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Dietary exposure to aflatoxin B(1) (AFB(1)) is a significant contributor to the incidence of hepatocellular carcinomas globally. AFB(1) exposure leads to the formation of AFB(1)-N(7)-guanine (AFB(1)-N(7)-Gua) and two diastereomers of the imidazole ring-opened 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B(1) (AFB(1)-FapyGua) in DNA. These adducts lead to G → T transversion mutations with the ring-opened adduct being more mutagenic than the cationic species. Accurate measurement of these three adducts as biomarkers in DNA and urine will help identify dietary exposure to AFB(1) as a risk factor in the development of hepatocellular carcinoma worldwide. Herein, we report an improved methodology for the measurement of AFB(1)-N(7)-Gua and the two diastereomers of AFB(1)-FapyGua using liquid chromatography-tandem mass spectrometry with isotope dilution. We measured the levels of these compounds in liver DNA of six control mice and six AFB(1)-treated mice. Levels varying from 1.5 to 45 lesions/10(6) DNA bases in AFB(1)-treated mice were detected depending on the compound and animal. No background levels of these adducts were detected in control mice. We also tested whether the AFB(1) treatment caused oxidatively induced DNA base damage using gas chromatography-tandem mass spectrometry with isotope dilution. Although background levels of several pyrimidine- and purine-derived lesions were detected, no increases in these levels were found upon AFB(1) treatment of mice. On the other hand, significantly increased levels of (5′R)- and (5′S)-8,5′-cyclo-2′-deoxyadenosines were observed in liver DNA of AFB(1)-treated mice. The impact of this work is expected to achieve the accurate measurement of three AFB(1)-DNA adducts and oxidatively induced DNA lesions as biomarkers of AFB(1) exposure as germane to investigations designed for the prevention of aflatoxin-related hepatocellular carcinomas and for determining the effects of genetic deficiencies in human populations.

Outdoor air pollution, a spatially and temporally complex mixture, is a human carcinogen. However, ambient measurements may not reflect subject-level exposures, personal monitors do not assess internal dose, and spot assessments of urinary biomarkers may not recapitulate chronic exposures. Nucleophilic sites in serum albumin – particularly the free thiol at Cys(34) – form adducts with electrophiles. Due to the 4-week lifetime of albumin in circulation, accumulating adducts can serve as intermediate- to long-residence biomarkers of chronic exposure and implicate potential biological effects. Employing nanoflow liquid chromatography-high resolution mass spectrometry (nLC-HRMS) and parallel reaction monitoring (PRM), we have developed and validated a novel targeted albumin adductomics platform capable of simultaneously monitoring dozens of Cys(34) adducts per sample in only 2.5 μL of serum, with on-column limits of detection in the low femtomolar range. Using this platform, we characterized the magnitude and impact of ambient outdoor air pollution exposures with three repeated measurements over 84 days in n=26 non-smoking women (n=78 total samples) from Qidong, China, an area with a rising burden of lung cancer incidence. In concordance with seasonally rising ambient concentrations of NO(2), SO(2), and PM(10) measured at stationary monitors, we observed elevations in concentrations of Cys(34) adducts of benzoquinone (p

We report validation of the first high-pressure liquid chromatography isotope-dilution mass spectrometry method to measure sulforaphane (SFN) and its glutathione-derived conjugates in human urine. As epidemiological evidence continues to mount that the consumption of a diet rich in cruciferous vegetables may reduce the risk of certain cancers, the development of analytical methodologies to accurately measure isothiocyanates (ITCs) and their subsequent metabolic products becomes paramount. SFN, the principal ITC produced by broccoli, is an effective chemopreventive agent with multiple modes of action. SFN and SFN conjugates have often been measured collectively utilizing a cyclocondensation assay with 1,2-benzenedithiol. More recently, some of the major SFN conjugates have been determined using mass spectrometry. Here, triple-quadrupole mass spectrometry has been coupled with the use of stable isotope-labeled internal standards of D8-SFN and all four D8-SFN mercapturic acid pathway conjugates to provide an accurate, precise, sensitive, and specific method for analysis of these compounds. Using urine samples collected during an earlier intervention with broccoli sprouts, the concentrations of SFN, SFN-cysteine, and the mercapturic acid SFN- N-acetylcysteine were sufficiently high such that as little as 50 nL of urine was required for analysis. Although each study participant received an equivalent dose of broccoli sprout preparation, the interindividual conversion of the precursor glucosinolate to SFN varied over 100-fold. These 98 urines provided an ideal sample set for examining the robustness of the assay. The mean urinary concentrations +/- standard deviations in overnight voids following ingestion of the first dose were 4.7 +/- 5.1, 0.03 +/- 0.05, 0.06 +/- 0.06, 18 +/- 15, and 42 +/- 23 nmol/mg creatinine for SFN, SFN-glutathione, SFN-cysteine-glycine, SFN-cysteine, and SFN- N-acetylcysteine, respectively. This method determines SFN and all four SFN glutathione-derived metabolites with minimal sample preparation and will be extremely useful in understanding the role of SFN-rich foods in preventing cancer and other chronic diseases.

Estrogen-DNA adducts are potential biomarkers for assessing the risk of developing of a number of hormonally modified cancers, including breast cancer. Formation of the 4-hydroxyestradiol-N(7)-guanine (4-OHE2-N(7)-guanine) adduct from the reaction of estradiol-3,4-quinone with DNA and its detection in vivo has been established. With the ultimate goal of exploring estrogen-DNA adducts as biomarkers in experimental and human investigations, the 4-OHE2-N(7)-guanine was synthesized, and preliminary studies demonstrated that this adduct was detectable in all 10 female human urine samples examined. Therefore, more extensive investigations were conducted to study this compound's chemical-physical properties and to examine the stability of 4-OHE2-N(7)-guanine under a range of pH conditions that might influence biomarker measurement. Under neutral to alkaline conditions, 4-OHE2-N(7)-guanine could completely oxidize to an 8-oxo-guanine derivative. This derivative was isolated by HPLC, and mass spectrometry confirmed the oxidized compound by demonstrating the formation of an m/ z 168 fragment, generated by oxygen addition to guanine. Furthermore, investigation of the 4-OHE2-N(7)-2'-deoxyguanosine nucleoside adduct showed that under alkaline conditions a formamidopyrimidine analogue was produced. The formamidopyrimidine derivative forms from ring opening of the guanine imidazole ring following C-8 oxidation in the N(7), N(9) disubstituted guanine. Formation of both of these oxidized estrogen-guanine DNA adducts has precedent with other chemical agents that covalently bind to the N(7) position in guanine. Therefore, the development and application of methods to measure estrogen-guanine adducts will need to also consider these new adducts, and the biological implications of these compounds will need to be explored to determine their contribution to estrogen toxicology.

The reduction of the aflatoxin B 1 (AFB 1) dialdehyde metabolite to its corresponding mono and dialcohols, catalyzed by aflatoxin B 1-aldehyde reductase (AFAR, rat AKR7A1, and human AKR7A3), is greatly increased in livers of rats treated with numerous chemoprotective agents. Recombinant human AKR7A3 has been shown to reduce the AFB 1-dialdehyde at rates greater than those of the rat AKR7A1. The activity of AKR7A1 or AKR7A3 may detoxify the AFB 1-dialdehyde, which reacts with proteins, and thereby inhibits AFB 1-induced toxicity; however, direct experimental evidence of this hypothesis was lacking. Two human B lymphoblastoid cell lines, designated pMF6/1A2/AKR7A1 and pMF6/1A2, were genetically engineered to stably express AKR7A1 and/or cytochrome P4501A2 (1A2). The pMF6/1A2/AKR7A1 cells were refractory to the cytotoxic effects of 3 ng/mL AFB 1, in comparison to pM6/1A2 cells, which were more sensitive. Diminished protection occurred at higher concentrations of AFB 1 in pMF6/1A2/AKR7A1 cells, suggesting that additional factors were influencing cell survival. COS-7 cells were transfected with either vector control, rat AKR7A1, or human AKR7A3, and the cells were treated with AFB 1-dialdehyde. There was a 6-fold increase in the dialdehyde LC 50, from 66 microM in vector-transfected cells to 400 microM in AKR7A1-transfected cells, and an 8.5-fold increase from 35 microM in vector-transfected cells to 300 microM in AKR7A3-transfected cells. In both cases, this protective effect of the AFAR enzyme was accompanied by a marked decrease in protein adducts. Fractionation of the cellular protein showed that the mitochondria/nuclei and microsomal fractions contained the highest concentration of protein adducts. The levels of human AKR7A3 and AKR7A2 were measured in 12 human liver samples. The expression of AKR7A3 was detectable in all livers and lower than those of AKR7A2 in 11 of the 12 samples. Overall, these results provide the first direct evidence of a role for rat AKR7A1 and human AKR7A3 in protection against AFB 1-induced cytotoxicity and protein adduct formation.

We report validation of the first isotope dilution mass spectrometry method for determination of aflatoxin B(1)-N(7)-guanine (AFB(1)-N(7)-Gua), a major human aflatoxin-DNA adduct that is excreted in the urine. Measurement of urinary AFB(1)-N(7)-Gua, a biomarker of the biologically effective dose following dietary aflatoxin B(1) (AFB(1)) exposure, has helped identify AFB(1) as a risk factor in the development of hepatocellular carcinoma, a common cancer worldwide. Triple-quadrupole mass spectrometry, coupled with the use of a stable isotope-labeled internal standard (AFB(1)-N(7)-(15)N(5)-Gua) and better solid phase extraction and immunoaffinity column chromatography, have enabled us to greatly improve accuracy, precision, specificity, and sensitivity over previously published determinations. The limit of quantitation for AFB(1)-N(7)-Gua was 0.8 pg/20 mL urine (0.07 pg/mg creatinine). The method was validated for accuracy and precision over the range of 0.8-25 pg/20 mL urine, with between-day and within-day reproducibility for analysis of six aliquots of a human urine sample containing 6.0 pg/20 mL measured at

Aromatic amines such as 2-naphthylamine and 4-aminobiphenyl are established human bladder carcinogens. Experimental evidence for carcinogenicity of monocylic aromatic amines is limited mostly to other organs, but a recent epidemiologic study of bladder cancer found that 2,6-dimethyl- (2,6-DMA), 3,5-dimethyl- (3,5-DMA), and 3-ethylaniline (3-EA) may play a significant role in the etiology of this disease in man. The present work was undertaken to test whether a genotoxic mechanism can account for the presumptive activity of 2,6-DMA, 3,5-DMA, and 3-EA by quantifying the binding of these compounds to DNA in vivo. Each of these three [(14)C]alkylanilines was administered at approximately 100 microg/kg to C57BL/6 mice, which were subsequently sacrificed 2, 4, 8, 16, and 24 h post-dosing. Bladder, colon, kidney, liver, lung, and pancreas were harvested from each animal, and DNA was isolated from each tissue. Adduct levels were determined by quantifying bound isotope using accelerator mass spectrometry. Adducts were detectable in the bladder and liver DNA samples from every animal at every time point at levels that ranged from 3 per 10(9) to 1.5 per 10(7) nucleotides. Adduct levels were highest in animals given 3,5-DMA and lowest in those given 3-EA. Levels in both bladder and liver declined by severalfold over the course of the experiment. Adducts were detected less frequently in the other four tissues. Taken together, the results strongly suggest that these three alkylanilines are metabolized in vivo to electrophilic intermediates that covalently bind to DNA and that adducts are formed in the DNA of bladder, which is a putative target organ for these alkylanilines.

Epidemiological studies have shown that exposure to aflatoxin B(1) (AFB(1)) and concurrent infection with hepatitis B lead to a multiplicative risk of developing liver cancer. This chemical-viral interaction can be recapitulated in the tree shrew (Tupia belangeri chinensis). As an initial characterization of this model, the metabolism of AFB(1) in tree shrews has been examined and compared to a sensitive bioassay species, the rat. Utilizing LC/MS/MS, an unreported product, aflatoxin M(1)-N(7)-guanine (AFM(1)-N(7)-guanine), was detected in urine and hepatic DNA samples 24 h after administration of 400 microg/kg AFB(1). In hepatic DNA isolated from tree shrews, AFM(1)-N(7)-guanine was the predominant adduct, 0.74 +/- 0.14 pmol/mg DNA, as compared to 0.37 +/- 0.07 pmol/mg DNA of AFB(1)-N(7)-guanine. Conversely, in rat liver, 6.56 +/- 2.41 pmol/mg DNA of AFB(1)-N(7)-guanine and 0.42 +/- 0.13 pmol/mg DNA of AFM(1)-N(7)-guanine were detected. Rats excreted 1.00 +/- 0.21 pmol AFB(1)-N(7)-guanine/mg creatinine and 0.29 +/- 0.10 pmol AFM(1)-N(7)-guanine/mg creatinine as compared to 0.60 +/- 0.12 pmol AFB(1)-N(7)-guanine/mg creatinine and 0.69 +/- 0.16 pmol AFM(1)-N(7)-guanine/mg creatinine excreted by the tree shrew. Furthermore, tree shrew urine contained 40 times more of the hydroxylated metabolite, AFM(1), than was excreted by rats. In vitro experiments confirmed this difference in oxidative metabolism. Hepatic microsomes isolated from tree shrews failed to produce aflatoxin Q(1) or aflatoxin P(1) but formed a significantly greater amount of AFM(1) than rat microsomes. Bioassays indicated that the tree shrew was considerably more resistant than the rat to AFB(1) hepatocarcinogenesis, which may reflect the significant differences in metabolic profiles of the two species.

Oxidation of the mycotoxin aflatoxin (AF) B1 yields the 8,9-epoxide, which nonenzymatically hydrolyzes rapidly to a dihydrodiol that in turn undergoes slow, base-catalyzed ring opening to a dialdehyde [Johnson, W. W., Harris, T. M., and Guengerich F. P. (1996) J. Am. Chem. Soc. 118, 8213-8220]. AFB1 dialdehyde does not bind to DNA but can react with protein lysine groups. One enzyme induced by cancer chemopreventive agents is AFB1 aldehyde reductase (AFAR), which catalyzes the NADPH-dependent reduction of the dialdehyde to a dialcohol. AFB1 dialdehyde is known to convert nonenzymatically to AFB1 dihydrodiol at neutral pH, and we reinvestigated the enzymatic reaction by preparing AFB1 dialdehyde at pH 10 and then used this to initiate reactions (at neutral pH) with rat and human AFAR isozymes. Two monoalcohols were identified as products, and their identities were established by NaB2H4 reduction, chemical cleavage, and mass spectrometry. The monoalcohol corresponding to reduction at C-8 formed first in reactions catalyzed by either the rat or the human AFAR. This C-8 monoalcohol was further reduced to AFB1 dialcohol by AFAR. The other monoalcohol (C-6a) was formed but not reduced to the dialcohol rapidly. Steady-state kinetic parameters were estimated for the reduction of AFB1 dialdehyde by rat and human AFAR to the monoalcohols. The apparent k(cat) and K(m) values were not adequate to rationalize the observed DeltaA(340) spectral changes in a kinetic model. Simulation fitting was done and yielded parameters indicative of greater enzyme efficiency. A survey of 12 human liver cytosol samples showed a variation of 2.3-fold in AFAR activity. Rats treated with AFB1 excreted the dialcohol and a monoalcohol in urine. The results of these studies are consistent with a role of (rat and human) AFAR in protection against AFB1 toxicity.

Polycyclic aromatic hydrocarbons, such as benzo[a]pyrene, are widespread environmental carcinogens of human concern. Several enzymatic systems have been shown to activate benzo[a]pyrene 7, 8-dihydrodiol, the proximate carcinogenic metabolite of benzo[a]pyrene, to a reactive species which produces both a chemiluminescence response and genotoxic lesions. The chemiluminescence response has been proposed to be the result of the formation of a dioxetane which upon ring opening forms a reactive dialdehyde intermediate. In in vitro incubations involving phorbol ester-stimulated human polymorphonuclear leukocytes or an isolated enzyme system consisting of myeloperoxidase, taurine, and hydrogen peroxide, a prolonged (>60 min) chemiluminescence response was observed from benzo[a]pyrene 7,8-dihydrodiol. HPLC analysis of the reaction mixture revealed the existence of a product which is dependent upon both taurine and the hydrocarbon. Characterization of this product using UV, NMR, and MS indicated that the product is a pyrene with two side chains resulting from bond breakage of a ring, yielding a dialdehyde. These side chains contain a portion of taurine covalently attached through imine formation with the aldehydes resulting from dioxetane ring opening. Replacement of taurine with either protein or DNA also produced a prolonged chemiluminescence response. These results demonstrate for the first time the formation of a novel electrophilic species from benzo[a]pyrene 7,8-dihydrodiol which along with an increased production of photons from this activation mechanism may lead to DNA and/or protein damage that is different from that elicited by diol epoxides.

Biologic effects of the hepatocarcinogenic mycotoxin aflatoxin B1 are principally induced by one of its metabolites, the exo-aflatoxin B1 epoxide which produces both DNA and protein adducts in vivo. Detoxication of the exo-aflatoxin B1 epoxide can be mediated in part by glutathione S-transferases whose induction could be important in chemoprotection interventions. Thus, biomarkers of the enzymatic conjugation of exo-aflatoxin B1 epoxide with glutathione may be important indices of protection against the toxic effects of this agent. Since glutathione conjugates undergo further metabolic processing in vivo to yield mercapturic acids, increased urinary excretion of exo-aflatoxin B1 mercapturate could be expected during chemoprotection intervention. To determine if this mercapturic acid could be used as a biomarker, techniques for its specific measurement were developed using monoclonal antibody immunoaffinity chromatography and reverse phase high-performance liquid chromatography with ultraviolet absorbance and mass spectral detection. First, a synthetic exo-aflatoxin B1 mercapturate was characterized using mass spectrometry, ultraviolet absorbance, circular dichroism spectrometry, and chemical derivatization. In vivo metabolite characterization was then facilitated by comparison with the synthetically prepared exo-aflatoxin B1 mercapturate and both aflatoxin B1-glutathione conjugate diastereoisomers. In rats, 1% of the aflatoxin dose was excreted as exo-aflatoxin B1 mercapturate within 24 h. The finding that exo-aflatoxin B1 mercapturate was excreted in urine in a dose-dependent manner provides the basis for investigating its applicability as a biomarker of glutathione S-transferase status in aflatoxin chemoprotection studies.

The glucuronidation of the food-borne heterocyclic amine 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) was investigated using hepatic microsomes from several species. PhIP-glucuronic acid conjugates were formed in an NADPH-free system using microsomes from rabbit, dog, guinea pig, and human. Rat, hamster, and mouse microsomes were incapable of directly producing PhIP-glucuronides. The PhIP-glucuronide generated with human microsomes could be resolved by reverse-phase HPLC from that produced with rabbit microsomes. In addition, the human PhIP-glucuronide was susceptible to enzymatic hydrolysis by beta-glucuronidase, whereas the rabbit PhIP-glucuronide did not undergo beta-glucuronidase catalyzed hydrolysis. Fast atom bombardment mass spectrometry of both glucuronides revealed the presence of ions with m/z 401 (M+H+). Rabbit PhIP-glucuronide had a lambda max of 316 nm, similar to that of the parent PhIP. By contrast, a spectral shift in UV absorbance was observed for the human PhIP-glucuronide, which had a lambda max of 305 nm. 1H-NMR spectroscopy and nuclear Overhauser enhancements established that rabbit PhIP-glucuronide was conjugated at the exocyclic amine nitrogen, whereas human PhIP-glucuronide was conjugated at the N3 imidazole ring nitrogen. Km values for PhIP were 0.2-0.3 mM in both species; however, rabbit microsomes exhibited a 22-fold higher Vmax. Collectively, these studies indicate that human and rabbit liver microsomes form structurally different glucuronides of PhIP and suggest the involvement of multiple isoforms of UDP-glucuronosyltransferase. Further, these data suggest that in certain species, including humans, the direct conjugation of PhIP with glucuronic acid may represent a primary route of PhIP metabolism and detoxication.

Aflatoxin Q1 8,9-oxide was synthesized and found to yield lower levels of N7-guanyl adducts than obtained from aflatoxin B1 8,9-oxide when mixed with calf thymus DNA or Salmonella typhimurium TA 98 cells. However, when S. typhimurium TA 98 was treated with the (analogous) epoxides of aflatoxin B1, aflatoxin G1, aflatoxin Q1, or sterigmatocystin, the ratios of revertants to N7-guanyl DNA adducts were similar. Aflatoxin Q1 and aflatoxin B1 8,9-oxide (trapped here as the glutathione conjugate) are the major oxidative products formed from aflatoxin B1 at all substrate concentrations in human liver microsomes, and cytochrome P-450 (P-450) 3A4 appears to be the dominant enzyme involved in both oxidations, as judged by studies involving correlation of activities in different liver samples, chemical inhibition, immunoinhibition, and reconstitution with purified hepatic and yeast recombinant P-450 3A4. Aflatoxin Q1 is not appreciably oxidized in human liver microsomes and is not very genotoxic. The postulated formation of both aflatoxin Q1 and aflatoxin 8,9-oxide from aflatoxin B1 can be rationalized by a model in which P-450 3A4 binds the substrate in either of two different configurations. This is further demonstrated by the dichotomous effect of 7,8-benzoflavone--this flavone stimulates 8,9-epoxidation while inhibiting the 3 alpha-hydroxylation reaction to form aflatoxin Q1. Thus, the 3 alpha-hydroxylation of aflatoxin B1 to aflatoxin Q1 is viewed as a potentially significant detoxication pathway.

The aflatoxin B 1 aldehyde reductases (AFARs), inducible members of the aldo-keto reductase superfamily, convert aflatoxin B 1 dialdehyde derived from the exo- and endo-8,9-epoxides into a number of reduced alcohol products that might be less capable of forming covalent adducts with proteins. An isotope dilution tandem mass spectrometry method for quantification of the metabolites, C-8 monoalcohol, dialcohol, and C-6a monoalcohol, was developed to ascertain their possible role as urinary biomarkers for application to chemoprevention investigations. This method uses a novel (13)C 17-aflatoxin B 1 dialcohol internal standard, synthesized from (13)C 17-aflatoxin B 1 biologically produced by Aspergillus flavus. Chromatographic standards of the alcohols were generated through sodium borohydride reduction of the aflatoxin B 1 dialdehyde. This method was then explored for sensitivity and specificity in urine samples of aflatoxin B 1-dosed rats that were pretreated with 3 H-1,2-dithiole-3-thione to induce the expression of AKR7A1, a rat isoform of AFAR. One of the two known monoalcohols and the dialcohol metabolite were detected in all urine samples. The concentrations were 203.5 +/- 39.0 ng of monoalcohol C-6a/mg of urinary creatinine and 10.0 +/- 1.0 ng of dialcohol/mg of creatinine (mean +/- standard error). These levels represented about 8.0 and 0.4% of the administered aflatoxin B 1 dose that was found in the urine at 24 h, respectively. Thus, this highly sensitive and specific isotope dilution method is applicable to in vivo quantification of urinary alcohol products produced by AFAR. Heretofore, the metabolic fate of the 8,9-epoxides that are critical for aflatoxin toxicities has been measured by biomarkers of lysine-albumin adducts, hepatic and urinary DNA adducts, and urinary mercapturic acids. This urinary detection of the alcohol products directly contributes to the goal of mass balancing the fate of the bioreactive 8,9-epoxides of AFB 1 in vivo.

Aflatoxin B1 (AFB1) is a major risk factor in the pathogenesis of liver cancer in Asia and sub-Saharan Africa. Biomarkers reflecting exposure will facilitate disease risk assessment and the efficacy of protective interventions in these populations. The Lys-AFB1 adduct in plasma albumin is a candidate biomarker for this role. Although aflatoxin albumin adducts are most frequently measured in epidemiological studies using an enzyme-linked immunosorbent assay, a more specific and 10-fold more sensitive isotopic dilution mass spectrometric assay for Lys-AFB1 has recently become available. Here, the dosimetry of chronically administered AFB1 at lower doses than have been previously studied was explored using this assay. AFB1 was administered to rats for nine consecutive days at eight dose levels ranging from 50 pg to 55 microg/kg body wt. Plasma samples were enzymatically digested and processed by solid phase extraction. Lys-AFB1 was isolated by HPLC and detected via selected reaction monitoring. The dose-response relationship was linear-quadratic exhibiting upward curvature at higher doses. The adduct yield [(pg Lys-AFB1/mg albumin)/(microg AFB1/kg body wt)] increased nonlinearly with the dose by 6-fold between the 0.05 and 55 microg AFB1/kg body wt groups and exhibited the onset of saturation in the highest dose group where the adduct yield was approximately 2%. Incomplete knowledge of the timing of exposure and the complexity of the underlying biology confound the precise determination of prior AFB1 exposures in humans; however, the dosimetry of AFB1 observed in chronically dosed rats conceptually suggests that measurements in humans may underestimate exposure if a constant fraction of the AFB1 dose, approximately 2%, is assumed to be converted to Lys-AFB1 without regard to the dose.

A liquid chromatography electrospray tandem mass spectrometry (LC-ESI-MS/MS) method for the measurement of aflatoxin biomarkers in urine has been developed and validated. The two major aflatoxin-DNA adducts formed in rat tissues, aflatoxin N(7)-guanine and its imidazole ring opened derivative, 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl formamido)-9-hydroxy-aflatoxin B(1), were detected and quantified in urine by the LC-ESI-MS/MS technique. Other metabolites derived from the conjugation and/or oxidation of aflatoxin B(1) measured in the urine of dosed rats included aflatoxin P(1), aflatoxin P(1)-glucuronide, aflatoxin Q(1), aflatoxin M(1), 8,9-dihydro-8,9-dihydroxy aflatoxin B(1), aflatoxin B(1)-mercapturic acid, the aflatoxin-cysteine glycine adduct derived from the aflatoxin-glutathione conjugate, aflatoxin M(1)P(1) and the aflatoxin B(1)-dialcohol. For in vivo studies to determine the dosimetry of certain aflatoxin metabolites, aflatoxin B(2) was used as an internal standard for recovery since this compound is not naturally produced in rats. In the final method using the internal standard, the coefficient of variation of six replicate analyses of in vivo rat urine samples for aflatoxin N(7)-guanine, aflatoxin B(1)-mercapturic acid, and aflatoxin M(1) was 12.5, 12.8, and 5.8%, respectively. Further, the LC-ESI-MS/MS method to detect aflatoxin N(7)-guanine in in vivo rat urine samples was at least 20-fold more sensitive than prior techniques. Using the LC-ESI-MS/MS technique, the dosimetry, on a weekly basis, of major urinary aflatoxin metabolites was assessed in animals chronically dosed over a 5-week period. Of particular importance was the application of this method to determine the modulation of levels of urinary aflatoxin metabolites by treatment with oltipraz, a chemopreventive agent that can completely ablate aflatoxin hepatocarcinogenesis in the rat. After 1 week, oltipraz administration diminished urinary aflatoxin N(7)-guanine, aflatoxin B(1)-mercapturic acid and aflatoxin M(1) levels by 83, 92, and 82%, respectively. The magnitude of this reduction was persistent at the day 14, 21, 28, and 35-day time points with the average decrease of aflatoxin N(7)-guanine, aflatoxin B(1)-mercapturic acid and aflatoxin M(1) being 73, 92, and 90%, respectively. Importantly, even under circumstances where the oltipraz intervention was most efficient in reducing aflatoxin metabolite levels, the LC-ESI-MS/MS method was still sensitive enough to detect the reduced biomarker content. This outcome has important translational implications for the application and analysis of the efficacy of primary and secondary prevention interventions in human populations where ambient exposure levels are low, but the toxicologic hazards of these exposures remain high.