Accelerator mass spectrometry allows for cellular quantification of doxorubicin at femtomolar concentrations.

Accelerator mass spectrometry (AMS) is a highly sensitive analytical methodology used to quantify the content of radioisotopes, such as (14)C, in a sample. The primary goals of this work were to demonstrate the utility of AMS in determining total cellular [(14)C]anthracycline concentrations following administration of doxorubicin (DOX) and to develop a sensitive assay that is superior to high performance liquid chromatography (HPLC) for the quantification of [(14)C]anthracycline at the tumor level. In order to validate the sensitivity of AMS versus HPLC with fluorescence detection, we performed three studies comparing the cellular accumulation of DOX: one in vitro cell line study, and two in vivo xenograft mouse studies. Using AMS, we quantified cellular [(14)C]anthracycline content up to 4 h following in vitro exposure at concentrations ranging from 0.2 pg/ml (345 fM) to 2 microg/ml (3.45 microM) [(14)C]DOX. The results of this study show that, compared to standard fluorescence-based HPLC, the AMS method was over five orders of magnitude more sensitive. Two in vivo studies compared the sensitivity of AMS to HPLC using a nude mouse xenograft model in which breast cancer cells were implanted subcutaneously. After sufficiently large tumors formed, [(14)C]DOX was administered intravenously at two dose levels. Additionally, we tested the AMS method in a nude mouse xenograft model of multidrug resistance (MDR) in which each mouse was implanted with both wild type and MDR+ cells on opposite flanks. The results of the second and third studies showed that [(14)C]anthracycline concentrations were significantly higher in the wild type tumors compared to the MDR+ tumors, consistent with the MDR model. Although this method does not discriminate between parent drug and metabolites, the extreme sensitivity of AMS should facilitate similar studies in humans to establish target site drug delivery and to potentially determine the optimal treatment dose and regimen.

Animal models and analytical approaches for understanding the relationships between wine and cancer.

We used two approaches for studying the relationships between wine consumption, wine composition and cancer In the first approach, a transgenic mouse model of human neurofibromatosis, combined with the use of well-defined, chemically purified diets, showed that red wine contains nonalcoholic components that can delay tumor onset. In additional studies, catechin, the main monomeric polyphenol of red wine, delayed tumor onset in this mouse model in a positive, linear relationship when incorporated into the diet at levels of 0.5-4 mmol/kg diet. In the second approach, low doses of the chemical carcinogen 2-amino-1-methyl-6-phenylimidazo(4, 5-b)pyridine (PhlP) were administered to rats, and formation of DNA adducts was evaluated by accelerator mass spectrometry. Consumption of red wine solids (the residue from red wine remaining after removal of alcohol and water) and the wine polyphenol quercetin did not influence PhlP-DNA adduct levels or induce liver enzymes (glutathione-S-transferase and quinone reductase). However, quercetin did alter distribution of PhlP in the rat tissues compared to control animals and animals fed other potential dietary chemopreventive agents, including phenylethyl isothiocyanate and sulforaphane. These studies demonstrate the feasibility of these approaches for studying the chemopreventive potential of dietary components at physiologic levels in

Determination of the tissue distribution and excretion by accelerator mass spectrometry of the nonadecapeptide 14C-Moli1901 in beagle dogs after intratracheal instillation.

Moli1901 is a 19 residue polycyclic peptide antibiotic which increases chloride transport and water mobilization in airway epithelium. These properties suggest that it may be a useful treatment for cystic fibrosis (CF). In this study, we used accelerator mass spectrometry (AMS) to quantify Moli1901 following administration of only 0.045 microCi of 14C-Moli1901 per dog. Limits of quantitation of AMS were 0.03 (urine) to 0.3 (feces) ng equiv. Moli1901/g. Administration of 14C-Moli1901 by intratracheal instillation (approximately 100 microg) into the left cranial lobe of the lung of beagle dogs resulted in retention of 64% of the dose in the left cranial lobe for up to 28 days. Whole blood and plasma concentrations of 14C were <5 ng/ml at all times after the dose. Concentrations of 14C in whole blood and plasma declined over the first day after the dose and rose thereafter, with the rise in plasma concentrations lagging behind those in whole blood. During the first 3 days after the dose, plasma accounted for the majority of 14C in whole blood, but after that time, plasma accounted for only 25-30% of the 14C in whole blood. Tissue (left and right caudal lung lobe, liver, kidney, spleen, brain) and bile concentrations were low, always less than 0.25% the concentrations found in the left cranial lung lobe. Approximately 13% of the dose was eliminated in urine and feces in 28 days, with fecal elimination accounting for about 10% of the dose. The data presented here are consistent with that obtained in other species. Moli1901 is slowly absorbed and excreted from the lung, and it does not accumulate in other tissues. Moli1901 is currently in the clinic and has proven to be safe in single dose studies in human volunteers and cystic fibrosis patients by the inhalation route. No information on the disposition of the compound in humans is available. This study in dogs demonstrates the feasibility of obtaining that information using 14C-Moli1901 and AMS.

Characterization of a peptide adduct formed by N-acetoxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a reactive intermediate of the food carcinogen PhIP.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a member of a class of compounds known as the heterocyclic amines (HCAs) that are formed in meat during cooking. It is a multi-organ carcinogen in rodents forms adducts and with DNA and protein. Although protein adducts are not thought to be involved in cancer development, they may be useful as internal dosimeters of PhIP exposure and bioactivation. Towards the goals of characterizing the adducts formed in humans and the development of an assay for quantitation of adduct levels, we have characterized a peptide adduct formed by the putative genotoxic metabolite, N-acetoxy-PhIP. A model peptide with the internal sequence Leu-Gln-Lys-Cys-Pro-Tyr, which is homologous to a potential target sequence for HCAs in human serum albumin, was reacted with N-acetoxy-PhIP and an adduct was identified and further characterized by LC-ESI-MS/MS. N-acetoxy-PhIP is covalently bound to the peptide via cysteine and the exocyclic amino group of PhIP. Future work is needed to establish if this adduct is formed and is stable in vivo in humans following exposure to PhIP.

The formation of AFB(1)-macromolecular adducts in rats and humans at dietary levels of exposure.

The levels of aflatoxin B(1)-DNA and aflatoxin B(1)-albumin adducts were investigated by accelerator mass spectrometry (AMS) in humans and rats following exposure to a known, dietary relevant amount of carbon-14 labeled aflatoxin B(1) ([(14)C]AFB(1)). The aims of the study were to: (a) investigate the dose-dependent formation of DNA and protein adducts at very low doses of AFB(1) (0.16 ng/kg-12.3 microg/kg) in the rat; (b) measure the levels of AFB(1)-albumin and AFB(1)-DNA adducts at known, relevant exposures in humans (c) study rat to human extrapolations of AFB(1)-albumin and DNA adduct levels. The results in the rat showed that both AFB(1)-albumin adduct and AFB(1)-DNA adduct formation were linear over this wide dose range. The order of adduct formation within the tissues studied was liver>kidney>colon>lung=spleen. Consenting volunteers received 1 microg ( approximately 15 ng/kg) of [(14)C]AFB(1) in a capsule approximately approximately 3.5-7 h prior to undergoing colon surgery. The mean level of human AFB(1)-albumin adducts was 38.8+/-19.55 pg [(14)C]AFB(1)/mg albumin/microg AFB(1)/kg body weight (b.w.), which was not statistically different to the equivalent dose in the rat (15 ng/kg) 42.29+/-7.13 pg [(14)C]AFB(1)/mg albumin/microg AFB(1)/kg b.w. There was evidence to suggest the formation of AFB(1)-DNA adducts in the human colon at very low doses. Comparison of the linear regressions of hepatic AFB(1)-DNA adduct and AFB(1)-albumin adduct levels in rat found them to be statistically similar suggesting that the level of AFB(1)-albumin adducts are useful biomarkers for AFB(1) dosimetry and may reflect the DNA adduct levels in the target tissue. [(14)C]AFB(1)-DNA and [(14)C]AFB(1)-albumin adducts were hydrolysed and analysed by HPLC to confirm that the [(14)C] measured by AMS was derived from the expected [(14)C]AFB(1) adducts.

Attomole quantitation of protein separations with accelerator mass spectrometry.

Quantification of specific proteins depends on separation by chromatography or electrophoresis followed by chemical detection schemes such as staining and fluorophore adhesion. Chemical exchange of short-lived isotopes, particularly sulfur, is also prevalent despite the inconveniences of counting radioactivity. Physical methods based on isotopic and elemental analyses offer highly sensitive protein quantitation that has linear response over wide dynamic ranges and is independent of protein conformation. Accelerator mass spectrometry quantifies long-lived isotopes such as 14C to subattomole sensitivity. We quantified protein interactions with small molecules such as toxins, vitamins, and natural biochemicals at precisions of 1-5%. Micro-proton-induced X-ray emission quantifies elemental abundances in separated metalloprotein samples to nanogram amounts and is capable of quantifying phopsphorylated loci in gels. Accelerator-based quantitation is a possible tool for quantifying the genome translation into proteome.

Synthesis and spectroscopic characterization of site-specific 2-amino-1-methyl-6-phenylimidazo.

The aim of the present study is to determine the chemical structure and conformation of DNA adducts formed by incubation of the bioactive form of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), N-acetoxy-PhIP, with a single-stranded 11mer oligodeoxyribonucleotide. Using conditions optimized to give the C8-dG-PhIP adduct as the major product, sufficient material was synthesized for NMR solution structure determination. The NMR data indicate that in duplex DNA this adduct exists in equilibrium between two different conformational states. In the main conformer, the covalently bound PhIP molecule intercalates in the helix, whilst in the minor conformation the PhIP ligand is probably solvent exposed. In addition to the C8-dG-PhIP adduct, at least eight polar adducts are found after reaction of N-acetoxy-PhIP with the oligonucleotide. Three of these were purified for further characterization and shown to exhibit lowest energy UV absorption bands in the range 342-347 nm, confirming the presence of PhIP or PhIP derivative. Accurate mass determination of two of the polar adducts by negative ion MALDI-TOF MS revealed ions consistent with a spirobisguanidino-PhIP derivative and a ring-opened adduct. The third adduct, which has the same mass as the C8-dG-PhIP oligonucleotide adduct, may contain PhIP bound to the N2 position of guanine.

In vivo human metabolism of [2-14C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).

To better understand the interactions of the pathways of activation and detoxification on the metabolism of the putative carcinogen, PhIP, we administered a dose of 70-84 microg [2-14C] PhIP (17.5 [microCi 14C) 48-72 h before scheduled colon surgery. Blood and urine collected for the next 48-72 h was evaluated by linear accelerator mass spectroscopy (AMS) and scintillation counting LC-MS to identify specific PhIP metabolites. The thermostable phenol sulfotransferase (SULT1A1) phenotype was correlated with the 4'-PhIP-SO4 levels in the urine at 0-4 h (R = 0.86, P = 0.059). The CYP1A2 activity had a negative correlation with PhIP serum levels at 1 h (R = 0.94, P = 0.06) and a positive correlation with urine N-OH-PhIP levels at 0-4 h (R = 0.85, P = 0.15). This low level radioisotope method of determining the influence of phenotype on metabolism will significantly improve our understanding of the interrelationships of these pathways and provide a critical foundation for the development of individual risk assessment.

Macromolecular adduct formation and metabolism of heterocyclic amines in humans and rodents at low doses.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are heterocyclic amines formed during the cooking of meat and fish. Both are genotoxic in a number of test systems and are carcinogenic in rats and mice. Human exposure to these compounds via dietary sources has been estimated to be under 1 microg/kg body wt. per day, although most laboratory animal studies have been conducted at doses in excess of 10 mg/kg body wt. per day. We are using accelerator mass spectrometry (AMS), a tool for measuring isotopes with attomole sensitivity, to study the dosimetry of protein and DNA adduct formation by low doses of MeIQx and PhIP in rodents and comparing the adduct levels to those formed in humans. The results of these studies show: 1, protein and DNA adduct levels in rodents are dose-dependent; 2, adduct levels in human tissues and blood are generally greater than in rodents administered equivalent doses; and 3, metabolite profiles differ substantially between humans and rodents for both MeIQx and PhIP, with more N-hydroxylation (bioactivation) and less ring oxidation (detoxification) in humans. These data suggest that rodent models do not accurately represent the human response to heterocyclic amine exposure.

Comparative biotransformation studies of MeIQx and PhIP in animal models and humans.

MeIQx and PhIP are putative carcinogenic heterocyclic amines formed during the cooking of meat and fish. Using accelerator mass spectrometry, we have investigated the metabolism and macromolecule binding of 14C-labelled MeIQx and PhIP in human cancer patients compared to the rat. Following oral administration of MeIQx and PhIP, more DNA adducts were formed in human colon tissue compared with rats. Differences were also observed between rats and humans in the metabolite profile and urine excretion for these compounds. These results suggest humans metabolise heterocyclic amines differently to laboratory rodents and question their use as models of human risk.

DNA and protein adduct formation in the colon and blood of humans after exposure to a dietary-relevant dose of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.

Epidemiology studies have indicated that certain dietary components, including well-cooked meat, are risk determinants for colon cancer. Cooked meat can contain significant quantities of heterocyclic aromatic amines (HCAs), which have been established as carcinogens in laboratory animals. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is usually the most mass-abundant HCA, with concentrations up to 480 ppb. We used accelerator mass spectrometry to establish whether DNA and protein adducts can be detected in humans exposed to a quantity of PhIP comparable with levels of exposure that occur in the diet. Five human volunteers were administered a dietary-relevant dose of [14C]PhIP (70-84 microg) 48-72 h before surgery for removal of colon tumors. Blood samples were collected at various time points, and albumin, hemoglobin, and WBC DNA were extracted for analysis by accelerator mass spectrometry. Tissue samples were collected during surgery and used to assess either tissue available doses of [14C]PhIP or adduct levels. The results of this study show: (a) PhIP is activated to a form that will bind to albumin, hemoglobin, and WBC DNA in peripheral blood. WBC DNA adducts were unstable and declined substantially over 24 h; (b) PhIP is bioavailable to the colon, with levels in normal tissue in the range 42-122 pg PhIP/g tissue; and (c) PhIP binds to both protein and DNA in the colon. DNA adduct levels in the normal tissue were 35-135 adducts/10(12) nucleotides, which was significantly lower than tumor tissue. The results of this study demonstrate that PhIP is bioavailable to the human colon following defined dietary-relevant doses and forms DNA and protein adducts.

The identification of [2-(14)C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine metabolites in humans.

[2-(14)C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine ([14C]PhIP), a putative human carcinogenic heterocyclic amine found in well-done cooked meat, was administered orally to three colon cancer patients undergoing a partial colonectomy. Forty-eight to seventy-two hours prior to surgery, subjects received a 70-84 microg dose of 14C. Urine and blood were analyzed by HPLC for PhIP and PhIP metabolites. Metabolites were identified based on HPLC co-elution with authentic PhIP metabolite standards, mass spectral analysis and susceptibility to enzymatic cleavage. In two subjects, approximately 90% of the administered [14C]PhIP dose was eliminated in the urine, whereas in the other, only 50% of the dose was found in the urine. One subject excreted three times more radioactivity in the first 4 h than did the others. Twelve radioactive peaks associated with PhIP were detected in the urine samples. The relative amount of each metabolite varied by subject, and the amounts of each metabolite within subjects changed over time. In all three subjects the most abundant urinary metabolite was identified as 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine-N2-glucuron ide (N-hydroxy-PhIP-N2-glucuronide), accounting for 47-60% of the recovered counts in 24 h. PhIP accounted for <1% of the excreted radiolabel in all three patients. Other metabolites detected in the urine at significant amounts were 4-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate, N-hydroxy-PhIP-N3-glucuronide and PhIP-N2-glucuronide. In the plasma, N-hydroxy-PhIP-N2-glucuronide accounted for 60, 18 and 20% of the recovered plasma radioactivity at 1 h post PhIP dose in subjects 1, 2 and 3 respectively. Plasma PhIP was 56-17% of the recovered dose at 1 h post exposure. The relatively high concentration of N-hydroxy-PhIP-N2-glucuronide and the fact that it is an indicator of bioactivation make this metabolite a potential biomarker for PhIP exposure and activation. Determining the relative differences in PhIP metabolites among individuals will indicate metabolic differences that may predict individual susceptibility to carcinogenic risk from this suspected dietary carcinogen.

Comparison of DNA-adduct and tissue-available dose levels of MeIQx in human and rodent colon following administration of a very low dose.

[2-14C]2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was administered orally (304 ng/kg body-weight dose based upon an average 70-kg-body-weight subject) to 5 human colon-cancer patients (58 to 84 years old), as well as to F344 rats and B6C3F1 mice. Colon tissue was collected from the human subjects at surgery and from the rodents 3.5 to 6 hr after administration. Colon DNA-adduct levels and tissue available doses were measured by accelerator mass spectrometry (AMS). The mean levels of MeIQx in the histologically normal colon tissue were not different among the human (97 +/- 26 pg MeIQx/g), rat (133 +/- 15 pg/g) or mouse (78 +/- 10 pg/g) tissues; and no difference existed between the levels detected in human normal and tumor tissue (101 +/- 15 pg/g). Mean DNA-adduct levels in normal human colon (26 +/- 4 adducts/10(12) nucleotides) were significantly greater (p < 0.01) than in rats (17.1 +/- 1 adduct/10(12) nucleotides) or mice (20.6 +/- 0.9 adduct/10(12) nucleotides). No difference existed in adduct levels between normal and tumor tissue in humans. These results show that MeIQx forms DNA adducts in human colon at low dose, and that the human colon may be more sensitive to the effects of MeIQx than that of mice or rats.

Attomole detection of 3H in biological samples using accelerator mass spectrometry: application in low-dose, dual-isotope tracer studies in conjunction with 14C accelerator mass spectrometry.

This is the first demonstration of the use of accelerator mass spectrometry (AMS) as a tool for the measurement of 3H with attomole (10(-18) mol) sensitivity in a biological study. AMS is an analytical technique for quantifying rare isotopes with high sensitivity and precision and has been most commonly used to measure 14C in both the geosciences and more recently in biomedical research. AMS measurement of serially diluted samples containing a 3H-labeled tracer showed a strong correlation with liquid scintillation counting. The mean coefficient of variation of 3H AMS based upon the analysis of separately prepared aliquots of these samples was 12%. The sensitivity for 3H detection in tissue, protein, and DNA was approximately 2-4 amol/mg of sample. This high sensitivity is comparable to detection limits for 14C-labeled carcinogens using 14C AMS and demonstrates the feasibility of 3H AMS for biomedical studies. One application of this technique is in low-dose, dual-isotope studies in conjunction with 14C AMS. We measured the levels of 3H-labeled 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 14C-labeled 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in rat liver tissue and bound to liver DNA and protein 4.5 h following acute administration of individual or coadministered doses in the range of 4-5100 pmol/kg of body weight. Levels of PhIP and MeIQx in whole tissue and bound to liver protein were dose-dependent. MeIQx-protein and -DNA adduct levels were higher than PhIP adduct levels, which is consistent with their respective carcinogenicity in this organ. Coadministration of PhIP and MeIQx did not demonstrate any measurable synergistic effects compared to administration of these compounds individually. These studies demonstrate the application of AMS for the low-level detection of 3H in small biological samples and for its use in conjunction with 14C AMS for dual-labeling studies.

Covalent binding of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline to albumin and hemoglobin at environmentally relevant doses. Comparison of human subjects and F344 rats.

Covalent binding of the food-borne heterocyclic amine 2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) to albumin and hemoglobin (Hb), 3.5-6.0 hr after oral administration of a single dose of either 21.3 or 228.0 microg of [14C]MeIQx (304 and 3257 ng/kg of body weight, respectively, based on a 70-kg subject weight), was studied in human volunteers using accelerator mass spectrometry. Human protein adduct levels were compared with data obtained for male F344 rats 4.5 hr after oral administration of 0.94-11,420 ng/kg of body weight [14C]MeIQx. Dose-dependent levels of MeIQx-albumin and MeIQx-Hb adducts were detected in both humans and rats. In each case, the regression coefficient (slope) of the dose-response curve was approximately 1. The highest levels of adduct formation per unit dose of MeIQx occurred with human albumin, followed by rat albumin, human Hb, and rat Hb (in that order). Although the human subjects were elderly and underwent colon resection surgery during the study period, the results indicate that formation of albumin and Hb adducts is dose dependent and that a trend exists for higher adduct levels per unit dose in humans, compared with F344 rats. Furthermore, MeIQx-albumin adducts are likely to provide a more sensitive marker of exposure to MeIQx than are MeIQx-Hb adducts.

Detectable levels of serum aflatoxin B1-albumin adducts in the United Kingdom population: implications for aflatoxin-B1 exposure in the United Kingdom.

This study aimed to estimate aflatoxin B1 (AFB1) exposure in the United Kingdom population by measuring levels of serum AFB1-albumin (alb), using immunoassay and high-performance liquid chromatography (HPLC) with fluorescence detection. A self-questionnaire on dietary habits from 104 volunteers (47 men and 57 women) in York was completed, and blood samples were collected. Serum alb was extracted, and AFB1-lysine (lys), the digest product of AFB1-alb, was isolated and measured. A sensitive ELISA (detection limit, approximately 1.4 pg of AFB1-lys) was developed. A good correlation was found between calibration of ELISA results and scintillation counting, for rats dosed with [3H]AFB1 (r = 0.972; P < 0.001). This ELISA was subsequently used to analyze human serum alb. For United Kingdom human sera, the mean adduct levels were 29.3 +/- 14.8 pg AFB1-lys equivalents (eq) mg albumin (males) and 26.9 +/- 14.4 pg AFB1-lys eq/mg alb (females). Confirmation of the ELISA data was sought using reversed-phase HPLC with fluorescence detection. HPLC chromatograms of digested York serum alb were compared to digested serum alb for humans from Qidong County, People's Republic of China, and from AFB1-dosed rats. These all gave similar HPLC profiles. Each sample contained fluorescent material that coeluted with and just before the AFB1-lys standard. Fluorescent fractions were found to be inhibitory in a separate anti-AFB1-lys ELISA, indicating that these earlier fluorescent peaks contained AFB1 residues. Our results suggest that measurable internal AFB1 exposure may be occurring in some United Kingdom individuals, albeit at lower levels than those seen for areas with high AFB1 exposure. The source of this exposure may reflect the known difficulties in accurately monitoring regulated imported foodstuffs and/or the lack of regulations on other potentially contaminated imports. However, no positive correlations were found between our AFB1-lys measurements and any dietary questionnaire information. Animal studies, as well as human studies, have been important in developing exposure and internal adduct relationships in humans. Based on this literature, our AFB1-alb data indicate a mean daily exposure of 3 microg of AFB1 and a mean internal dose in liver DNA of 5.9 adducts/10(7) nucleotides. We believe this may be an overestimate of the AFB1 exposure level in the United Kingdom, and further studies are needed to accurately relate external dose and internal AFB1 biomarkers in humans.

Metabolism of the food-borne mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in humans.

The metabolism of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was investigated in five human volunteers given a dietary equivalent of 14C-labeled MeIQx. The amount of the dose excreted in urine ranged from 20.2% to 58.6%, with unmetabolized MeIQx accounting for 0.7-2.8% of the dose. Five principal metabolites were detected in urine, and four of the derivatives were characterized by on-line UV spectroscopy and by HPLC-MS following immunoaffinity chromatography. Two metabolites were identified as the phase II conjugates N2-(3,8-dimethylimidazo[4,5-f]quinoxalin-2-yl)sulfamic acid (MeIQx-N2-SO3(-)) and N2-(beta-1-glucosiduronyl)-2-amino-3,8-dimethylimidazo[4,5-f ]quinoxaline (MeIQx-N2-Gl). Two other metabolites were the cytochrome P450-mediated (P450) oxidation products 2-amino-8-(hydroxymethyl)-3-methylimidazo[4,5-f]quinoxaline (8-CH2OH-MeIQx), and N2-(beta-1-glucosiduronyl)-N-hydroxy-2-amino-3,8-dimethylimidaz o[4,5-f]quinoxaline (NOH-MeIQx-N2-Gl). The latter product is a conjugate of the genotoxic metabolite 2-(hydroxyamino)-3,8-dimethylimidazo-[4,5-f]quinoxaline (NHOH-MeIQx). A large interindividual variation was observed in the metabolism and disposition of MeIQx; these four metabolites and unchanged MeIQx combined accounted for 6.3-26.7% of the total dose. The remaining principal metabolite found in all subjects accounted for 7.6-28% of the dose. It has not been previously identified in rodents or nonhuman primates, and its structure remains unknown. P450-mediated ring oxidation of MeIQx at the C-5 position, a major pathway of detoxication in rodents, was not detected in humans. Both 8-CH2OH-MeIQx formation and NHOH-MeIQx formation are catalyzed by P450 1A2 and may be useful biomarkers of P450 1A2 activity in humans. The levels of NHOH-MeIQx-N2-Gl found in human urine ranged from 1.4% to 10.0% of the dose, which is significantly higher than that formed in rodents and nonhuman primates undergoing cancer bioassays. Thus, bioactivation of MeIQx by P450-mediated N-oxidation is extensive in humans.

Application of accelerated mass spectrometry (AMS) in DNA adduct quantification and identification.

DNA adducts are nucleotide bases that have been covalently modified by reactive electrophilic chemical intermediates, and have been extensively researched for their role in mutagenesis and carcinogenesis. However, many DNA adduct measurement techniques have difficulty in the quantification of adducts at realistic human exposure levels. We are using the extremely sensitive analytical technique of accelerator mass spectrometry (AMS) to study adducts either at low dose or directly in humans. AMS is a technique for measuring isotope ratios with high selectivity, attomole sensitivity (10(-18) mol) and precision of 0.5-10%, depending on isotope level and preparation method. This sensitivity and precision is being used to study the dose-response, toxicokinetics, and toxicodynamics of DNA adduct formation and removal following administration of very low doses of chemicals.

MeIQx-DNA adduct formation in rodent and human tissues at low doses.

Heterocyclic amines, such as 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), are mutagenic/carcinogenic compounds formed during the cooking of protein-rich foods. Human exposure to MeIQx has been estimated to range from ng/person/day to a few microgram/person/day. In contrast, animal studies have been conducted at doses in excess of 10 mg/kg/day. In order to determine the relevance of high-dose animal data for human exposure, the dose-response curves for [14C]-MeIQx have been determined in rodents at low doses under both single-dose and chronic dosing regimens using the high sensitivity of accelerator mass spectrometry (AMS). To make a direct species comparison, rodent and human colonic MeIQx-DNA adduct levels have been compared following oral administration of [14C]-MeIQx. The results of these studies show: (1) total MeIQx levels are highest in the liver > kidney > pancreas > intestine > blood; (2) MeIQx levels in the liver plateau after 7 days of chronic feeding; (3) hepatic MeIQx-DNA adducts begin to plateau after 2-4 weeks and reach steady-state levels between 4 and 12 weeks on chronic exposures; (4) hepatic DNA adducts generally increase as a linear function of administered dose for a single-dose exposure and as a power function for chronic feeding over a dose range spanning 4 orders of magnitude; (5) human colon DNA adduct levels are approximately 10 times greater than in rodents at the same dose and time point following exposure; and (6) > or = 90% of the MeIQx-DNA adduct in both rodent and human colon appears to be the dG-C8-MeIQx adduct. These studies show that MeIQx is readily available to the tissues for both humans and rodents and that adduct levels are generally linear with administered dose except at high chronic doses where adduct levels begin to plateau slightly. This plateau indicates that linear extrapolation from high-dose studies probably underestimates the amount of DNA damage present in the tissues following low dose. Further, if adducts represent the biologically effective dose, these data show that human colon may be as sensitive to the genotoxic effects of MeIQx as rat liver. The significance of these endpoints to tumor response remains to be determined.

Biomonitoring of possible human exposure to environmental genotoxic chemicals: lessons from a study following the wreck of the oil tanker Braer.

In January 1993 the oil tanker Braer ran aground in the Shetland Islands, Scotland. Approximately 80,000 tons of crude oil were released. Exceptionally high winds caused extensive pollution and exposure of the local population to crude oil. We describe the study which was immediately set in place to examine the exposed population for evidence of genotoxic exposure. Blood samples were taken and primary DNA damage was measured in the mononuclear cell fraction by the butanol modification of the 32P-postlabelling method. Mutation was measured at the hprt locus in T lymphocytes. No evidence of genotoxicity was obtained for either end point, but nevertheless, we believe that useful lessons were learnt, which should be incorporated into the design of future studies: (1) A rapid response is essential, and even if sufficient funds are not immediately available, it is still worth attempting to obtain samples quickly and use cryopreservation, also to attempt to estimate exposure. (2) Adequate numbers of volunteers must be sought, together with enough controls, not just to allow meaningful analysis but to overcome loss of samples and failure of things to go according to plan. (3) Points concerning laboratory practice include: (i) samples should be coded, (ii) clearly defined and proven protocols should be used, (iii) irreplaceable samples should not be used for method development, (iv) should a problem become apparent during the study, work on such samples should cease immediately until the problem is solved, (v) all critical experimental components should be pretested against a laboratory standard. (4) The study design should include replicate experiments to monitor experimental variability and reproducibility, as well as internal standards and cryopreserved "in house" samples. Care must be taken that samples from any one exposure group are spread between a number of independent experiments and that each experiment includes samples from a number of exposure groups. (5) A computerised data base should be maintained with full details of experimental variables, donor attributes, and raw data so that any contribution of experimental artefacts to "outlier" results can be monitored. (6) Because of the nature of the statistical variation for many environmental genotoxicity end points, only a large-scale study is likely to be capable of yielding useful information.