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DNA adduction in context: native metabolic activation of a foodborne procarcinogen and adducted oligonucleotide sequencing monitored by LC-MS/MS
Abstract
DNA adducts are purported to be useful biomarkers of environmental exposure to a variety of agents. On a daily basis, potential procarcinogenic and carcinogenic exposures from food, the environment and endogenous routes serve to create a burden of exposure that over 20 to 30 years ultimately leads to cumulative genetic damage and cancer. In order to appreciate the magnitude of the genotoxic exposure problem relating to DNA damage, Chapter 1 provides a review of one class of procarcinogens related to foodborne exposure: heterocyclic aromatic amines. Their formation, metabolism and quantitation are detailed in order to establish the basis for evaluating this class of compounds in this thesis. Having established the importance of testing this class of compounds, Chapter 2 details the development of a cell-based method for the native metabolic activation of the heterocyclic aromatic amine 2-amino-1-methyl-6-phenyl-imidazo[4,5-ß]pyridine (PhIP). The metabolically competent human lymphoblast cells (MCL-5) are recombinantly engineered to contain 5 Cytochrome P450 enzymes capable of Phase I redox metabolism. Dosing studies involving variable concentrations of PhIP from 5-40 µM incubated over 24 hours, a 10 µM dosing of PhIP incubated over 36 hours and a 5 µM dosing of PhIP for 6 weeks were done to evaluate the formation of PhIP-dG DNA adducts under normal cell growth conditions. An LC-MS/MS method was developed and validated for the quantitation of PhIP-dG adducts isolated from the MCL-5 cell DNA using a unique nanoelectrospray LC source. Chapters 3 and 4 continue the theme of this thesis by evaluating the sequence specific context of DNA adduction within oligonucleotides. In these chapters, methodology was developed to establish the appropriate mobile phase, column stationary phase and nanospray emitter type in order to analyze adducted oligonucleotides on a LCQ Classic ion trap. Because previous literature reports utilized an ion pairing reagent that was detrimental to the LCQ Classic, new LC conditions were required. Chapter 4 in this thesis used the LC-MS/MS method developed in Chapter 3 for the analysis of BPDE adducts formed in a 14-mer oligonucleotide sequence in which CpG dinucleotide sites were varied from monomethylated primary strands to permethylated oligomers. Adduct quantity as a function of adduct position in the 14-mer primary strand was determined without the need for digestion of the oligonucleotide. Chapter 5 evaluates the future direction of DNA adduct work for PhIP in which the quantity of PhIP-dG DNA adduct is related to gene expression changes in a bronchial epithelial cell line. Serial dilutions studies show a distinct concentration at which no transcriptional effects are observed as well as no DNA adducts detected. Additional recommendations are provided for the expansion of the use of monolithic columns for several challenging oligonucleotide problems.
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The cytochrome P450 enzymes are of great importance and interest because they catalyze reactions which have profound effects on the biological activities of drugs, environmental chemistry, and endogenous compounds. As a consequence, the literature on these enzymes is too voluminous to read, scan, or even cite. And while the pace accelerates and the scope broadens to include more biochemical aspects such as amino acid sequencing and three-dimensional structural models of isoenzymes, the bench scientists struggles to perceive where the new information fits and what applicability it has, if any, zo his/her work. Pharmacologists and toxicologists also feel swamped by the exponential increase in cytochrome P450 publications. The present paper is designed to assist by providing a framework for the prediction and interpretation of new data. It is an effort to summarize the functions of known human cytochrome P450s in terms of their specific catalytic activities, substrates, inducers, and inhibitors. Summaries are presented in tables for ready access of information. (Tables 5-21) The literature through 1996 is covered in the reference section. In some instances, references are to reviews that should be consulted for citations of the original literature.
(Full text of the paper available through Informa Healthcare Publisher only, or download updated article: Summary of information on human CYP enzymes: human P450 metabolism data. Drug Metabolism Reviews 01/2002; 34(1-2):83-448.)
In this update we provide a list of the 71 P450 genes and the four P450 pseudogenes that have been characterized as of September 30, 1988. The chromosomal locations of many of these genes are also summarized. A modest revision of the initially proposed nomenclature of the P450 superfamily (Nebert et al., DNA 6, 1-11, 1987) is described specifically for the human and mouse chromosomal loci. The motivation for this revision is to conform to the rules of nomenclature for human and mouse genes. Recommendations for the naming of chromosomal loci include the root symbol "CYP" for human ("Cyp" for mouse), denoting "cytochrome P450." We recommend that this root also be used for other organisms. For a chromosomal locus, the root symbol is followed by an Arabic numeral designating the P450 family, a letter indicating the subfamily, and an Arabic numeral representing the individual gene within the family or subfamily. Numbers of the individual genes usually will be assigned in the order the genes are identified. This system is consistent with our earlier proposed nomenclature for P450 families and gene products from all eukaryotes and prokaryotes.
In lung and liver cancers, p53 mutations are mostly G:C to T:A transversions. This type of mutation is known to be induced by benzo(a)pyrene and aflatoxin B1 which are associated with the etiology of lung and liver cancers, respectively. Using a novel assay based on DNA polymerase fingerprint analysis, we identified p53 nucleotides targeted by these carcinogens. Thirteen of 14 nucleotide residues of the p53 gene which underwent G:C to T:A mutations in lung cancers were targeted by benzo(a)pyrene. Similarly, aflatoxin B1 formed adducts at a mutational hotspot specific for liver cancer. The same nucleotide (third base of codon 249), which mutates rarely in lung cancers, was not a target for benzo(a)pyrene. These in vitro observations indicate that p53 mutational hotspots identified in different tumors are selected targets specifically for the etiologically defined environmental carcinogens.
The reaction of the (+)- and (-)-enantiomers of BDPE trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene) with the oligodeoxynucleotide d(ATATGTATA) in aqueous buffer solutions gives rise predominantly to trans and cis addition products at the exocyclic amino group of the single deoxyguanosine residue. The trans/cis ratios are 7:1 in the case of (+)-BPDE, and 2:1 in the case of (-)-BPDE, while the reaction yields correspond to 34 and 15% respectively, of modified strands. These relatively high reaction efficiencies, at least for this particular type of oligonucleotide sequence, offer the possibilities of synthesizing relatively large amounts of well-defined covalent BPDE-oligonucleotide adducts (with different sequences of nucleotides flanking the modified base) for detailed spectroscopic and biochemical studies.
In the C4-photocycloaddition at 365 nm between ³H-psoralen and native DNA, the furocoumarin reacts with the pyrimidine bases of the macromolecule mostly in the 4′-5′-positions (fluorescent photocompounds). The non-fluorescent cycloadditions (addition in the 3—4 position) form in a ratio of 1: 3 compared with the preceding ones.
Heterocyclic aromatic amines (HAAs) are mutagenic and carcinogenic compounds found in meats cooked at high temperatures. Although chicken is consumed in large quantities in the United States, there is little information on its HAA content. The objective of this study was to measure the five predominant HAAs (IQ, MeIQ, MeIQx, DiMeIQx, and PhIP) in chicken cooked by various methods to different degrees of doneness. Chicken breasts were panfried, oven-broiled, or grilled/barbecued. Whole chickens were roasted or stewed. Skinless, boneless chicken breasts were cooked to three degrees of doneness: just until done, well done, or very well done. High levels of PhIP (ranging from 12 to 480 ng/g cooked meat) were found in chicken breasts when panfried, oven-broiled, and grilled/barbecued but not in while roasted or stewed chicken. PhIP concentration increased in skinless, boneless chicken breast with longer cooking time, higher internal temperature, and greater degree of surface browning. PhIP concentration was also high in chicken breasts cooked with skin and bones. MeIQx and DiMeIQx levels increased with the degree of doneness, whereas IQ and MeIQ were not detectable in any of these chicken samples. Certain cooking methods produce PhIP, a known colon and breast carcinogen in rodents and possibly a human carcinogen, at substantially higher levels in chicken than has been reported previously in red meat.
Multiply charged anions derived from electrospray ionization of the sodium salts of various small oligonucleotides (n = 4-8) have been subjected to tandem mass spectrometry (MS/MS) in a quadrupole ion trap. All ions were observed to dissociate with high efficiencies even under conditions not ordinarily conducive for the observance of high MS/MS efficiency. Large fractions of the total product ion signal could be attributed to single-cleavage reactions with the parent ion charge shared among the two product ions in various combinations. In every case, the most facile reaction was observed to be the loss of the adenine anion. This reaction was then observed to be followed by cleavage of the 3' C-O bond of phosphodiester linkage of the sugar from which the adenine had been lost.
Treatment of the acetylated derivative (3b), which was prepared from uridine in 86% overall yield, with tri(1H-1,2,4-triazol-1-yl)phosphine oxide gave compound (6a) in high yield, and with 3-nitro-1,2,4-triazole and diphenyl phosphorochloridate it gave compound (6b) in high yield. When the former product (6a) was allowed to react with ammonia, methylamine, dimethylamine, and morpholine at room temperature, and the products further deacetylated if necessary, ara-C (1; R1= R2= H) and its corresponding 4-N-alkyl derivatives (1; R1= H, R2= Me), (1; R1= R2= Me), and [1; R1,R2=–(CH2)2O(CH2)2–] were obtained in very high yields. 4-N-Phenyl-ara-C (1; R1= H, R2= Ph) was obtained in high yield when compound (6a) or (6b) was heated with aniline in pyridine solution and the products then deacetylated. The nitro-compound (6b) was converted into the ara-C derivative (1; R1= H, R2= CH2CO2Me), and the sulphide (7) was obtained following the deacetylation of the products of the reaction between the 1,2,4-triazolyl derivative (6a), toluene-p-thiol, and triethylamine.
Capillary liquid chromatography/microelectrospray-mass spectrometry (capillary LC/mu ESI-MS) was used to quantify DNA adducts of the heterocyclic aromatic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in livers of male Fischer-344 rats. Animals received a single oral dose of either 0.05, 0.50, 1.0, or 10 mg/kg IQ and were sacrificed 24 h following treatment. The major lesion identified at all doses was N-(deoxyguanosine-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (dG-C8-IQ), The capillary LC/mu ESI-MS method provided the means for quantifying 17.5 fmol of dG-C8-IQ (2.0 adducts in 10(8) nucleosides) (S/N 10) in 300 mug of liver DNA with an intra- and interday precision of 3.5 and 6.6% (RSD), respectively, dG-C8-IQ was quantified with a mean intra-and interday accuracy of 105 +/- 26 and 106 +/- 28 (SD) based on back-calculated adduct masses from five standard curves analyzed over a four-week period. This is the first report on development of a capillary LC/mu ESI-MS method to quantify dG-CS-IQ adducts in liver DNA of rats following dosing with IQ at different levels. Furthermore, the ability to accurately and precisely quantify dG-C8-IQ at a level of 2.0 adducts in 108 nucleosides in vivo makes this method well suited for use in future studies relating carcinogen exposure to risk in humans.
We provide here a list of 481 P450 genes and 22 pseudogenes, plus all accession numbers that have been reported as of October 18,1995. These genes have been described in 85 eukaryote (including vertebrates, invertebrates, fungi, and plants) and 20 prokaryote species. Of 74 gene families so far described, 14 families exist in all mammals examined to date. These 14 families comprise 26 mammalian subfamilies, of which 20 and 15 have been mapped in the human genome and the mouse genome, respectively. Each subfamily usually represents a cluster of tightly linked genes widely scattered throughout the genome, but there are exceptions. Interestingly, the CYP51 family has been found in mammals, filamentous fungi and yeast, and plants - attesting to the fact that this P450 gene family is very ancient. One functional CYP51 gene and two processed pseudogenes, which are the first examples of intronless pseudogenes within the P450 superfamily, have been mapped to three different human chromosomes
This revision supersedes the four previous updates in which a nomenclature system, based on divergent evolution of the superfamily, has been described. For the gene, we recommend that the italicized root symbol 'CYP' for human ('Cyp' for mouse and Drosophila), representing 'cytochrome P450' be followed by an Arabic number denoting the family, a letter designating the subfamily (when two or more exist), and an Arabic numeral representing the individual gene within the subfamily. A hyphen is no longer recommended in mouse gene nomenclature. 'P' ('ps' in mouse and Drosophila) after the gene number denotes a pseudogene; 'X' after the gene number means its use has been discontinued. If a gene is the sole member of a family, the subfamily letter and gene number would be helpful but need not be included. The human nomenclature system should be used for all species other than mouse and Drosophila. The cDNAs, mRNAs and enzymes in all species (including mouse) should include all capital letters, and without italics or hyphens. This nomenclature system is similar to that proposed in our previous updates
(C) Lippincott-Raven Publishers.
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.
Heterocyclic aromatic amines (HAAs) are sometimes formed in meats and fish cooked at high temperatures. In the present study, the effects of cooking methods by deep-fat frying, pan-frying, grilling and barbecuing on the formation of HAAs of fillets of rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta fario) were investigated. Barbecued brown trout (1 g) was estimated to contain 0.12 ng of IQ (2-amino-3-methylimidazo[4,5-f]quinoline), 0.02 ng 4,8-DiMeIQx (2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline). Grilled rainbow trout (1 g) was estimated to contain 0.02 ng 4,8-DiMeIQx. MeIQ (2-amino-3,4-dimethylimidazo[4,5-f]quinoline), MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline) and PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) were not detectable in all cooked fish.
Despite the tremendous sensitivity and lower sample requirements for nanospray vs. conventional electrospray, metallized nanospray emitters have suffered from one of two problems: low mechanical stability (leading to emitter failure) or lengthy, tedious production methods. Here, we describe a simple alternative to metallized tips using polyaniline (PANI), a synthetic polymer well known for its high conductivity, anticorrosion properties, antistatic properties, and mechanical stability. A simple method for coating borosilicate emitters (1.2 mm o.d.) pulled to fine tapers (4 ± 1 μm) with water-soluble and xylene-soluble dispersions of conductive polyaniline (which allows for electrical contact at the emitter outlet) is described. The polyaniline-coated emitters show high durability and are resistant to electrical discharge, likely because of the thick (yet optically transparent) coatings; a single emitter can be used over a period of days for multiple samples with no visible indication of the destruction of the polyaniline coating. The optical transparency of the coating also allows the user to visualize the sample plug loaded into the emitter. Examples of nanospray using coatings of the water-soluble and xylene-soluble polyaniline dispersions are given. A comparison of PANI-coated and gold-coated nanospray emitters to conventional electrospray ionization (ESI) show that PANI-coated emitters provide similar enhanced sensitivity that gold-coated emitters exhibit vs. conventional ESI.
Two isomeric oligodeoxynucleotide hexamers, 5′-d(N-6meATGCAT)-3′ and 5′-d(ATG5meCAT)-3′, were subjected to analysis by electrospray and ion trap mass spectrometry. In the case of the isomer with a modified adenine, location of the modified base in the sequence was straightforward and a triple mass spectrometry experiment provided information on the identity of the modification. In contrast, the isomer with the methylated cytosine did not yield definitive information on the location or identity of the modification. Tandem mass spectrometry data in this case could indicate that the modification was present on either the third or fourth nucleoside. The two isomers represent extremes in the facility with which modified bases can be identified and located in a small oligonucleotide via multiple mass spectrometry of multiply charged anions. A preference for loss of particular bases strongly influences which structurally diagnostic ions are formed upon collisional activation. The likelihood for locating and identifying a modified base is dependent, therefore, upon the likelihood that the base is lost directly from the parent ion.
Food-related mutagens play a major role in human carcinogenesis. Heterocyclic aromatic amines which are formed in meat or fish during cooking contribute to food-related carcinogenesis, at least in rodents. The mechanism of formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was clarified by using 13C-labelled phenylalanine as a reaction partner in a model system containing additionally creatinine. Isolation of the labelled reaction product (PhIP) and 13C-NMR experiments showed that the carbon atoms of phenylalanine form a part of the pyridine moiety. Carbon atoms C-5, C-6 and C-7 in PhIP originated quantitatively from phenylalanine, leading to the conclusion that PhIP is formed by a defined mechanism and that two phenylalanine molecules are needed to form PhIP in this model system. In the proposed mechanism phenylacetaldehyde plays a key role which undergoes an aldol condensation with creatinine. The six-membered pyridine ring is completed by formation of a Schiff’s base and cyclisation.
Ion-pair reversed-phase high-performance liquid chromatography was successfully coupled to negative-ion electrospray ionization mass spectrometry by using 60 × 0.20 mm i.d. capillary columns packed with 2.3-μm micropellicular, octadecylated poly(styrene/divinylbenzene) particles as stationary phase and gradients of acetonitrile in 50 mM aqueous triethylammonium bicarbonate as mobile phase. Systematic variation of the eluent composition, such as concentration of ion-pair reagent, anion in the ion-pair reagent, solution pH, and acetonitrile concentration led to the conclusion that most parameters have opposite effects on chromatographic and mass spectrometric performances. The use of acetonitrile as sheath liquid enabled the rapid and highly efficient separation and detection of phosphorylated and nonphosphorylated oligonucleotides ranging in size from 8 to 40 nucleotides. High-quality full-scan mass spectra showing little cation adduction were acquired from which the molecular masses of the separated oligonucleotides were calculated with an accuracy of 0.011%. With calibration curves being linear over at least 2 orders of magnitude, the lower limits of detection for a oligodeoxythymidine 16-mer were 104 fmol with full scan and 710 amol with selected-ion-monitoring data acquisition. The potential of ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry was demonstrated for mixed-sequence oligomers by the characterization of a reaction mixture from solid-phase synthesis of a 40-mer oligonucleotide.
A new interface procedure has been developed that allows, for the first time, the high-efficiency analysis of synthetic oligonucleotides up to 75 bases by reversed-phase HPLC and on-line electrospray ionization mass spectrometry. For oligonucleotides up to 30 bases in length, single-base resolution can be obtained with low levels of cation adduct formation in the negative ion electrospray mass spectra. A key part of the method uses 1,1,1,3,3,3-hexafluoro-2-propanol as an additive to the HPLC mobile phase, adjusted to pH 7.0 with triethylamine. This novel additive results in both good HPLC separation and efficient electrospray ionization. The broad potential of this new method is demonstrated for synthetic homopolymers of thymidine (PolyT), fragments based on the pBR322 plasmid sequence, and phosphorothioate ester antisense oligonucleotides. This approach will be of particular utility for the characterization of DNA probes and PCR primers and quality control of antisense compounds such as phosphorothioates and their metabolites, as well as of materials used in clinical trials.
In the lacI gene of Escherichia coli spontaneous base substituion hotspots occur at 5-methylcytosine residues. The hotspots disappear when the respective cytosines are not methylated. We suggest that the hotspots may result from the spontaneous deamination of 5-methylcytosine to thymine, which is not excised by the enzyme DNA-uracil glycosidase.
Nach der Incubation von 3H-Benzo[a]pyren in Rinder-Bronchialgewebe läßt sich im Explantat ein Nucleinsäure-Addukt des Epoxids (I) nachweisen.
The covalent binding of the N-acetoxy-, N-hydroxy-, and nitro derivatives of the food-borne carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to 2'-deoxyribonucleosides or DNA was investigated in vitro and in vivo. N-Acetoxy-PhIP reacted with deoxyguanosine (dG), but not with the other deoxyribonucleosides, to form N-(deoxyguanosin-8-yl)-PhIP (dG-C8-PhIP), whose structure was determined by NMR and mass spectral analyses and by ultraviolet absorption and pH-solvent partitioning characteristics. While reaction of N-acetoxy-PhIP with calf thymus DNA at pH 5.0 yielded 5.38 +/- 1.16 nmol of bound PhIP residues/mg of DNA, N-hydroxy-PhIP gave only 0.13-0.23 nmol binding/mg of DNA under identical reaction conditions. Nitro-PhIP produced no detectable binding under these conditions. HPLC analysis of 1-butanol extracts of enzymatically hydrolyzed DNA that had been modified by N-acetoxy-PhIP in vitro showed a major adduct which coeluted with and had an ultraviolet absorption and a mass spectrum that were identical to that of authentic dG-C8-PhIP. 32P-Postlabeling analysis of DNA isolated from colon, pancreas, lung, heart, and liver of rats treated orally with PhIP revealed the presence of a major PhIP-DNA adduct. This adduct had chromatographic properties identical to that of the 32P-labeled bis(phosphate) derivative of dG-C8-PhIP and represented 35-45% of the total adducts.(ABSTRACT TRUNCATED AT 250 WORDS)
Direct genomic sequencing revealed that cytosine residues known to have undergone a germ-line mutation in the low density lipoprotein receptor gene or somatic mutations in the p53 tumor suppressor gene were methylated in all normal human tissues analyzed. Thus, these mutations should be scored as transitions from 5-methylcytosine to thymine rather than from cytosine to thymine. Methylated cytosines occur exclusively at CpG dinucleotides, which, although markedly underrepresented in human DNA, are sites for more than 30 percent of all known disease-related point mutations. Thus, 5-methylcytosine functions as an endogenous mutagen and carcinogen in humans, in that methylation seems to increase the potential for mutation at cytosine residues at least by a factor of 10.
The effect of glucose on the formation of the food mutagen PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) was studied in a model system. When a mixture of creatine (0.9 mmol), phenylalanine (0.9 mmol) and glucose (0.45 mmol) was heated in diethylene glycol and water (3 ml, 5:1) for 10 min at 180 or 225 degrees C several mutagens were produced. Identification by HPLC, UV absorption spectroscopy and mass spectrometry revealed the presence of PhIP as well as 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and minor amounts of 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline. Heating the system without glucose produced PhIP as a single mutagen, but in considerably lower amount. An inhibiting effect of glucose in high concentrations was demonstrated. When glucose was added in more than or equimolar amounts of the other two reactants, the formation of mutagens was markedly reduced. Tyrosine heated under the same conditions, with creatine and glucose, showed mutagenic activity. However, no PhIP nor any other known food mutagen was identified from the tyrosine mixture.
A human B-lymphoblastoid cell line, designated MCL-5, constitutively expressing human cytochrome P-450 CYP1A1 and also expressing five transfected human cDNAs encoding drug-metabolizing enzymes, has been developed. cDNAs encoding CYP1A2, CYP2A6, and microsomal epoxide hydrolase (mEH) were introduced by using a vector conferring hygromycin B resistance, and cDNAs encoding CYP2E1 and CYP3A4 were introduced by using a vector conferring resistance to 1-histidinol. MCL-5 cells stably expressed all five cDNAs and the native CYP1A1 as determined by measurement of form-specific enzyme activity levels. The mutagenicity of seven model procarcinogens to MCL-5 cells was examined at the hypoxanthine guanine phosphoribosyltransferase (hprt) and thymidine kinase (tk) loci. Exposure to benzo[a]pyrene (BP), 3-methylcholanthrene (3MC), N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), aflatoxin B1, (AFB1), 2-(acetylamino)fluorene (AAF), or benzidine (BZD) induced a statistically significant increase in mutant frequency. Linear interpolation of the concentration of procarcinogen necessary to produce a doubling of the mutant fraction at the hprt locus in MCL-5 cells and the parent AHH-1 cell line revealed that, for each of the chemicals examined, except BZD, MCL-5 cells were significantly more sensitive than the parent AHH-1 cells. The increase in sensitivity to mutagenicity ranged from 3-fold for AAF to greater than 40,000-fold for NDMA. MCL-5 cells have great potential as a screening system for the analysis of human procarcinogen/promutagen activation.
Chemical and physical carcinogens leave footprints of their activities on DNA because of the patterns of base changes they induce. Additionally, the conversion of 5-methylcytosine to thymine in CpG sequences leads to a characteristic mutation which can be used to estimate the contribution of endogenous processes to human mutations. Knowledge of the pattern mutations found in genes commonly mutated in human cancer, such as the p53 tumor suppressor gene, allows for predictions to be made on the likelihood of an exogenous DNA-damaging agent being involved. Working from gene to carcinogen is likely to have a profound impact on our understanding of the origins of human cancer.
Fast atom bombardment (FAB) and tandem mass spectrometry (MS/MS) are shown to be useful methods for the detection and structural characterization of nanogram amounts of amino polyaromatic hydrocarbon-nucleoside DNA adducts. The positive ion spectra of four aromatic amine guanosine adducts were studied in detail. The FAB spectra of these adducts exhibit an [MH]+ ion and a more abundant aglycon fragment ion, [AH2]+, which results from the loss of the deoxyribose sugar. The sensitivity of the adducts to FAB was enhanced by preparing trimethylsilyl (TMS) ether derivatives. High-quality full-scan spectra could be obtained on less than 70 ng of the derivatized adducts without signal averaging. With a B/E-linked scan of the [MH]+ ion for the TMS2 species, these same adducts could be detected by examination of their metastable ion spectra at levels as low as 4-5 ng (S/N greater than 10). Collision-induced dissociation (CID) of the [MH]+ ion yields the aglycon fragment and an ion, S1, which results from cleavage through the sugar. The CID spectrum of the aglycon [AH2]+ ion is much more useful, providing structural information relating to the base, the polyaromatic hydrocarbon, and, possibly, the site of covalent attachment. Differentiation of isomeric aminophenanthrene-guanine adducts was demonstrated on the basis of the CID spectra of their respective [AH2]+ ions. The use of TMS derivatives also improves the sensitivity of these methods.
Rat liver microsomes metabolized the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to the genotoxic metabolite 2-hydroxamino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (2-hydroxamino-PhIP) and to the detoxified product 2-amino-4'-hydroxy-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (4'-hydroxamino-PhIP). A 25-fold higher rate of metabolism was measured in microsomes from polychlorinated-biphenyl-treated
rats (94 nmol/mg proteins/30 min) in comparison with those from untreated rats. Other effective inducers of PhIP metabolism
were β-naphthoflavone and isosafrole (ISF), whereas phenobarbital was ineffective. About twice as much 2-hydroxamino-PhIP as 4'-hydroxy-PhIP
was formed in microsomes irrespective of the inducer the rats had been treated with. The metabolism was dependent on NADPH
and was abolished by the cytochrome P450 inhibitor α-naphthoflavone. In a reconstituted enzyme system purified rat cytochrome P450 IA2 (P450ISF-G) had the highest N-hydroxylation rate (30 nmol/nmol P450/30 min) closely followed by the rat cytochrome P450 IA1 (P450BNF-B). Less activity was seen with rat P450 IIC11 (P450UT-A) and rabbit P450 IA2 (P450 LM4). Rat P450 IIE1 (P450j), P450 IIE1 (P450PB-B) and rabbit P450 IIB4 (P450 LM-2) and P450 IIE1 (P450 LM3a) were essentially inactive. Rat P450 IA1 (P450BNF-B) produced five times more 4'-hydroxy-PhIP (32 ± 2 nmol/nmol P450/30 min) than did P450 IA2(P450ISF-G). Hence, the measured ratio of activation to detoxication for rat P450 IA2 (P450ISF-G) enzyme was 7-fold higher than that of the other active P45O enzymes.
The characterization of eight benzo[a]pyrene-deoxyribonucleoside adducts derived from reaction of the anti-dihydrodiol epoxide and deoxyguanylic and deoxyadenylic acids is described. It is reported that the epoxide ring is opened by the purine amino groups to yield similar amounts of both cis and trans products. NMR data show that the 7- and 8-hydroxyl groups are pseudodiaxial in the cis products and pseudodiequatorial in the trans products, and we suggest that these products arise from reaction with the diaxial and diequatorial conformers of the dihydrodiol epoxides, respectively. The chiral nature of the interactions of these metabolites with DNA restricts the range of products formed with this macromolecule, and a trans product with deoxyguanosine is the major product formed with either enantiomer of the anti-dihydrodiol epoxide.
1. The P450 gene superfamily is presently known to contain more than 78 members, divided into 14 families. 2. The superfamily has undergone divergent evolution, and the ancestral gene is probably more than 2 billion years old. 3. The recent 'burst' in new P450 genes, particularly in the II family during the past 800 million years, appears to be the result of 'animal-plant warfare'. 4. Due to the presence or absence of a particular P450 gene in one species but not the other, it may not be correct to extrapolate toxicity or cancer data from rodent to human. 5. Increases in the P450 gene product (enzyme induction) almost always reflect an elevated rate in gene transcription, although there are several exceptions. 6. The mechanisms of P450 gene regulation (induction) by classes of inducers might become better understood through the comparison of different phyla that differ in response to a particular class of inducers. 7. Amongst several carefully selected phyla, delineation between which electron donor (presence of Fe2S2 protein or NADPH-P450 oxidoreductase, or both) interacts with P450 may provide valuable information about the evolution of eukaryotes from prokaryotes.
Electrophoric derivatives of 5-methylcytosine and 5-hydroxymethyluracil nucleobases are determined using high-performance liquid chromatography-mass spectrometry coupled via a moving-belt interface. Standards as well as samples derived from DNA are analysed. As little as 9.9 pg (signal-to-noise ratio 5) and 180 fg (signal-to-noise ratio 10) of the respective nucleobases are detected in the electron-capture negative chemical ionization mode, and linear responses are observed over a moderate dynamic range. In a comparison study, liquid chromatography-electron-capture negative chemical ionization mass spectrometry is found to have a sensitivity comparable to gas chromatography-electron-capture negative chemical ionization mass spectrometry for 5-hydroxymethyluracil. A detection limit of 60 fg (signal-to-noise ratio 5) by gas chromatography-mass spectrometry is only three-fold better than the amount detected by liquid chromatography-mass spectrometry using the same mass spectrometer.
Wood workers have been previously reported to be at higher risk for the development of cancers of certain sites, including the nasal cavity, paranasal sinuses, lung, stomach, and lymphatic and hemopoietic tissues. Wood work involves exposure to a variety of potential carcinogens, including wood dust itself, chemicals applied to the wood, and other carcinogenic agents that are associated with wood work. We report a series of case-control studies based on the New Zealand Cancer Registry. These studies involved 19,904 male patients registered with cancer from 1980 to 1984 who were 20 years of age or older at the time of registration. For each cancer site studied, the registrants for all other sites (except lung cancer) formed the control group. The following four cancer sites were found to be associated with wood work: lip, nasopharynx, lung, and liver. There was little evidence of increased risks for other cancer sites. Among wood workers, sawmillers experienced the highest risks for lung cancer (odds ratio, 1.76; 95% confidence interval, 1.23 to 2.52) and liver cancer (odds ratio, 3.55; 95% confidence interval, 1.09 to 0.14). Carpenters showed increased risks for lip cancer (odds ratio, 2.28; 95% confidence interval, 1.23 to 4.14) and lung cancer (odds ratio, 1.27; 95% confidence interval, 1.05 to 1.54). The increased risk of nasopharyngeal cancer was strongest for foresters and loggers (odds ratio, 6.02; 95% confidence interval, 1.01 to 28.41).
Chinese hamster ovary cells were exposed to N-hydroxy-2-aminofluorene, N-hydroxy-N'-acetylbenzidine and 1-nitrosopyrene, and the resulting DNA adducts, sister-chromatid exchanges (SCEs) and mutations at the hypoxanthine-guanine phosphoribosyl transferase locus were quantified. Each agent produced a major DNA adduct substituted through the C8 of deoxyguanosine. When the data from all three agents were combined, both mutation and SCE induction correlated strongly with the concentration of DNA adducts. However, significant differences were found in the relationships between adduct formation and the biological responses produced by the individual agents. While N-hydroxy-N'-acetylbenzidine induced the most mutations per adduct, N-hydroxy-2-aminofluorene caused the greatest number of SCEs per adduct. The data support the involvement of C8-deoxyguanosine adducts in mutation and SCE induction, and indicate that the structure of the group adducted to DNA may be an important factor in determining the magnitude of these biological responses. These findings also suggest that SCE and mutation induction are independent expressions of DNA damage.
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.
When a mixture of creatinine, phenylalanine and glucose in diethylene glycol-water solution was heated for 2 h at 128 degrees C, a mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was found to be produced, identified by high-performance liquid chromatography, and UV-visible and mass spectrometries. The yield of PhIP was 3.6 nmoles/mmole original creatinine. PhIP was originally isolated from cooked beef, and this study shows creatinine, phenylalanine and glucose to be the probable precursors of PhIP in beef.
Substitution of a methyl group in the 5-position of pyrimidines increases melting temperatures and modifies biological properties of DNA. Increased DNA stability is often attributed to hydrophobic interactions between water and the methyl group. However, we present evidence that the major effect of methyl substitution is to increase the molecular polarizability of the pyrimidine, thereby increasing the base stacking. Experimentally determined base stacking interaction constants for free bases in water are shown to correlate well with calculated molecular polarizability and DNA melting temperatures.
A new mutagenic compound has been isolated from ground beef which was fried at 300 degrees C for 5.5 min on each side. The new mutagen was purified using an aqueous acid extraction, XAD-2 adsorption-solvent elution, a series of preparative and analytical h.p.l.c. purification steps, and monitored with the Ames/Salmonella assay. This study reveals a new mutagen member of the amino-imidazoazaarene class of aromatic amines, having a mol. w of 224, and a formula of C13H12N4 as determined by high-resolution mass spectrometry. N.m.r. spectrometry supports the structure, 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP), for the new mutagen. The 1-methyl and 3-methyl synthesized isomers of PhIP were compared to the purified mutagen. The two isomers had identical mass spectra to the purified compound, but only the 1-methyl isomer showed similar u.v. and n.m.r. spectra. The two synthetic isomers were separable by h.p.l.c. and the beef derived component co-eluted with the 1-methyl-PhIP isomer. PhIP has a specific activity in the Ames/Salmonella assay of 1950 revertants/microgram. Although it is not as mutagenic as other compounds isolated from fried beef (e.g. MeIQx, 58 000 revertants/microgram) it is the most abundant mutagenic compound by mass in fried beef. PhIP is present at approximately 15 p.p.b. of the original weight of uncooked beef (accounting for 75% of the mass of genotoxic material) and contributes 18% of the total mutagenicity of the fried beef.
The present arrangement collects particles and semivolatiles of main- and sidestream smoke and allows a recovery of the trapped substances nearly quantitatively and without impurities. The fractionation procedure allows to separate various groups of carcinogens such as PAH, aza-arenes and aromatic amines for analytical and biological studies. Sidestream smoke contains ten times more polycyclic aromatic hydrocarbons (PAH) compared with mainstream smoke. This holds also true for aza-arenes and amines. PAH of the gaseous phases include only 1% of the particle-bound PAH.
18 Carcinogens, including aflatoxin B(1), benzo(a)pyrene, acetylaminofluorene, benzidine, and dimethylamino-trans-stilbene, are shown to be activated by liver homogenates to form potent frameshift mutagens. We believe that these carcinogens have in common a ring system sufficiently planar for a stacking interaction with DNA base pairs and a part of the molecule capable of being metabolized to a reactive group: these structural features are discussed in terms of the theory of frameshift mutagenesis. We propose that these carcinogens, and many others that are mutagens, cause cancer by somatic mutation. A simple, inexpensive, and extremely sensitive test for detection of carcinogens as mutagens is described. It consists of the use of a rat or human liver homogenate for carcinogen activation (thus supplying mammalian metabolism) and a set of Salmonella histidine mutants for mutagen detection. The homogenate, bacteria, and a TPNH-generating system are all incubated together on a petri plate. With the most active compounds, as little as a few nanograms can be detected.
Several carcinogenic metabolites of the carcinogen 2-acetyl-aminofluorene, especially 2-nitrosofluorene and N-hydroxy-2-aminofluorene, are potent frameshift mutagens for Salmonella typhimurium. 2-Nitrosonaphthalene, 2-nitrosophenanthrene, 4-nitroso-trans-stilbene, 4-nitrosobiphenyl, and 4-nitrosoazobenzene, all of which are metabolites or likely metabolites of carcinogenic aromatic amines, are also potent frameshift mutagens. These compounds may be frameshift mutagens of the class that intercalates into DNA and then reacts covalently with the DNA; various ultimate carcinogens may be of this type. The utility of a set of bacterial strains for detecting carcinogens as mutagens is shown.
The simplest interpretation of the author's and other's work on carcinogens as mutagens is that carcinogens cause cancer because of their action as mutagens. The principle of the mutagenesis for a large group of the carcinogens can be explained by the theory of frameshift mutagenesis. A simple bacterial test can be used to see if a compound is likely to be a carcinogen for humans (mammalian testing is, of course, expensive and takes years). It is thought that the prediction of what will be carcinogenic can be put on a more rational basis.
A nonanucleotide, d(G1G2T3C4[BaP]A5C6G7A8G9), in which (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene (7-hydroxyl group and epoxide oxygen are trans) is covalently bonded to the exocyclic N6-amino group of deoxyadenosine (dA5) through trans addition at C10 of the epoxide (to give a 10S adduct) has been synthesized. The solution structure of the duplex, d(G1G2T3C4[BaP]A5C6G7A8G9).d(C10T11C12G13G14G15A16C17C18+ ++), containing a dG mismatch opposite the modified dA (designated 10S-[BaP]dA.dG 9-mer duplex) has been investigated using a combination of 1D and 2D (including COSY, PECOSY, TOCSY, NOESY, and indirect detection of 1H-31P HETCOR) NMR spectroscopies. The NMR results together with restrained molecular dynamics/energy minimization calculations show that the modified dA5 adopts a syn glycosidic torsion angle whereas all other nucleotide residues adopt anti glycosidic torsion angles. The sugar ring of dA5 is in the C3'-endo conformation, and the sugar rings of the other residues are in the C2'-endo conformation. The hydrocarbon attached at dA5 orients toward the 3' end of the modified strand (i.e., dC6 direction) and intercalates between and parallel to bases of dG13 and dG14 of the complementary strand directly opposite dC6 and dA5, respectively. The edge of the hydrocarbon bearing H11 and H12 is positioned between the imino protons of dG13 and dG14 in the interior of the duplex, whereas H4 and H5 at the opposite edge are positioned near the sugar H1' and H2" protons of dG13 and facing the exterior of the duplex. The mismatched AG base pair is stabilized by dAsyn-dGanti base pairing in which the imino proton and the O6 of dG14 are hydrogen bonded to N7- and the single N6-amino proton, respectively, of the modified dA5. The modified DNA duplex remains in a right-handed helix, which bends at the site of intercalation about 20 to 30 degrees away from the helical axis and toward the direction of the modified strand.
Frequent consumers of meat have an increased risk of colorectal cancer and possibly also of breast, stomach, pancreas and urinary bladder cancer. Bacon, 'Falusausage', ground beef, meatballs, pork belly, pork chops and sliced beef account for more than one-third of the intake of fried meat of the population of Stockholm of age 50-75. These dishes were fried at four temperatures (150, 175, 200 and 225 degrees C) representing normal household cooking practices in Stockholm. Heterocyclic amines in these dishes were analysed using solid-phase extraction and HPLC. The heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were recovered. The formation of IQ was favoured by moderate cooking temperatures; IQ was detected in one meat sample cooked at 150 degrees C and in some pan residues. The yield of MeIQx, DiMeIQx and PhIP increased with the temperature. For several of the meat dishes, the content of heterocyclic amines in the pan residue was as large or larger than for corresponding piece of meat. The highest levels of MeIQx were 23.7 ng/g in the meat and 23.3 ng/g in the pan residue. Corresponding data for DiMeIQx were 2.7 and 4.1 ng/g and for PhIP 12.7 and 82.4 ng/g. The study leaves little doubt that mutagenic heterocyclic amines are ingested by the population of Stockholm, and added to previous epidemiological studies from the same area, the combined data are consistent with human carcinogenicity of heterocyclic amines. However, analytical epidemiological studies are needed before any statement on causality can be made.
CpG dinucleotides are the target of about one third of transition mutations found in human genetic diseases and tumors. Methylation at these sites is thought to be the cause of these genetic changes through spontaneous deamination of 5-methylcytosine. In order to define the contribution of 5-methylcytosine to the spectrum of p53 mutations in human cancers, we have determined the complete DNA methylation pattern along exons 5-8 of the human p53 gene by ligation-mediated polymerase chain reaction genomic sequencing. The study was conducted with nine different types of normal human tissue and cell lines, including skin fibroblasts, keratinocytes, lung epithelial cells, mammary epithelial cells and colonic mucosa cells. We found that the p53 sequences along exons 5-8 are completely methylated at every CpG site, including 46 different sites on both DNA strands. This methylation pattern is tissue-independent suggesting that tissue-specific methylation does not contribute to the differential mutation patterns seen in tumors. The occurrence of mutational hotspots at specific CpG sites is not related to selective methylation of only a subset of CpGs but may rather depend on a selection bias for particular amino acid changes. Our results are not inconsistent with theories that mutations in tumors with high CpG mutation rates, like colon cancer, are caused by spontaneous deamination of 5-methylcytosine and mutations in tumors with a lack of CpG involvement reflect superimposed fingerprints from exogenous carcinogens. However, given the lack of tissue specificity of methylation, alternative explanations (eg targeting of methylated CpG sites by tissue-selective carcinogens) should be considered to explain the high percentage of CpG mutations in some tumor types.
Sample stacking is used to improve the detection limits of capillary zone electrophoresis coupled to continuous flow fast atom bombardment mass spectrometry for the analysis of DNA adducts. It was found that, with stacking, the concentration detection limit of deoxynucleotide adducts could be improved by as much as 3 orders of magnitude, thereby bringing it into the 10(-8) M range. In addition, the mass spectrometric mass detection limits of a model acetylaminofluorene deoxyguanosine 5'-monophosphate adduct were found to be in the low picomole range for full scanning and the low femtomole range for multiple reaction monitoring of a selected fragmentation. The techniques have been applied to the analysis of adducts formed in the in vitro reaction of N-acetoxy-N-acetyl-2-aminofluorene with DNA.
A nonanucleotide in which (-)-(7S,8R,9R,10S)-7,8-dihydroxy-9,10-epoxy- 7,8,9,10-tetrahydrobenzo[a]pyrene (7-hydroxy group and epoxide oxygen are trans) is covalently bonded to the exocyclic N6-amino group of deoxyadenosine through trans addition at C10 of the epoxide (10R adduct) has been synthesized. The modified oligonucleotide d(GGTCA*CGAG) was incorporated into the duplex d(GGTCA*CGAG).d(CTCGGGACC), containing a dG mismatch opposite the modified base (dA*). Proton assignments for the solution structure of the duplex containing the 10R adduct were made using 2D TOCSY and NOESY NMR spectra. The complete hybrid relaxation matrix program, MORASS2.0, was used to generate NOESY distance constraints for iterative refinement using distance-restrained molecular dynamics calculations with AMBER4.0. The iteratively refined structure showed the hydrocarbon intercalated from the major groove immediately below the dC4-dG15 base pair and oriented toward the 5'-end of the modified strand. The modified dA is in an anti configuration, with the dG of the GA mismatch turned out into the major groove. Chemical shifts of the hydrocarbon protons and unusual chemical shifts of sugar protons were accounted for by this orientation of the adduct. The information available currently provides the foundation for the rational explanation of observed benzo[a]pyrene (BaP) structures and predictions for other BaP dG and dA 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).
Cooking of protein-rich foods may induce the formation of a series of heterocyclic aromatic amines (HAAs) that have been found to be mutagens and carcinogens. Despite very potent mutagenic activity found in the Salmonella/microsomal assay, this test cannot predict carcinogenic potency of HAAs in rodents and monkeys. Doses used in the feeding studies with animals exceeded by several orders of magnitude the levels of HAAs found in human diet, being approximately 500,000-3,000,000-fold higher than the human dietary levels. A comparison of these levels and their relevance for humans is presented. Differences in metabolic fate of different HAAs due to species and sex of the animals are discussed. These differences could account for the variable cancer-producing potential in different species. A number of still unresolved variables (such as the levels of HAAs in foods, bioavailability, possible synergistic effect from mixtures of HAAs, and metabolic fate and detoxification) preclude reliable assessment of the potential health hazard from HAAs in foods. The difference in the ability of human and animal liver microsomes to bioactivate HAAs and to form DNA adducts causes further uncertainty. The differences due to the hepatic cytochrome P-450-mediated activation of HAA among rats, monkeys and humans may influence susceptibility to cancer from these agents. HAAs occur only in traces in the human diet; however, they are present in many foods consumed daily. The levels of 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP) are approximately 100-fold higher than the levels of 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). However, their mutagenic potential in the Ames Salmonella assay is reversed. Other in vitro tests, however, indicate similar genotoxicity between IQ, MeIQx and PhIP. Although most feeding studies with HAAs have been conducted with IQ and MeIQ, evidence obtained from a variety of studies indicates the possibility that PhIP may have an active role in the aetiology of human cancer and, therefore, its role as such should be evaluated. The influence of trace levels of HAAs on human health remains to be confirmed.
The mutagenic heterocyclic amines 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) were measured in ground-beef patties fried at 150, 190 and 230 degrees C for 2-10 min on each side. Heterocyclic amines were purified using solid-phase extraction and analysed by HPLC. Recovery-corrected amounts of each heterocyclic amine were determined by the method of standard addition based on spiked samples with recoveries ranging from 40 to 70%. Mutagenic activity measured by the Ames/Salmonella test was determined for each sample. The amounts of MeIQx, PhIP, DiMeIQx and IQ increased with time and temperature of cooking. 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and 2-amino-9H-pyrido[2,3-b]indole (A alpha C) were not detected in any sample. The mutagenic activity response measured for the meat extracts (TA98 revertants) was similar to the mutagenic activity calculated from the mass of heterocyclic amines present. The rate of formation of PhIP in a model system containing creatinine and phenylalanine heated in 80% diethylene glycol was compared with PhIP formation during meat frying. The apparent heats of activation were 6.5 kcal/mol in the model system compared with 6.0 kcal/mol in the fried meat patties. The increase in PhIP and MeIQx formation fitted an exponential function over the range 0 to 11 min and from 150 to 230 degrees C. This report shows clearly that increases in cooking temperature and time can have a profound effect on the amounts of heterocyclic amines generated and subsequently consumed in the diet.