Oxidation of 2,6-dimethylaniline by recombinant human cytochrome P450s and human liver microsomes.

2,6-Dimethylaniline (2,6-DMA) is classified as a rodent nasal cavity carcinogen and a possible human carcinogen. The major metabolite of 2,6-DMA in rats and dogs is 4-amino-3,5-dimethylphenol (DMAP) but oxidization of the amino group to produce metabolites such as N-(2,6-dimethylphenyl)hydroxylamine (DMHA) is also indicated by the occurrence of hemoglobin adducts of 2,6-DMA in human and rats. Previous studies have shown a large interindividual variability in human 2,6-DMA hemoglobin adduct levels. In the present study, 2,6-DMA oxidation in vitro by human liver microsomes and recombinant human P450 enzymes was investigated to assess whether the hemoglobin adduct variability could be attributed to metabolic differences. At micromolar concentrations, the only product detectable (UV) was DMAP, while at 10 nM, DMHA was a substantial product. 2E1 and 2A6 were identified as the major P450s in human liver microsomes responsible for the production of DMAP by using P450-specific chemical inhibitors and mouse monoclonal antibodies that selectively inhibit human P450 2E1 and 2A6. 2A6 was identified as the major P450 responsible for the N-hydroxylation. Native P450 2E1 and human liver microsomes catalyzed the rearrangement of DMHA to DMAP independent of NADPH. Consistent with a mechanism involving oxygen rebound to the heme iron center, labeled oxygen was not incorporated into DMAP from either 18O2 gas or H2 18O in this rearrangement. Results presented here suggest much of the observed interindividual variability of 2,6-DMA hemoglobin adduct levels could be due to differences in the relative amounts of hepatic 2E1 and 2A6.

Site-selective nitration of tyrosine in human serum albumin by peroxynitrite.

Peroxynitrite, which is formed in biological systems by the reaction of nitric oxide with superoxide anion, is a highly reactive molecule that can lead to cell injury or cell death. Reactions of peroxynitrite under physiological conditions include nitration of tyrosine-containing proteins or peptides, and we have been investigating the behavior of human serum albumin following exposure to peroxynitrite. Peroxynitrite, at relative concentrations ranging from 0.2 to 50 with respect to protein, was added to human serum albumin in buffer at pH 7.2. The resulting mixtures were dialyzed to remove small molecules, dried under vacuum, and then digested with trypsin. The digests were analyzed by high performance liquid chromatography with UV detection at 230 and 354 nm, the latter wavelength being selective for nitrotyrosine. At the higher relative concentrations of peroxynitrite, the 354-nm chromatograms contained a large number of peaks, including at least nine with molecular weights corresponding to nitration of nominal tryptic peptides. Following treatment with the lower relative concentrations of peroxynitrite, however, the 354-nm chromatograms were dominated by only two nitrated peptides; these were identified by comparison of LC retention times and collision-induced decomposition mass spectra as nitro-Y(411)TK(413) and nitro-Y(138)LYEIAR(144). Each of these tyrosines resides in a known reactive site within the protein, i.e., subdomains IIIA and IB, respectively.

Gender- and smoking-related bladder cancer risk.

BACKGROUND: There is growing evidence that, when smoking habits are comparable, women incur a higher risk of lung cancer than men. Because smokers are also at risk for bladder cancer, we investigated possible sex differences in the susceptibility to bladder cancer among smokers. METHODS: A population-based, case--control study was conducted in Los Angeles, CA, involving 1514 case patients with bladder cancer and 1514 individually matched population control subjects. Information on tobacco use was collected through in-person interviews. Peripheral blood was collected from study participants to measure 3- and 4-aminobiphenyl (ABP)-hemoglobin adducts, a marker of arylamine exposure. Data were analyzed to determine whether the risk of bladder cancer differs between male and female smokers and whether female smokers exhibit higher levels of ABP-hemoglobin adducts than male smokers with comparable smoking habits. All statistical tests were two-sided. RESULTS: Cigarette smokers had a statistically significant 2.5-fold higher risk (95% confidence interval = 2.1 to 3.0) of bladder cancer than never smokers. Use of filtered versus nonfiltered cigarettes, low-tar versus higher tar cigarettes, or the pattern of inhalation did not modify the risk. The risk of bladder cancer in women who smoked was statistically significantly higher than that in men who smoked comparable numbers of cigarettes (P =.016 for sex-lifetime smoking interaction). Consistent with the sex difference in smoking-related bladder cancer risk, the slopes of the linear regression lines of the 3- and 4-ABP--hemoglobin adducts by cigarettes per day were statistically significantly steeper in women than in men (P values for sex differences <.001 and.006, respectively). CONCLUSION: The risk of bladder cancer may be higher in women than in men who smoked comparable amounts of cigarettes.

Quantification of (7S,8R)-dihydroxy-(9R,10S)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene adducts in human serum albumin by laser-induced fluorescence: implications for the in vivo metabolism of benzo[a]pyrene.

The ubiquitous environmental carcinogen benzo[a]pyrene (BaP) is metabolized in vivo in humans to its ultimate carcinogenic form of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). Mouse skin tumorigenicity studies indicate that the (7R,8S,9S,10R) enantiomer of BPDE, (7R,8S)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(7R,8S,9S,10R)-BPDE], is a potent tumor initiator, whereas the (7S,8R,9R,10S) enantiomer of BPDE, (7S,8R)-dihydroxy-(9R,10S)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(7S,8R,9R,10S)-BPDE], may act as a tumor promoter. In vitro experiments have shown that human liver microsomes are capable of metabolizing BaP to both the (7R,8S,9S,10R) and (7S,8R,9R,10S) enantiomers of BPDE. However, the metabolism of BaP to (7S,8R,9R,10S)-BPDE has not been demonstrated in humans in vivo. The adducts formed between human serum albumin (HSA) and the (7S,8R,9R,10R) and (7R,8S,9S,10R) enantiomers of BPDE have been described previously. (7S,8R,9R,10S)-BPDE forms a stable adduct at histidine146 of HSA, whereas (7R,8S,9R,10R)-BPDE forms a relatively unstable ester adduct at aspartate187 or glutamate188 of HSA. Using high-performance liquid chromatography with laser-induced fluorescence (LIF) detector, we quantified the level of (7S,8R,9R,10S)-BPDE adducts at histidine146 in HSA isolated from 63 healthy males who were population control subjects for an ongoing case-control study of bladder cancer. By design, roughly half of the participants were lifelong nonsmokers (n = 35), whereas the remaining 28 participants were current smokers of varying intensities. HP-BPDE adducts were detected in 60 of the 63 samples (95%) by HPLC-LIF. Adduct levels ranged from undetectable (<0.04 fmol/mg HSA) to 0.77 fmol/mg HSA. The samples had a mean and median (7S,8R,9R,10S)-BPDE-HSA adduct level of 0.22 and 0.16 fmol of adduct/mg albumin, respectively. Mean adduct levels did not differ between smokers and nonsmokers (P = 0.72). Occupational exposure to polycyclic aromatic hydrocarbons was unrelated to adduct level (P = 0.62). Intake frequencies of two food items showed statistically significant associations with adduct levels. Consumption of sweet potatoes was negatively related to adduct level (P = 0.029), whereas intake of grapefruit juice was positively related to adduct level (P = 0.045). None of the three indices of residential ambient air pollution under study showed a statistically significant association with adduct levels.

N-acetyltransferase 2 phenotype but not NAT1*10 genotype affects aminobiphenyl-hemoglobin adduct levels.

Aminobiphenyls (ABPs) in tobacco have been implicated in bladder cancer etiology in smokers. N-Acetylation of ABPs in the liver, predominantly by the N-acetyltransferase 2 (NAT2) isozyme, represents a detoxification pathway, whereas O-acetylation of N-hydroxy-ABPs in the bladder, predominantly by the N-acetyltransferase 1 (NAT1) isozyme, represents a bioactivation pathway. We and others have demonstrated that NAT2 phenotype affects 3- and 4-ABP-hemoglobin adduct levels (higher levels in slow acetylators), which are considered valid biomarkers of the internal dose of ABP to the bladder. We have also shown that NAT1 genotype (NAT1*10 allele) is associated with increased DNA adduct levels in urothelial tissue and higher risk of bladder cancer among smokers. It is not known whether NAT1*10 genotype influences ABP-hemoglobin adduct levels. Therefore, we assessed 403 primarily non-Hispanic white residents of Los Angeles County for their NAT2 acetylator phenotype, NAT1*10 acetylator genotype, and 3- and 4-ABP-hemoglobin adduct levels. Eighty-two subjects were current tobacco smokers of varying intensities. Tobacco smokers had significantly higher mean 3- and 4-ABP-hemoglobin adduct levels relative to nonsmokers. The levels increased with increased amounts smoked per day (two-sided, P < 0.0001 in all cases). With adjustment for NAT1 genotype and race, the smoking-adjusted geometric mean level of 3-ABP-hemoglobin adducts in NAT2 slow acetylators was 47% higher than that in NAT2 rapid acetylators (P = 0.01). The comparable value for 4-ABP-hemoglobin adducts was 17% (P = 0.02). In contrast, no association between NAT1*10 genotype and 3- or 4 ABP-hemoglobin adduct levels was observed after adjustment for NAT2 phenotype, smoking, and race. The present study suggests that the impact of the NAT1*10 genotype on 3- and 4-ABP-hemoglobin adducts is noninformative on the possible association between NAT1 activity and bladder cancer risk.

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.

DNA adduct formation by secondary metabolites of cyclopenta[cd]pyrene in vitro.

In this study, calf thymus DNA was reacted in vitro with cyclopenta[cd]pyrene 3,4-epoxide (CPPE) or its metabolites, 3,4-dihydroCPP-3,4-diol (CPP-3,4-diol) and 4-hydroxy-3,4-dihydroCPP (4-OH-DCPP), activated with sulfotransferase. The adducts formed were analyzed by HPLC with fluorescence detection following enzymatic digestion of DNA to deoxynucleosides. We have shown previously that the major CPPE-reacted DNA adducts are cis-3-(deoxyguanosin-N2-yl)-4-hydroxy-3,4-dihydroCPP. Sulfotransferase activation of trans-CPP-3,4-diol yielded two adducts that were identical to the products resulting from the reaction of CPPE with DNA, while cis-CPP-3,4-diol gave very low covalent binding. Two adducts formed by sulfotransferase activation of 4-OH-DCPP were identical to the products of the reaction of synthetic 4-NaO3S-O-DCPP or sulfotransferase-activated 4-OH-DCPP with deoxyguanosine. These results indicate that guanine is the predominant site of CPP adduct formation in DNA, and that the 4-hydroxy-3-dGuo adducts can arise by reaction of DNA with either CPPE or sulfotransferase-activated trans-CPP-3,4-diol.

Identification of subdomain IB in human serum albumin as a major binding site for polycyclic aromatic hydrocarbon epoxides.

Covalent adducts between serum albumin and low molecular weight organic electrophiles are formed with a high degree of regioselectivity mostly for nucleophilic amino acid residues located in subdomains IIA and IIIA. Previous studies have indicated that diol epoxide metabolites of polycyclic aromatic hydrocarbons (PAH) may target residues in a different subdomain. The regioselectivity of PAH epoxide and diol epoxide binding was examined in this study by reaction of human serum albumin in vitro with the racemic trans,anti-isomers of 7,8-dihydrobenzo[a]pyrene-7,8-diol 9,10-epoxide (1), 2,3-dihydrofluoranthene-2,3-diol 1,10b-epoxide (2), 1,2-dihydrochrysene-1,2-diol 3,4-epoxide (5), 6-methyl-1,2-dihydrochrysene-1,2-diol 3,4-epoxide (6), 5-methyl-1,2-dihydrochrysene-1,2-diol 3,4-epoxide (7), 3,4-dihydrobenzo[c]phenanthrene-3,4-diol 1,2-epoxide (8), 11,12-dihydrobenzo[g]chrysene-11,12-diol 13,14-epoxide (9), and 11,12-dihydrodibenzo[a,l]pyrene-11,12-diol 13,14-epoxide (10) and the racemic epoxides cyclopenta[cd]pyrene 3,4-epoxide (3) and benzo[a]pyrene 4,5-epoxide (4) followed by determination of the linkage site. Adducted albumin was digested enzymatically, and digests were chromatographed by reversed-phase HPLC to purify peptide adducts, which were analyzed by electrospray ionization collision-induced dissociation (CID) tandem mass spectrometry. Product ion spectra revealed that adducts fragmented predominantly by cleavage of the peptide-PAH bond with retention of charge by the peptide as well as by the hydrocarbon. Peptide sequences were determined by MS/MS analysis of the peptide ions formed by in-source CID to cleave the adduct bond. Longer peptide sequences established site selectivity by virtue of their uniqueness, while shorter sequences revealed the reactant amino acid within the site. Epoxide 4 and diol epoxides 1, 2, 5, and 6 reacted predominantly with His146; epoxide 3 and diol epoxides 7-9 reacted predominantly with Lys137. Both residues are situated in subdomain IB. The binding site for 10 could not be determined uniquely, but one of the several possibilities was Lys159, which is also located in subdomain IB. The results, taken together with previous findings, demonstrate that the reaction of polycyclic aromatic hydrocarbon epoxides with human serum albumin is highly selective for a small number of residues in subdomain IB.

Characterization of DNA adducts formed by cyclopenta[cd]pyrene epoxide.

Cyclopenta[cd]pyrene (CPP) is a ubiquitous environmental pollutant whose 3,4-epoxide (CPPE) is generally regarded as its ultimate carcinogenic metabolite. The present study was undertaken to determine the structures of major DNA adducts formed by CPPE in vitro. Incorporation of specific radiolabeled bases into calf thymus DNA prior to reaction with CPPE demonstrated that the major adducts were formed by guanine, while minor adducts were formed by adenine and cytosine. Unmodified DNA was reacted with [3H]CPPE and the deoxynucleoside adducts obtained were compared chromatographically with the products obtained by reaction of CPPE with 2'-deoxyguanosine (dGuo). Two dGuo adducts from the latter reaction were identified by 1H-NMR, fast atom bombardment mass spectrometry, and circular dichroism as diastereoisomers of cis-3-(deoxyguanosin-N2-yl)-4-hydroxy-3,4-dihydroCPP. Other products that may have included the isomeric trans-N2-dGuo adduct were formed in the reaction. The major adduct fraction in the DNA digest, accounting for over 70% of the total, was chromatographically indistinguishable from the two cis dGuo-N2 adducts. A second DNA adduct fraction was observed, which appeared also to be formed by reaction with guanine as indicated by experiments in which DNA containing [3H]guanine was reacted with unlabeled CPPE. The results confirm that guanine is the major target in DNA for reaction with CPPE and are the first proof of structure for a CPPE-deoxynucleoside adduct.

Glutathione S-transferase M1 genotype affects aminobiphenyl-hemoglobin adduct levels in white, black and Asian smokers and nonsmokers.

Cigarette smoking is the major cause of bladder cancer in men in the United States, and the arylamines contained in cigarettes smoke, including 4-amino-biphenyl (4-ABP), are believed to play an important role in the induction of bladder cancer among smokers. N-acetylation, which is catalyzed by the genetically controlled hepatic N-acetyltransferase enzyme displaying two phenotypes (slow versus rapid), is a detoxification pathway for arylamines with regard to bladder carcinogenesis. In Los Angeles, CA, non-Hispanic white (white), black, and Asian males have comparable smoking habits and yet dramatically different risks of bladder cancer (31 of 100,000 in whites, 16 of 100,000 in blacks, and 13 of 100,000 in Chinese and Japanese). Previously, we have demonstrated that the prevalence of slow acetylators (the high-risk phenotype) was highest in whites (54%), intermediate in blacks (34%), and lowest in Asians (14%). We also showed that mean 3- and 4-ABP hemoglobin adduct levels were significantly higher in cigarette smokers relative to nonsmokers, and that the level increased with increasing number of cigarettes smoke/day. Most importantly, slow acetylators consistently exhibited higher mean levels of ABP hemoglobin adducts relative to rapid acetylators, regardless of race and level of cigarette smoking. We assessed 151 residents of Los Angeles County (CA) who were either white, black, or Asian (Chinese or Japanese) and over the age of 30 years for their glutathione S-transferase M1 (GSTM1) genotype (null versus non-null), acetylator phenotype (slow versus rapid), levels of 3- and 4-ABP hemoglobin adducts, and current use of tobacco products. Whites (27%) had the highest prevalence of the highest risk profile (slow acetylator, GSTM1 null), followed by blacks (15%) and Asians (2.7%), and the difference was statistically significant (P = 0.006). Whites also had less than one-half the prevalence of the "protective" profile (rapid acetylator, GSTM1 non-null) relative to blacks and Asians (23 versus 57%; P = 0.0001). Regardless of race and level of cigarette smoking, mean levels of 3- and 4-ABP hemoglobin adducts were higher in subjects possessing the higher risk (GSTM1/acetylator profile. Mean level of 4-ABP hemoglobin adduct (adjusting for race, cigarette smoking, and acetylator phenotype) was significantly higher in subjects possessing the GSTM1-null versus GSTM1-non-null genotype (46.5 versus 36.0 pg/g Hb; P = 0.037). The comparable difference in mean levels of 3-ABP hemoglobin adduct was borderline significant (1.6 versus 1.1 pg/g Hb; P = 0.07). Thus, our results suggest that GSTM1 is involved in the detoxification of 3- and 4-ABP and may contribute to the racial variation in bladder cancer incidence among white, black, and Asian males in Los Angeles, CA.

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.

Altered aromatic amine metabolism in epileptic patients treated with phenobarbital.

The fate of carcinogens differs among individuals who have different activities of drug-metabolizing enzymes that are important in activating and detoxifying carcinogens. A drug that profoundly alters the metabolism of the drugs and carcinogens is the anticonvulsive agent phenobarbital. To investigate why epileptic patients appear to have a low risk of cancer of the urinary bladder, and on the basis of the observation that levels of aromatic amine-hemoglobin adducts are strongly associated with various risk factors for cancer at that site, we determined aromatic amine-hemoglobin adducts in 62 epileptic patients as a surrogate measure of the reaction of carcinogenic metabolites with DNA in target tissue. Although adducts were detected in all subjects, the levels were proportional to daily tobacco consumption. When the subjects were stratified into groups smoking 20 g tobacco/day or more, smoking <20 g/day, and not smoking, an effect of medication was detected. Epileptic patients treated chronically with phenobarbital or primidone, which is effectively metabolized to phenobarbital, were found to have lower levels of 4-aminobiphenyl adducts than patients on the other treatment (P = 0.02; ANOVA). In nonsmokers, no effect of medication could be demonstrated above background variation; however, an increasing effect was seen with tobacco consumption with only one-half the increase in adducts per g of tobacco smoked as epileptic patients on other treatment. The difference in the increases (slopes of regression lines) was highly significant statistically. This reduction in the level of hemoglobin-aromatic amine adducts is probably due to induction of detoxification enzymes in the patients treated with phenobarbital.

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.

Conservation of histone carcinogen adducts during replication: implications for long-term molecular dosimetry.

The effect of cell replication on histone-carcinogen adducts was investigated by determining the specific adduct levels as a function of time following carcinogen treatment of human TK6 cells grown in culture. Core histones isolated from cells treated with aflatoxin B1 or r-7,t-8 dihydroxy-t-9,t-10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene exhibited a decrease over five generations in specific adduct level that did not exceed the decrease expected as a result of dilution with newly synthesized protein except during the early phase (< 1 generation) of the experiment when loss of chemically unstable adducts might occur. Similar kinetics without the initial, more rapid phase was observed when cells were treated with N-nitroso-N-methylurea. Multigeneration stability of aflatoxin B1 and N-nitroso-N-methylurea adducts that formed on histone H1 was also observed; in these experiments it was not possible to determine if there was an initial phase in the kinetics. These experiments indicate that cell replication does not result in the repair or removal of adducted histones, establishing the feasibility of using histone-carcinogen adducts for molecular dosimetry purposes.

Enantiospecificity of covalent adduct formation by benzo[a]pyrene anti-diol epoxide with human serum albumin.

Human serum albumin was reacted with the (+)- and (-)-enantiomers of r-7,t-8-dihydroxy-t-9,t-10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene to determine if the chiral nature of the protein influences adduct formation. The alkylated proteins were analyzed directly by fluorescence line narrowing spectroscopy, and their spectra were compared to those of the model synthetic adducts N tau-(7,8,9-trihydroxy-r-7,t-8,t-9,c-10-tetrahydrobenzo[a]pyren-10- yl)histidine and 7,8,9-trihydroxy-r-7,t-8,t-9,c-10-tetrahydrobenzo[a]pyren- 10-yl N-t-BOC-alaninate ester. The results from these analyses indicated that different adducts were formed by the enantiomers of the diol epoxide. The adducted proteins were also enzymatically digested, and the 8,9-cis-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-containing adducts and hydrolysis products were isolated by boronate affinity chromatography. Diode array UV, fast atom bombardment, and on-line atmospheric pressure ionization-mass spectral analysis of the HPLC purified products indicated that the more mutagenic and tumorigenic (+)-enantiomer forms carboxylic ester adducts with the protein at either Asp(187) or Glu(188), while the (-)-enantiomer forms N tau-histidine adducts at His(146). This previously unrealized enantiospecificity of the reaction of benzo[a]pyrene anti-diol epoxide with human serum albumin has important consequences for the application of the adducts as biomarkers of internal exposure.

Molecular dosimetry of aromatic amines in human populations.

Certain aromatic amines carcinogenic for the human urinary bladder, such as 4-aminobiphenyl, undergo hepatic metabolic activation to N-hydroxylamines, which are transported to the bladder. During the transport process, these reactive species come in contact with hemoglobin and react with this blood protein. The principal hemoglobin adduct formed is a cysteine sulfinamide, and quantitative methods have been developed for the analysis of sulfinamide adducts at the levels present in ordinary human blood specimens. N-acetylation is an alternative metabolic fate to N-hydroxylation. The amount of hemoglobin adduct is decreased to the extent that this pathway is increased relative to N-hydroxylation. Thus, the hemoglobin adduct is sensitive to dose, cytochrome P-450-mediated activation, and N-acetyltransferase-mediated detoxification. In addition, it has been shown that DNA adduct concentration of 4-aminobiphenyl present in human bladder epithelial cells is significantly associated with hemoglobin adduct levels. Thus, the hemoglobin adduct of 4-aminobiphenyl, and perhaps several other aromatic amines, is a good dosimeter for the target tissue dose of the ultimate carcinogenic metabolite of these amines. Several studies have been undertaken in which the hemoglobin adducts of aminobiphenyls in human blood specimens were determined quantitatively. Information concerning exposure status and acetylator phenotype of the same individuals was obtained simultaneously. The results of these studies indicate that the hemoglobin adduct of 4-aminobiphenyl is closely associated with three major risk factors for bladder cancer: cigarette smoking, type of tobacco smoked, and acetylator phenotype. They also support a major etiologic role for aromatic amines in much of human bladder cancer.

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)

Metabolism of benz[a]anthracene by human liver microsomes.

The metabolism of benz[a]anthracene (BA) by human hepatic microsomes was investigated. Only dihydrodiols were observed when BA was the substrate. No tetrahydrotetrols were detected, indicating lack of diol epoxide formation. The BA-dihydrodiols identified by GCMS analysis and comparison to authentic standards were BA-8,9-dihydrodiol (42.4% of total metabolites), BA-5,6-dihydrodiol (25%), BA-10,11-dihydrodiol (24.8%), BA-3,4-dihydrodiol (5.3%), and BA-1,2-dihydrodiol (< 1.5%). BA-dihydrodiols were also used individually as substrates. Only BA-1,2-dihydrodiol, the least abundant isomer produced from BA, was converted efficiently to a tetrahydrotetrol (> 72% conversion). BA-10,11-dihydrodiol was converted to BA-8,9,10,11-tetrahydrotetrols in < 12% yield. BA-10,11- and BA-3,4-dihydrodiols were not converted to tetrahydrotetrols.