of beef, creatine and milk, simulating a meat recipe previously shown to produce this compound. High-resolution mass spectra indicate a mol. wt of 202 and the formula C10H10N4O. Mutagenic activity was 10,000 TA1538 revertants per microgram in the Ames Salmonella test. From the NMR data, peaks showing an amino group at 6.812 p.p.m. and an N-methyl group at 3.520 p.p.m. were found, typical for the amino-imidazo structure commonly found in cooked meats. Additional signals at 2.440 p.p.m. (three protons), 7.653 (one proton) and 6.531 (one proton) were observed. A structure of 2-amino-(1 or 3),6-dimethylfuro[2,3(or 3,2)-e]imidazo- [4,5-b]pyridine was proposed based on the mass and NMR spectral data and comparison to literature compounds. The methylimidazofuropyridine reported here is probably related to another mutagen in cooked meats with a mol. wt of 216 with an additional methyl group on either the furan or pyridine rings.

Animals↗
PubMed1990

Metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) by liver microsomes and isolated rabbit cytochrome P450 isozymes.

The cytochrome P450-dependent metabolism of the heterocyclic amine mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) has been determined. We investigated the in vitro metabolism of PhIP by polycyclic hydrocarbon-induced mouse and rabbit liver microsomes, and by purified rabbit liver P450 isozymes. Following a 60 min incubation, 3-methylcholanthrene-induced mouse microsomes converted 36% of the PhIP to two major metabolites, N-hydroxy-PhIP and 4'-hydroxy-PhIP, with 43% total metabolism. Rabbit P450 form 6 and form 4 produced the same two major metabolites (20 and 5% total metabolism respectively). Additional metabolites were produced in low yields and amounts varied depending on the isozyme used (1-5%). Metabolites were not detected in incubations of PhIP with P450 forms 2 and 3C. N-Hydroxy-PhIP was found to be directly mutagenic to Salmonella TA98, while the 4'-hydroxy-PhIP was not mutagenic either with or without additional metabolic activation. These data suggest that the cytochrome P450IA isozymes are involved in the metabolism of PhIP by rabbit liver and that formation of N-hydroxy-PhIP is involved in the mutagenicity of PhIP.

Animals↗