Trials and Tribulations in the First Three Years in Operation of the SSAMS for Biomedical 14C-AMS at LLNL.

We report on the first several years of operation of our recently installed 250 kV SSAMS at LLNL, purchased to replace our 1-MV AMS system for the measurement of 14C from labeled biochemical samples. We have modified the ion source region to improve ion output. Additionally, the SSAMS required significant software modifications to the data acquisition system in order to accurately measure 14C at the high-count rates typically encountered with labeled biochemical samples. We found that the data can be corrected assuming a nonparalyzable dead time response with a single event dead time of 6 µs. Since operation began, we have measured over 13,000 graphitic unknowns and over 1900 standards with an overall precision of 1.0%. We have optimized our system for the analysis of CO2 gas samples. We compared aliquots of identical samples measured as solid graphite and as liquid drops. Excellent agreement was found between the two, although the average precision of the graphite targets was an order of magnitude better than the liquid drop analysis due to the much larger number of 14C atoms available for measurement.

Mass Spectrometric Characterization of an Acid-Labile Adduct Formed with 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and Albumin in Humans.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a carcinogenic heterocyclic aromatic amine formed during the high-temperature cooking of meats. The cytochrome P450-mediated N-hydroxylation of the exocyclic amine group of PhIP produces 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine, an electrophilic metabolite that forms adducts with DNA and proteins. Previous studies conducted by our laboratory showed that the reaction of N-oxidized PhIP metabolites with human albumin in vitro primarily occurs at the Cys34 residue, to produce an acid-labile linked sulfinamide adduct. On the basis of these findings, we developed a sensitive ultraperformance liquid chromatography-mass spectrometry method to measure acid-labile albumin-PhIP adducts in human volunteers administered a dietary-relevant dose of 14C-labeled PhIP [Dingley, K. H., et al. (1999) Cancer Epidemiol., Biomarkers Prev. 8, 507-512]. Mild acid treatment of albumin (0.1 N HCl, 37 °C for 1 h) or proteolytic digestion with Pronase [50 mM ammonium bicarbonate buffer (pH 8.5) at 37 °C for 18 h] released similar amounts of covalently bound PhIP, which was characterized by multistage scanning and quantified by Orbitrap mass spectrometry. The amount of [14C]PhIP recovered by acid treatment of albumin 24 h following dosing accounted for 7.2-21.3% of the [14C]PhIP bound to albumin based on accelerator mass spectrometry measurements. 2-Amino-1-methyl-6-(5-hydroxy)phenylimidazo[4,5-b]pyridine, a hydrolysis product of the Cys34 S-N linked sulfenamide adduct of PhIP, was not detected in either acid-treated or protease-treated samples. These findings suggest that a portion of the PhIP bound to albumin in vivo probably occurs as an acid-labile sulfinamide adduct formed at the Cys34 residue.

Operation of the "Small" BioAMS Spectrometers at CAMS: Past and Future Prospects.

A summary of results from the solid samples run on our compact 1 MV AMS system over its 13.5 years of operation is presented. On average 7065 samples per year were measured with that average dropping to 3278 samples per year following the deployment of our liquid sample capability. Although the dynamic range of our spectrometer is 4.5 orders in magnitude, most of the measured graphitic samples had 14C/C concentrations between 0.1 and 1 modern. The measurements of our ANU sucrose standard followed a Gaussian distribution with an average of 1.5082 ± 0.0134 modern. The LLNL biomedical AMS program supported many different types of experiments, however, the large majority of samples measured were derived from animal model systems. We have transitioned all of our biomedical AMS measurements to the recently installed 250 kV SSAMS instrument with good agreement compared in measured 14C/C isotopic ratios between sample splits. Finally, we present results from replacement of argon stripping gas with helium in the SSAMS with a 22% improvement in ion transmission through the accelerator and high-energy analyzing magnet.

An Interface for the Direct Coupling of Small Liquid Samples to AMS.

We describe the moving wire interface attached to the 1-MV AMS system at LLNL's Center for Accelerator Mass Spectrometry for the analysis of nonvolatile liquid samples as either discrete drops or from the direct output of biochemical separatory instrumentation, such as high-performance liquid chromatography. Discrete samples containing at least a few 10s of nanograms of carbon and as little as 50 zmol 14C can be measured with a 3-5% precision in a few minutes. The dynamic range of our system spans approximately 3 orders in magnitude. Sample to sample memory is minimized by the use of fresh targets for each discrete sample or by minimizing the amount of carbon present in a peak generated by an HPLC containing a significant amount of 14C. Liquid Sample AMS provides a new technology to expand our biomedical AMS program by enabling the capability to measure low-level biochemicals in extremely small samples that would otherwise be inaccessible.

Model-Based, Closed-Loop Control of PZT Creep for Cavity Ring-Down Spectroscopy.

Cavity ring-down spectrometers typically employ a PZT stack to modulate the cavity transmission spectrum. While PZTs ease instrument complexity and aid measurement sensitivity, PZT hysteresis hinders the implementation of cavity-length-stabilized, data-acquisition routines. Once the cavity length is stabilized, the cavity's free spectral range imparts extreme linearity and precision to the measured spectrum's wavelength axis. Methods such as frequency-stabilized cavity ring-down spectroscopy have successfully mitigated PZT hysteresis, but their complexity limits commercial applications. Described herein is a single-laser, model-based, closed-loop method for cavity length control.

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.

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.

Solution structure of the 2-amino-1- methyl-6-phenylimidazo[4,5-b]pyridine C8-deoxyguanosine adduct in duplex DNA.

The carcinogenic heterocyclic amine (HA) 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is formed during the cooking of various meats. To enable structure/activity studies aimed at understanding how DNA damaged by a member of the HA class of compounds can ultimately lead to cancer, we have determined the first solution structure of an 11-mer duplex containing the C8-dG adduct formed by reaction with N-acetoxy-PhIP. A slow conformational exchange is observed in which the PhIP ligand either intercalates into the DNA helix by denaturing and displacing the modified base pair (main form) or is located outside the helix in a minimally perturbed B-DNA duplex (minor form). In the main base-displaced intercalation structure, the minor groove is widened, and the major groove is compressed at the lesion site because of the location of the bulky PhIP-N-methyl and phenyl ring in the minor groove; this distortion causes significant bending of the helix. The PhIP phenyl ring interacts with the phosphodiester-sugar ring backbone of the complementary strand and its fast rotation with respect to the intercalated imidazopyridine ring causes substantial distortions at this site, such as unwinding and bulging-out of the strand. The glycosidic torsion angle of the [PhIP]dG residue is syn, and the displaced guanine base is directed toward the 3' end of the modified strand. This study contributes, to our knowledge, the first structural information on the biologically relevant HA class to a growing body of knowledge about how conformational similarities and differences for a variety of types of lesions can influence protein interactions and ultimately biological outcome.

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.

Bioanalytical applications of accelerator mass spectrometry for pharmaceutical research.

Accelerator mass spectrometry (AMS) is a mass spectrometric method for quantifying isotopes. It has had great impact in the geosciences and is now being applied in the biomedical fields. AMS measures radioisotopes such as 14C, 3H, 41Ca, and 36Cl, and others, with attomole sensitivity and high precision. Its use is allowing absorption, distribution, metabolism and elimination studies, as well as detailed pharmacokinetics, to be carried out directly in humans with very low chemical or radiological hazard. It is used in combination with standard separation methodologies, such as chromatography, in identification of metabolites and molecular targets for both toxicants and pharmacologic agents. AMS allows the use of very low specific activity chemicals (< 1 mCi/mmol), creating opportunities to use compounds not available in a high specific activity form, such as those that must be biosynthesized, produced in combinatorial libraries, or made through inefficient synthesis. AMS is allowing studies to be carried out with agents having low bioavailability, low systemic distributions, or high toxicity where administered doses must be kept low (<1 microg/kg). It may have uses in tests for idiosyncratic metabolism, drug interaction, or individual susceptibility, among others. The ability to use very low chemical doses, low radiological doses, small samples and conduct multiple dose studies may help move drug candidates into humans faster and safer than before. The uses of AMS are growing and its potential for drug development is only now beginning to be realized.

Quantitation of benzo[a]pyrene-DNA adducts by postlabeling with 14C-acetic anhydride and accelerator mass spectrometry.

Quantitation of carcinogen-DNA adducts provides an estimate of the biologically effective dose of a chemical carcinogen reaching the target tissue. In order to improve exposure-assessment and cancer risk estimates, we are developing an ultrasensitive procedure for the detection of carcinogen-DNA adducts. The method is based upon postlabeling of carcinogen-DNA adducts by acetylation with 14C-acetic anhydride combined with quantitation of 14C by accelerator mass spectrometry (AMS). For this purpose, adducts of benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide (BPDE) with DNA and deoxyguanosine (dG) were synthesized. The most promutagenic adduct of BPDE, 7R,8S,9R-trihydroxy-10S-(N(2)-deoxyguanosyl)-7,8,9, 10-tetrahydrobenzo[a]pyrene (BPdG), was HPLC purified and structurally characterized. Postlabeling of the BPdG adduct with acetic anhydride yielded a major product with a greater than 60% yield. The postlabeled adduct was identified by liquid chromatography-mass spectrometry as pentakis(acetyl) BPdG (AcBPdG). Postlabeling of the BPdG adduct with 14C-acetic anhydride yielded a major product coeluting with an AcBPdG standard. Quantitation of the 14C-postlabeled adduct by AMS promises to allow detection of attomolar amounts of adducts. The method is now being optimized and validated for use in human samples.

Methods of DNA adduct determination and their application to testing compounds for genotoxicity.

At the International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC (March 25-26, 1999), a working group considered the uses of DNA adduct determination methods for testing compounds for genotoxicity. When a drug or chemical displays an unusual or inconsistent combination of positive and negative results in in vitro and in vivo genotoxicity assays and/or in carcinogenicity experiments, investigations into whether or not DNA adducts are formed may be helpful in assessing whether or not the test compound is a genotoxin. DNA adduct determinations can be carried out using radiolabeled compounds and measuring radioactive decay (scintillation counting) or isotope ratios (accelerator mass spectrometry) in the isolated DNA. With unlabeled compounds adducts may be measured by (32)P-postlabeling analysis of the DNA, or by physicochemical methods including mass spectrometry, fluorescence spectroscopy, or electrochemical detection, or by immunochemical methods. Each of these approaches has different strengths and limitations, influenced by sensitivity, cost, time, and interpretation of results. The design of DNA binding studies needs to be on a case-by-case basis, depending on the compound's profile of activity. DNA purity becomes increasingly important the more sensitive, and less chemically specific, the assay. While there may be adduct levels at which there is no observable biological effect, there are at present insufficient data on which to set a threshold level for biological significance.

Species and strain comparisons in the macromolecular binding of extremely low doses of [14C]benzene in rodents, using accelerator mass spectrometry.

The kinetics of macromolecular binding of a 5 micrograms/kg body wt dose of [14C]benzene was studied over 48 h in B6C3F1, DBA/2, and C57BL/6 mice and Fischer rats to determine if adduct levels reflect known differences in metabolic capacity, genotoxicity, and carcinogenic potency. Previous studies have suggested that differences in benzene toxicity among strains result from differences in metabolism. Rats and mice were administered [14C]benzene (i.p.), followed by removal of liver and bone marrow at time intervals up to 48 h postexposure. Protein and DNA were isolated and analyzed by accelerator mass spectrometry. Area under the curves for protein and DNA adducts in bone marrow were greatest in B6C3F1 mouse > DBA/2 mouse > C57BL/6 mouse > Fischer rat. These data are consistent with the hypothesis that metabolic capacity contributes to the difference in benzene's carcinogenicity among species. Additionally, these data suggest that target organ adduct levels correlate with tumorigenicity and thus may be indicative of an individuals risk.

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.

Dose-dependent binding of ortho-phenylphenol to protein but not DNA in the urinary bladder of male F344 rats.

ortho-Phenylphenol (OPP) is a widely used fungicide and antibacterial agent that is also known to be highly effective in inducing bladder tumors in male F344 rats. At present, neither the role of the urinary bladder in the bioactivation of OPP metabolites nor the nature of the molecular target is understood. To address these issues, we investigated the relationship between OPP dosage and macromolecular adduct formation in the urinary bladder of male F344 rats. Male F344 rats were treated with 0, 15, 50, 125, 250, 500, 1000 mg/kg of OPP and its radiocarbon analogue via oral gavage. The dosed rats were euthanized after 24 h, and the proteins were extracted from the liver, kidney, and bladder. The amount of radioactivity associated with the extracted protein was quantified using highly sensitive accelerator mass spectrometry. Protein binding in liver and kidney exhibited a linear or modest curvilinear relationship over the dose range studied. In the urinary bladder, however, a pronounced nonlinear relationship between protein adduct levels and administered dose was observed. The measured protein adduct levels were in agreement with the predicted concentrations of phenylbenzoquinone based on a proposed mechanism involving free phenylhydroquinone autoxidation in the urine. Unlike protein binding, DNA adducts measured from the same bladder samples did not show a significant difference from the control group. These data are consistent with the hypothesis that OPP is an indirect acting carcinogen, and that regenerative hyperplasia due to OPP-metabolite cytotoxicity and/or binding of OPP metabolites to protein targets may play an important role in OPP-induced bladder carcinogenesis.

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.