Peroxynitrite-induced reactions of synthetic oligo 2'-deoxynucleotides and DNA containing guanine: formation and stability of a 5-guanidino-4-nitroimidazole lesion.

Peroxynitrite is a strong oxidizing agent that is formed in the reaction of nitric oxide and superoxide anion. It is capable of oxidizing and nitrating a variety of biological targets including DNA, and these modifications may be responsible for a number of pathological conditions and diseases. A recent study showed that peroxynitrite reacts with 2',3',5'-tri-O-acetylguanosine to yield a novel compound, tri-O-acetyl-1-(beta-D-erythro-pentafuranosyl)-5-guanidino-4-nitroimidazole, and, unlike other peroxynitrite-mediated guanine oxidation products, it is a stable and significant component formed even at low peroxynitrite concentrations. In this work, we studied the in vitro formation of the guanine-derived product, 5-guanidino-4-nitroimidazole, in synthetic oligonucleotides and DNA treated with peroxynitrite. When calf thymus DNA or oligonucleotides were reacted with peroxynitrite at ambient temperature, the modified base 5-guanidino-4-nitroimidazole was generated along with several other products. The oligonucleotides containing the 5-guanidino-4-nitroimidazole modification were purified by reverse-phase and anion-exchange HPLC and characterized by matrix-assisted laser desorption mass spectrometry. 5-Guanidino-4-nitroimidazole formation in peroxynitrite-treated DNA was characterized after enzymatic digestion of the reacted DNA to the nucleoside level. HPLC purification and electrospray ionization mass spectrometry (with selected reaction monitoring) enabled the analysis of this modified nucleoside with high sensitivity. The yield of 5-guanidino-4-nitroimidazole formed in single-stranded DNA was approximately 10-fold higher than that found in duplex DNA. With calf thymus DNA, 5-guanidino-4-nitroimidazole was dose-dependently formed at low peroxynitrite concentrations. In stability tests, a synthetic oligonucleotide containing the 5-guanidino-4-nitroimidazole modification was only partially cleaved by hot piperidine and was a weak substrate for Fpg glycosylase repair enzyme; in addition, this site was not cleaved by endonuclease III. These results suggest that nuclear DNA containing 5-guanidino-4-nitroimidazole may not be quickly repaired by DNA repair enzyme systems. Finally, primer extension experiments revealed that this lesion is a potential DNA replication blocker when polymerization is catalyzed by polymerase alpha and polymerase I (Klenow fragment, lack of exonuclease activity) but not with human polymerase beta. Replication fidelity experiments further showed that 5-guanidino-4-nitroimidazole may cause G-->T and G-->C transversions in calf thymus polymerase alpha and E. coli polymerase I.

Excretion of the N(2)-glucuronide conjugate of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine in urine and its relationship to CYP1A2 and NAT2 activity levels in humans.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a mutagenic and carcinogenic heterocyclic aromatic amine formed in meat products during cooking. The genotoxity of PhIP requires an initial cytochrome P450-mediated N-oxidation followed by N-O-esterification catalyzed generally by N-acetyltransferases and sulfotransferases. This study examined the urinary excretion of N(2)-(beta-1-glucos-iduronyl)-2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine-the major human urinary N-oxidation metabolite of PhIP-and determined its relationship to individual activity levels of cytochrome P4501A2 (CYP1A2) and N-acetyltransferase (NAT2). The subjects (33 males and 33 females) in the dietary study were phenotyped for their CYP1A2 and NAT2 activity prior to consumption of meat-based diet, and urine collections were obtained 0-12 and 12-24 h after ingestion of the meal. Acidic hydrolysis of N(2)-(beta-1-glucosiduronyl)-2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine and its d(3)-analog to form their respective deaminated products 2-hydroxy-1-methyl-6-phenylimidazo[4,5-b]pyridine (2-OH-PhIP) was used in the assay. The products after derivatization were analyzed by capillary gas chromatography-negative ion chemical ionization mass spectrometry with selective ion monitoring. The amount of N(2)-(beta-1-glucosiduronyl)-2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine measured as the acid hydrolysis product 2-OH-PhIP in the 0-12 h urine was 20.2 +/- 8.0% (mean +/- SD) of the ingested dose; the median was 18.8% and the range varied from 5.4 to 39.6% within the group. In a subset (n = 18) of samples from individual urine collected from the 12-24 h period, an average value of 4.4 +/- 2.5% (+/- SD) of the dose was recovered. The excretion of N(2)-(beta-1-glucosiduronyl)-2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine in the 0-12 h urine was significantly related to the quantity of PhIP ingested for all subjects (r = 0.52, P <0.0001). Linear regression analysis of the relationship between the excretion level of N(2)-(beta-1-glucosiduronyl)-2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine, adjusted for meat intake and CYP1A2 activity in the combined group of males and females showed a low association (r = 0.25, P = 0.05). There was no association between the amount of N(2)-(beta-1-glucosiduronyl)-2-hydroxyamino-1-methyl-6-phenylimid-azo[4,5-b]pyridine in urine and NAT2 activity levels of the subjects nor with the age of the subjects. N(2)-(beta-1-glucosi-duronyl)-2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine comprised a significant proportion of the ingested dose in some individuals; however, considerable variation was found within the group. The results indicate that interindividual differences in the rates of N-oxidation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, as well as phase II glucuronidation reactions regulate the formation of this metabolite in humans.

N-oxidative metabolism of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in humans: excretion of the N2-glucuronide conjugate of 2-hydroxyamino-MeIQx in urine.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), a major heterocyclic aromatic amine (HAA) formed in cooked meats, is metabolically transformed to mutagenic/carcinogenic intermediates. Cytochrome P4501A2 (CYP1A2)-mediated N-hydroxylation followed by phase II O-esterification by N-acetyltransferase (NAT2) are generally regarded as activation processes in which MeIQx and other HAAs are converted to genotoxic species. In this study, we determined the relationship between the activities of these two enzymes and the urinary excretion level of the N2-glucuronide conjugate of 2-hydroxyamino-MeIQx--N2-(beta-1-glucosiduronyl)-2-hydroxyam ino-3,8-dimethylimidazo[4,5-f]quinoxaline (N-OH-MeIQx-N2-glucuronide)--among healthy subjects fed a uniform diet containing high-temperature cooked meat. The individuals (n = 66) in the study ate meat containing known amounts of MeIQx, and urine was collected from 0 to 12 h after the meal. After addition of the deuterium-labeled internal standard to urine, N-OH-MeIQx-N2-glucuronide was isolated using solid-phase extraction and immunoaffinity separation. The isolated conjugate was converted to the deaminated product 2-hydroxy-3,8-dimethylimidazo[4,5-f]quinoxaline (2-OH-MeIQx) by heating with acetic acid. 2-OH-MeIQx and its deuterated analogue were derivatized to form the corresponding 3,5-bis(trifluoromethyl)benzyl ether derivatives and analyzed by capillary gas chromatography-negative ion chemical ionization mass spectrometry using selected ion monitoring procedures. The subjects in the study excreted an average of 9.4 +/- 3.0% (+/-SD) of an ingested dose of MeIQx as N-OH-MeIQx-N2-glucuronide in urine; the range varied from 2.2 to 17.1%. A significant correlation was found between the level of N-OH-MeIQx-N2-glucuronide in urine and the amount of MeIQx ingested (r(s) = 0.44; P = 0.0002). The excretion level of N-OH-MeIQx-N2-glucuronide in urine was not associated with the enzyme activities of NAT2 or CYP1A2. This is expected with the latter enzyme because the metabolism of MeIQx is first order and very rapid at the amounts ingested. The amount of N-OH-MeIQx-N2-glucuronide in urine was not correlated with the age or sex of the individuals. Our results indicate that biotransformation of MeIQx via CYP1A2 oxidation to form the N-hydroxylamine followed by N2-glucuronidation is a general pathway of MeIQx metabolism in humans; the variability in the excreted levels of N-OH-MeIQx-N2-glucuronide is probably due to interindividual differences in UDP-glucuronosyltransferase activity and/or excretion pathways.

Biomonitoring of heterocyclic aromatic amine metabolites in human urine.

Human exposure to heterocyclic aromatic amines such as MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline) may be monitored by measuring the levels of the heterocyclic aromatic amine in urine. In order to investigate the contribution of N-oxidation to the metabolism of MeIQx in vivo, we developed a biomonitoring procedure for the analysis and quantification of the N2-glucuronide conjugate of 2-hydroxyamino-3,8-dimethylimidazo[4,5-f]quinoxaline in human urine. Subjects (n = 66) in the dietary study ingested a uniform diet of cooked meat containing known amounts of MeIQx, and urine was collected after consumption of the test meal. A method based on solid-phase extraction and immunoaffinity separation was used to isolate N2-(beta-1-glucosiduronyl)-2-hydroxyamino-3,8-dimethylimidazo++ +[4,5-f]quinoxaline and its stable isotope-labeled internal standard from urine. The isolated conjugate was converted to the deaminated product 2-hydroxy-3,8-dimethylimidazo[4,5-f]quinoxaline by treatment with acetic acid under moderate heating. 2-Hydroxy-3,8-dimethylimidazo[4,5-f]quinoxaline and the [2H3]methyl analog were derivatized to form the corresponding 3,5-bis(trifluoromethyl)benzyl ether derivatives and quantified by capillary gas chromatography-negative ion chemical ionization mass spectrometry employing selected ion monitoring procedures. The amounts of N2-(beta-1-glucosiduronyl)-2-hydroxyamino-3,8-dimethylimidazo++ +[4,5-f]quinoxaline recovered in urine collected 0-12 h after the test meal accounted for 2.2-17.1% of the ingested dose, with a median value of 9.5%. The variability in the proportion of the dose excreted among the subjects may be reflective of several factors, including interindividual variation in the enzymic activity of CYP1A2 and/or conjugation reactions of the N-hydroxylamine metabolite with N-glucuronosyltransferase(s).

Urinary excretion of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in White, African-American, and Asian-American men in Los Angeles County.

Meats, such as beef, pork, poultry, and fish, cooked at high temperatures produce heterocyclic aromatic amines, which have been implicated indirectly as etiological agents involved in colorectal and other cancers in humans. This study examined the urinary excretion of a mutagenic/carcinogenic heterocyclic aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), among 45 African-American, 42 Asian-American (Chinese or Japanese), and 42 non-Hispanic white male residents of Los Angeles who consumed an unrestricted diet. Total PhIP (free and conjugated) was isolated from overnight urine collections, purified by immunoaffinity chromatography, and then quantified by high-pressure liquid chromatography combined with electrospray ionization mass spectrometry. Geometric mean levels of PhIP in Asian-Americans and African-Americans were approximately 2.8-fold higher than in whites. The urinary excretion levels of PhIP were not associated with intake frequencies of any cooked meat based on a self-administered dietary questionnaire, in contrast to our earlier finding (Ji et al., Cancer Epidemiol. Biomark. Prev., 3: 407-411, 1994) of a positive and statistically significant association between bacon intake and the urinary level of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) among this same group of study subjects. Although there is a statistically significant association between urinary levels of PhIP and MeIQx (2-sided P = 0.001), 10 subjects (8%) displayed extreme discordance between urinary PhIP and MeIQx levels. Several factors, including variable contents of heterocyclic aromatic amines in food, enzymic and interindividual metabolic differences, and analytical methodology determine the degree of concordance between the urinary excretion levels of PhIP and MeIQx. Accordingly, urinary excretion levels of a single heterocyclic aromatic amine can only serve as an approximate measure of another in estimating exposure to these compounds in humans consuming unrestricted diets.

Urinary excretion of unmetabolized and phase II conjugates of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in humans: relationship to cytochrome P4501A2 and N-acetyltransferase activity.

Cooking meat, fish, or poultry at high temperature gives rise to heterocyclic aromatic amines (HAAs), which may be metabolically activated to mutagenic or carcinogenic intermediates. The enzymes cytochrome P4501A2 (CYP1A2) and N-acetyltransferase (NAT2) are principally implicated in such biotransformations. We have determined the relationship between the activity of these two enzymes and the urinary excretion of unmetabolized and Phase II conjugates of the two HAAs MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline) and PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) in individuals fed a uniform diet containing high-temperature cooked meat. The subjects in the study ate meat containing known amounts of MeIQx and PhIP, and urine collections were made 0-12 and 12-24 h after a meal. MeIQx and PhIP were measured in urine after acid treatment that quantitatively hydrolyzes the Phase II conjugates to the respective parent amine. The extracts containing the HAAs were purified by immunoaffinity chromatography and analyzed by liquid chromatography using electrospray ionization-tandem mass spectrometry. The MeIQx content in the 0-12 h urine increased after acid hydrolysis by a factor of 3-21-fold. After acid treatment, the total amount of MelQx (unmetabolized plus the N2-glucuronide and sulfamate metabolites) excreted in the 0-12 h urine was 10.5 +/- 3.5% (mean +/- SD) of the dose, whereas the total amount of PhIP [unmetabolized plus acid-labile conjugate(s)] in the 0-12 h period was 4.3 +/- 1.7% (mean +/- SD) of the dose. The total amount of PhIP in the 12-24 h urine after acid treatment was 0.9 +/- 0.4% (mean +/- SD) of the dose. Linear regression analysis of the amounts of MeIQx and PhIP excreted in the 0-12 h period expressed as a percentage of the ingested dose, for all subjects, gave a low but significant correlation (r = 0.37, P = 0.005). Linear regression analyses showed that lower total MeIQx (unmetabolized plus the N2-glucuronide and sulfamate metabolites) in urine was associated with higher CYP1A2 activity, whereas total PhIP (unmetabolized plus conjugated) in urine showed no association to CYP1A2 activity. These results indicate that in humans, MeIQx metabolism and disposition are more strongly influenced by CYP1A2 activity than are those of PhIP. Linear regression analysis found no association between NAT2 activity and the levels (unmetabolized plus acid-labile conjugates) of MeIQx or PhIP excreted in urine.

Lysine adducts between methyltetrahydrophthalic anhydride and collagen in guinea pig lung.

The formation of adducts between methyltetrahydrophthalic anhydride (MTHPA), an important industrial chemical and potent allergen, and collagen from guinea pig lung tissue was investigated. Collagen peptides were obtained from the lung tissue by homogenization, defatting, washing, and digestion with collagenase. In experiments in vitro, lung tissue was exposed to 8.4 mumol (50 microCi) of 14C MTHPA. The amount of adducts was 97 nmol MTHPA/g of wet tissue as determined from the bound radioactivity. In a study in vivo, four guinea pigs were injected intratracheally with 8.4 mumol of 14C MTHPA each. The amount of adducts was 0-1.2 nmol MTHPA/g of wet tissue (determined by bound radioactivity). N epsilon-methyltetrahydrophthaloyl-L-lysine (MTHPL) was synthesized and characterized by NMR, UV, and mass spectrometry (MS). A method to analyze MTHPL, after derivatization with methanol and pentafluorobenzoyl chloride, using gas chromatography-MS was developed. Analysis of Pronase-digested MTHPA-exposed lung tissue showed a concentration of 19 nmol MTHPL/g wet lung in vitro and between 0 and 0.15 nmol MTHPL/g wet lung in vivo. Thus, 20% in vitro and 12-15% in vivo of the bound radioactivity was found as adducts with lysine. These results are a first step toward studies of allergenic epitopes in proteins and methods for biological monitoring of exposure to acid anhydrides.

MeIQx (2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline): metabolism in humans and urinary metabolites in human populations.

The metabolism of the heterocyclic amines has been extensively studied in rodents and limited studies have been conducted in nonhuman primates. A study has been undertaken on the metabolism of 2-amino-3,8-dimethylimadazo[4,5-f]quinoxaline (MeIQx) in human subjects consuming normal cooked foods. A variety of fish and meat products has been cooked in several ways and fed to human volunteers. Urine was collected and analyzed according to methods developed for this purpose. Earlier rodent studies using radioactive MeIQx had suggested that the principal urinary excretion products included unmetabolized MeIQx, MeIQx sulfamate and MeIQx N-glucuronide. Conditions were developed for HPLC analysis of the free amine and its conjugates in human urine and for total hydrolysis of the conjugates to the free amine. Extraction and cleanup from urine were made possible by the availability of suitable monoclonal antibodies to MeIQx which could be used for immunoaffinity chromatography. For sensitive detection of the amounts present in human urine, gas chromatographymass spectrometry (GC-MS) was used in the negative chemical ionization mode. These studies have demonstrated that the distribution of metabolites in humans strongly resembles that in the rat. The sulfamate and N-glucuronide appear to be predominant human metabolites, while no evidence could be found of N-acetyl MeIQx. Subsequent to the studies just described, the methods developed were applied to urines collected in an epidemiological study on aromatic amine metabolism in Los Angeles. The study includes African American, White, and Asian people who have been phenotyped for caffeine acetylator status.

Species differences in metabolism of heterocyclic aromatic amines, human exposure, and biomonitoring.

Heterocyclic aromatic amines (HAAs) are animal carcinogens and suspected human carcinogens which are formed in cooked foods at the low parts per billion level. HAAs in cooked meats were purified by either immunoaffinity chromatography or solid phase tandem extraction, which allowed for the simultaneous analysis of 11 HAAs by HPLC. The metabolism of two prominent HAAs, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), was investigated in animal models and in vitro with human tissues to develop strategies for human biomonitoring. MeIQx and IQ are rapidly absorbed from the gastrointestinal tract of rodents and transformed into several detoxification products which are excreted in urine and feces. Metabolites result from cytochrome P450-mediated ring oxidation at the C-5 position followed by conjugation to sulfate or beta-glucuronic acid. Other major metabolites include the phase II conjugates, N2-glucuronide and N2-sulfamate. A metastable N2-glucuronide conjugate of the genotoxic metabolite of N-hydroxy-MeIQx was also detected in urine and bile. The binding of both carcinogens to blood proteins was low and suggests that human biomonitoring through protein adducts may be difficult. These metabolic pathways exist in nonhuman primates and several of these pathways also occur in vitro with human liver. The urinary excretion of MeIQx in seven human subjects following consumption of cooked beef or fish ranged between 2 and 22 ng in 12 hr when determined by negative ion chemical ionization GC-MS. After acid hydrolysis of urine, the amount of MeIQx increased 4- to 10-fold in 6 of the 7 subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Urinary excretion of 2-amino-3,8-dimethylimidazo-[4,5-f]quinoxaline in white, black, and Asian men in Los Angeles County.

The heterocyclic aromatic amines produced by high temperature cooking of foods containing creatin(in)e and amino acids (such as beef, pork, poultry and fish) are a class of potent animal carcinogens and have been implicated indirectly in human colon and pancreas carcinogenesis. We studied the urinary excretion of a mutagenic heterocyclic aromatic amine compound, 2-amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MelQx), among 47 black, 41 Asian (Chinese or Japanese), and 43 non-Hispanic white (white) male residents of Los Angeles County by quantitative analysis of total free and conjugated MelQx in pooled overnight urine collections. Significant interracial differences were observed. Geometric mean level in blacks was 1.3- and 3.0-fold higher than that in Asians and whites, respectively. Urinary level of MelQx was positively associated with intake frequencies of bacon, pork/ham and sausage/luncheon meats among study subjects, consistent with the observation that in Los Angeles, blacks had the highest consumption of these three food groups among the three races. Among men in Los Angeles County, the incidence rates of pancreas and colon cancers, which have been shown to be positively related to intake of fried meats, are 50% and 20% higher in blacks relative to Asians and whites, respectively. Our results are, therefore, consistent with the hypothesis that exposure to heterocyclic aromatic amines is related to risk of pancreas and colon cancers, and may in part explain the higher incidence among blacks relative to Asians and whites in Los Angeles.

Human urinary excretion of sulfamate and glucuronide conjugates of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx).

The contribution of Phase II conjugation reactions to human disposition of 2-amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MelQx) was investigated by analysis of urine for MelQx and its sulfamate and glucuronide metabolites. Subjects consumed pan-fried fish, beef, or bacon and collected 0-12 and 12-24-h postconsumption urine samples. MelQx content of the samples was determined both with and without acid treatment that quantitatively hydrolyzes the Phase II conjugates. The amount of unconjugated MelQx in the urine of seven subjects ranged between 2 and 36 ng in the first 12-h sample and was undetectable in the second. Hydrolysis increased the MelQx content 3-13-fold in the urine of six subjects, while the urine of one subject showed no significant change. Unconjugated MelQx excreted in urine was found to range between 0.5 and 4.7% of the ingested dose. In acid-treated urine the amount of MelQx was found to range between 1 and 14% of the ingested dose. A method for isolating the acid-labile conjugates in urine was developed, which included the following steps: acetone/methanol precipitation; solid-phase extraction; ion exchange fractionation, normal phase aminopropyl fractionation, and reverse phase high pressure liquid chromatography separation of the metabolites. Acidic hydrolysis of the fractions obtained in the last step, followed by gas chromatography-mass spectrometry analysis of the MelQx produced, was used to confirm the presence of the sulfamate and the glucuronide metabolites in human urine. The results provide evidence that glucuronidation and amine sulfamation are significant pathways of detoxification of MelQx in humans. In addition, the increased amount of MelQx released after acid hydrolysis facilitates the quantitative analysis of urinary MelQx.

Heterocyclic aromatic amine formation in grilled bacon, beef and fish and in grill scrapings.

The heterocyclic aromatic amines (HAAs) 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 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 alpha carboline (A alpha C) were quantitated in grilled bacon and beef. The levels of PhIP in bacon ranged from < 0.1 to 52 p.p.b., MeIQx was detected at levels ranging from 0.9 to 18 p.p.b. Both 4,8-DiMeIQx and A alpha C were found at < 1 p.p.b. In grilled meat patties, MeIQx and PhIP were detected at levels ranging from 0.8 to 3.2 p.p.b., while 4,8-DiMeIQx and A alpha C were below the limit of detection (approximately 0.5 p.p.b.). HAAs were below the limit of detection in grilled fish. The bacon fat drippings and the pan scrapings obtained from grilled meat and fish also contained significant amounts of HAAs and indicated that either these carcinogens are released with the fat during grilling or that HAAs are formed directly in the released fat and juices. Several of these carcinogens were detected in the pan scrapings at concentrations 10- to 100-fold higher than in cooked meats. PhIP was detected at 144 p.p.b. in combined grilled meat and fish scrapings, followed by A alpha C at 77 p.p.b., MeIQx at 29 p.p.b. and 4,8-DiMeIQx at 4 p.p.b. The co-mutagens harman and norharman were also detected in cooked meats and fish at amounts ranging from 5 to 30 p.p.b. Fat drippings and grill residue scrapings are often used as a base for gravies and sauces. Thus, cooking practices and dietary habits have a strong impact on HAA exposure.

Characterization of various classes of protein adducts.

Analysis of the types of protein adducts formed by chemical carcinogens indicate that adducts may be categorized into various classes according to the nature of the carcinogen as well as the amino acid with which they react. Tryptophan(214) of serum albumin was previously shown to react specifically with N-sulfonyloxy-N-acetyl-4-aminobiphenyl. The same residue is now shown to also react with the sulfate esters of N-hydroxy-N-acetyl-2-aminofluorene and N-hydroxy-N,N'-diacetylbenzidine. Thus, Trp-214 appears to be a binding site for a variety of activated N-aryl hydroxamic acids. Epoxides and diol epoxides derived from polynuclear aromatic hydrocarbons alkylate carboxylic groups in hemoglobin and serum albumin. Because the esters formed are readily hydrolyzed to dihydrodiols and tetrahydrotetrols which can be determined by GC-MS, it is possible to analyze for a wide range of polyaromatic hydrocarbon (PAH) epoxide adducts. With this approach it was shown that human subjects experiencing exposure to ambient levels of environmental PAH do take up and metabolize chrysene and benzo[a]pyrene. Feral, bottom-dwelling fish inhabiting contaminated waters were also examined. Globin adducts containing certain dihydroxy groups such as those arising in anti-diol epoxide adducts were concentrated by boronate affinity chromatography and further analyzed by HPLC with diode-array UV/visible detection. Four compounds were detected that exhibited spectra characteristic of a polynuclear chromophore. Two of these appeared to be isomers. Further instrumental analysis is needed to elucidate the structure of these unknown putative adducts.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of the food-borne carcinogens 2-amino-3-methylimidazo-[4,5-f]quinoline and 2-amino-3,8- dimethylimidazo[4,5-f]-quinoxaline in the rat as a model for human biomonitoring.

Metabolism of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazol[4,5-f]quinoxaline (MeIQx) and their binding to blood proteins were examined in the rat to develop methods of human biomonitoring. Hemoglobin and serum albumin were among many blood proteins modified. Approximately 0.01% of the dose for both compounds was bound to these proteins, and induction of cytochrome P-450 with polychlorobiphenyls resulted in decreased levels of adduction. Hemoglobin sulfinic acid amide adducts could not be detected for either amine, however, as much as 10% of the IQ bound to albumin was characterized as an N2-cysteine(34)sulfinyl-IQ linkage. Human dosimetry of these carcinogens through such adducts may prove difficult due to the low levels of protein binding. Major routes of detoxification of both contaminants included cytochrome P-450-mediated ring hydroxylation at the C-5 position followed by conjugation to glucuronic or sulfuric acid. Direct conjugation to the exocyclic amine group through N-glucoronidation and sulfamate formation were other important routes of inactivation, but N-acetylation was a minor pathway. The N-glucoronide conjugate of the mutagenic metabolite N-hydroxy-MeIQx was also detected in urine. Rats given MeIQx at 10 micrograms/kg excreted 20% of the dose in urine within 24 hr and the remainder was recovered in feces. The N2-glucuronide was the major metabolite found in urine and accounted for 4% of the total dose. The other metabolites cited above also were excreted in urine at amounts ranging from 0.5 to 3% of the dose, whereas 0.5 to 2% was detected as unmetabolized MeIQx.(ABSTRACT TRUNCATED AT 250 WORDS)

Urinary markers for exposures to alkylating or nitrosating agents.

Investigation of urinary markers as indices of endogenous nitrosation and of gastric cancer etiology has been a major focus of our work. As part of this effort, studies have been carried out on a Colombian population at high risk for gastric cancer. In this group, nitrosoproline excretion was highly correlated with nitrate excretion in the subpopulation with advanced gastric pathology, but not in control subpopulations with more normal stomachs. Neither urinary 7-methylguanine nor 3-methyladenine was strongly related to gastric pathology or to urinary nitrate or nitrosoproline levels. More recently, as evidence has accumulated concerning the importance of nitric oxide as a cellular messenger, we have begun research toward developing markers for the presence of nitric oxide and for endogenous nitrosation via this compound. Nitric oxide is formed from arginine by activated endothelial cells as a messenger for vasodilation. We have shown that prolonged exercise leads to increased urinary nitrate and that when 15N-arginine is ingested by humans, 15N-nitrate levels increase in 24-hr urine collections. Nitrosohydroxyethylglycine and 3-nitrotyrosine were evaluated as indices for the formation of N-nitrosomorpholine and for the nitration of protein, respectively, under experimental conditions (e.g., immunostimulation) expected to enhance nitric oxide formation. Nitrotyrosine has not proved useful as a biomarker for nitration/nitrosation reactions in immunostimulated rats. Immunostimulation of rats following administration of morpholine led to increases in urinary nitrate and nitrosohydroxyethylglycine. This procedure, however, would not be appropriate for humans due to the toxicity of morpholine and the carcinogenicity of N-nitrosomorpholine.

Urinary excretion of nitrate, N-nitrosoproline, 3-methyladenine, and 7-methylguanine in a Colombian population at high risk for stomach cancer.

Urinary excretion levels of nitrate and N-nitrosoproline were determined in 160 individuals in a Colombian population at high risk for gastric cancer. In 156 of these subjects urinary levels of 3-methyladenine and 7-methylguanine were determined. Gastric biopsy specimens were obtained from 118 individuals and were histologically characterized according to pathological criteria into the following groups: normal, superficial gastritis, chronic atrophic gastritis, chronic atrophic gastritis with intestinal metaplasia, and dysplastic. The histological changes were correlated with the four variables listed above. There were no significant differences in the excretion of nitrate, N-nitrosoproline, 3-methyladenine, or 7-methylguanine in subjects with different pathological changes. A statistically significant correlation was present between nitrate and N-nitrosoproline excretion in the total population group (r = 0.297, P = 0.0001). A highly significant correlation (r = 0.56, P = 0.0002) was noted for urinary nitrate and N-nitrosoproline excretion in individuals with intestinal metaplasia and dysplasia. An increase in the urinary excretion of 3-methyladenine and 7-methylguanine was associated with tobacco smoking in the total population group.

Comparison of urinary nitrate, N-nitrosoproline, 7-methylguanine and 3-methyladenine levels in a human population at risk for gastric cancer.

We have measured the urinary excretion of nitrate, N-nitrosoproline, 3-methyladenine and 7-methylguanine in a human population at high risk for gastric cancer. A strong correlation was observed between nitrate and N-nitrosoproline excretion (p less than 0.00000); statistically significant correlations (p less than 0.05) were also observed for N-nitrosoproline with 3-methyladenine (p = 0.003) and with 7-methylguanine (p = 0.03) and for 7-methyl-guanine with nitrate (p = 0.02), although these correlations were sensitive to outliers. Smokers appeared to excrete slightly higher levels of 3-methyladenine than nonsmokers; no other difference was observed between smokers and nonsmokers or between drinkers and nondrinkers. The correlation between N-nitrosoproline excretion and nitrate excretion supports the hypothesis that elevated endogenous nitrate levels may contribute to elevated endogenous formation of N-nitroso compounds.

The determination of urinary 3-methyladenine by immunoaffinity chromatography-monoclonal antibody-based ELISA: use in human biomonitoring studies.

A mouse monoclonal antibody (Mab) was prepared which showed high specificity for a potential marker of exposure to methylating carcinogens such as 3-methyladenine (3-MeAde). In a low-temperature (4 degrees C) ELISA a linear calibration curve was obtained between 3 and 50 fmol/well. In combination with an immunoaffinity (IA) column prepared from a 3-MeAde rabbit antiserum, the ELISA was used to determine 3-MeAde in urine. The IA-ELISA method was validated by comparison with results obtained by an IA-GC-MS method. The effect of consuming a low 3-MeAde diet on urinary 3-MeAde excretion was investigated in a human volunteer. Urine collected during a 'normal' diet exhibited the characteristic variation in 3-MeAde levels previously observed (9.5 micrograms/24 h, SD = 4.4, n = 5). In contrast, 3-MeAde excretion was markedly lower and less variable on days when the diet was closely controlled (0.63 microgram/24 h, SD = 0.08, n = 3). Dietary intake of 3-MeAde on the latter days was between 0.37 and 0.43 microgram/day, indicating that most, if not all, of the 3-MeAde seen in previous experiments was derived from the diet. The origin of dietary 3-MeAde is not known, but may be related to fumigant use. Dietary manipulation affords the possibility of carrying out model studies, in volunteers, on 3-MeAde intake and formation in vivo.