Benzo[a]pyrene toxicokinetics in humans following dietary supplementation with 3,3'-diindolylmethane (DIM) or Brussels sprouts.

Utilizing the atto-zeptomole sensitivity of UPLC-accelerator mass spectrometry (UPLC-AMS), we previously demonstrated significant first-pass metabolism following escalating (25-250 ng) oral micro-dosing in humans of [14C]-benzo[a]pyrene ([14C]-BaP). The present study examines the potential for supplementation with Brussels sprouts (BS) or 3,3'-diindolylmethane (DIM) to alter plasma levels of [14C]-BaP and metabolites over a 48-h period following micro-dosing with 50 ng (5.4 nCi) [14C]-BaP. Volunteers were dosed with [14C]-BaP following fourteen days on a cruciferous vegetable restricted diet, or the same diet supplemented for seven days with 50 g of BS or 300 mg of BR-DIM® prior to dosing. BS or DIM reduced total [14C] recovered from plasma by 56-67% relative to non-intervention. Dietary supplementation with DIM markedly increased Tmax and reduced Cmax for [14C]-BaP indicative of slower absorption. Both dietary treatments significantly reduced Cmax values of four downstream BaP metabolites, consistent with delaying BaP absorption. Dietary treatments also appeared to reduce the T1/2 and the plasma AUC(0,∞) for Unknown Metabolite C, indicating some effect in accelerating clearance of this metabolite. Toxicokinetic constants for other metabolites followed the pattern for [14C]-BaP (metabolite profiles remained relatively consistent) and non-compartmental analysis did not indicate other significant alterations. Significant amounts of metabolites in plasma were at the bay region of [14C]-BaP irrespective of treatment. Although the number of subjects and large interindividual variation are limitations of this study, it represents the first human trial showing dietary intervention altering toxicokinetics of a defined dose of a known human carcinogen.

Comparative Pharmacokinetics of High and Low Doses of the Herbicide Propanil in Mice.

We have documented that the herbicide propanil is immunotoxic in mice, and our in vitro tissue culture experiments largely recapitulate the in vivo studies. Laboratory studies on environmental contaminants are the most meaningful when these studies are conducted using concentrations that approximate levels in the environment. Many techniques to measure the distribution and pharmacokinetics (PK) on compounds rely on techniques, such as liquid scintillation counting (LSC) of radio-labeled starting compound, that require concentrations higher than environmental levels. The aim of this study was to compare tissue PK after exposure to propanil concentrations more relevant to levels of exposure to agricultural workers and the general population to concentrations previously reported for laboratory studies. To this end, we conducted a study to measure propanil distribution in three immune organs, using ultrasensitive accelerator mass spectrometry (AMS). We used two doses: the lower dose modeled levels expected in the environment or long-term occupational exposure to low doses, while the higher dose was to model the effects of an accidental exposure. Our results showed that the distribution and PK profiles from these two different concentrations was markedly different. The profile of the high dose (concentration) exposure was indicative of saturation of the detoxifying capability of the animal. In contrast, at the lower environmentally relevant concentration, in vivo concentrations of propanil in spleen, liver, and blood dropped to a very low level by 720 min. In conclusion, these studies highlight the differences in PK of propanil at these two doses, which suggests that the toxicity of this chemical should be re-investigated to obtain better data on toxic effects at doses relevant for humans.

Toward Predicting Acute Myeloid Leukemia Patient Response to 7 + 3 Induction Chemotherapy via Diagnostic Microdosing.

Acute myeloid leukemia (AML) is a rare yet deadly cancer of the blood and bone marrow. Presently, induction chemotherapy with the DNA damaging drugs cytarabine (ARA-C) and idarubicin (IDA), known as 7 + 3, is the standard of care for most AML patients. However, 7 + 3 is a relatively ineffective therapy, particularly in older patients, and has serious therapy-related toxicities. Therefore, a diagnostic test to predict which patients will respond to 7 + 3 is a critical unmet medical need. We hypothesize that a threshold level of therapy-induced 7 + 3 drug-DNA adducts determines cytotoxicity and clinical response. We further hypothesize that in vitro exposure of AML cells to nontoxic diagnostic microdoses enables prediction of the ability of AML cells to achieve that threshold during treatment. Our test involves dosing cells with very low levels of 14C-labeled drug followed by DNA isolation and quantification of drug-DNA adducts via accelerator mass spectrometry. Here, we have shown proof of principle by correlating ARA-C- and DOX-DNA adduct levels with cellular IC50 values of paired sensitive and resistant cancer cell lines and AML cell lines. Moreover, we have completed a pilot retrospective trial of diagnostic microdosing for 10 viably cryopreserved primary AML samples and observed higher ARA-C- and DOX-DNA adducts in the 7 + 3 responders than nonresponders. These initial results suggest that diagnostic microdosing may be a feasible and useful test for predicting patient response to 7 + 3 induction chemotherapy, leading to improved outcomes for AML patients and reduced treatment-related morbidity and mortality.

Correlation of Platinum Cytotoxicity to Drug-DNA Adduct Levels in a Breast Cancer Cell Line Panel.

Platinum drugs, including carboplatin and oxaliplatin, are commonly used chemotherapy drugs that kill cancer cells by forming toxic drug-DNA adducts. These drugs have a proven, but modest, efficacy against several aggressive subtypes of breast cancer but also cause several side effects that can lead to the cessation of treatment. There is a clinical need to identify patients who will respond to platinum drugs in order to better inform clinical decision making. Diagnostic microdosing involves dosing patients or patient samples with subtherapeutic doses of radiolabeled platinum followed by measurement of platinum-DNA adducts in blood or tumor tissue and may be used to predict patient response. We exposed a panel of six breast cancer cell lines to 14C-labeled carboplatin or oxaliplatin at therapeutic and microdose (1% therapeutic dose) concentrations for a range of exposure lengths and isolated DNA from the cells. The DNA was converted to graphite, and measurement of radiocarbon due to platinum-DNA adduction was quantified via accelerator mass spectrometry (AMS). We observed a linear correlation in adduct levels between the microdose and therapeutic dose, and the level of platinum-DNA adducts corresponded to cell line drug sensitivity for both carboplatin and oxaliplatin. These results showed a clear separation in adduct levels between the sensitive and resistant groups of cell lines that could not be fully explained or predicted by changes in DNA repair rates or mutations in DNA repair genes. Further, we were able to quantitate oxaliplatin-DNA adducts in the blood and tumor tissue of a metastatic breast cancer patient. Together, these data support the use of diagnostic microdosing for predicting patient sensitivity to platinum. Future studies will be aimed at replicating this data in a clinical feasibility trial.

A diagnostic microdosing approach to investigate platinum sensitivity in non-small cell lung cancer.

The platinum-based drugs cisplatin, carboplatin and oxaliplatin are often used for chemotherapy, but drug resistance is common. The prediction of resistance to these drugs via genomics is a challenging problem since hundreds of genes are involved. A possible alternative is to use mass spectrometry to determine the propensity for cells to form drug-DNA adducts-the pharmacodynamic drug-target complex for this class of drugs. The feasibility of predictive diagnostic microdosing was assessed in non-small cell lung cancer (NSCLC) cell culture and a pilot clinical trial. Accelerator mass spectrometry (AMS) was used to quantify [14 C]carboplatin-DNA monoadduct levels in the cell lines induced by microdoses and therapeutic doses of carboplatin, followed by correlation with carboplatin IC50 values for each cell line. The adduct levels in cell culture experiments were linearly proportional to dose (R2 = 0.95, p < 0.0001) and correlated with IC50 across all cell lines for microdose and therapeutically relevant carboplatin concentrations (p = 0.02 and p = 0.01, respectively). A pilot microdosing clinical trial was conducted to define protocols and gather preliminary data. Plasma pharmacokinetics (PK) and [14 C]carboplatin-DNA adducts in white blood cells and tumor tissues from six NSCLC patients were quantified via AMS. The blood plasma half-life of [14 C]carboplatin administered as a microdose was consistent with the known PK of therapeutic dosing. The optimal [14 C]carboplatin formulation for the microdose was 107 dpm/kg of body weight and 1% of the therapeutic dose for the total mass of carboplatin. No microdose-associated toxicity was observed in the patients. Additional accruals are required to significantly correlate adduct levels with response.

Quantifying Carbon-14 for Biology Using Cavity Ring-Down Spectroscopy.

A cavity ring-down spectroscopy (CRDS) instrument was developed using mature, robust hardware for the measurement of carbon-14 in biological studies. The system was characterized using carbon-14 elevated glucose samples and returned a linear response up to 387 times contemporary carbon-14 concentrations. Carbon-14 free and contemporary carbon-14 samples with varying carbon-13 concentrations were used to assess the method detection limit of approximately one-third contemporary carbon-14 levels. Sources of inaccuracies are presented and discussed, and the capability to measure carbon-14 in biological samples is demonstrated by comparing pharmacokinetics from carbon-14 dosed guinea pigs analyzed by both CRDS and accelerator mass spectrometry. The CRDS approach presented affords easy access to powerful carbon-14 tracer techniques that can characterize complex biochemical systems.

Use of Accelerator Mass Spectrometry in Human Health and Molecular Toxicology.

Accelerator mass spectrometry (AMS) has been adopted as a powerful bioanalytical method for human studies in the areas of pharmacology and toxicology. The exquisite sensitivity (10-18 mol) of AMS has facilitated studies of toxins and drugs at environmentally and physiologically relevant concentrations in humans. Such studies include risk assessment of environmental toxicants, drug candidate selection, absolute bioavailability determination, and more recently, assessment of drug-target binding as a biomarker of response to chemotherapy. Combining AMS with complementary capabilities such as high performance liquid chromatography (HPLC) can maximize data within a single experiment and provide additional insight when assessing drugs and toxins, such as metabolic profiling. Recent advances in the AMS technology at Lawrence Livermore National Laboratory have allowed for direct coupling of AMS with complementary capabilities such as HPLC via a liquid sample moving wire interface, offering greater sensitivity compared to that of graphite-based analysis, therefore enabling the use of lower 14C and chemical doses, which are imperative for clinical testing. The aim of this review is to highlight the recent efforts in human studies using AMS, including technological advancements and discussion of the continued promise of AMS for innovative clinical based research.

Disposition of the Dietary Mutagen 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline in Healthy and Pancreatic Cancer Compromised Humans.

Pancreatic cancer is the fourth leading cause of cancer death in the U.S. Once diagnosed, prognosis is poor with a 5-year survival rate of less than 5%. Exposure to carcinogenic heterocyclic amines (HCAs) derived from cooked meat has been shown to be positively associated with pancreatic cancer risk. To evaluate the processes that determine the carcinogenic potential of HCAs for human pancreas, 14-carbon labeled 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), a putative human carcinogenic HCA found in well-done cooked meat, was administered at a dietary relevant dose to human volunteers diagnosed with pancreatic cancer undergoing partial pancreatectomy and healthy control volunteers. After (14)C-MeIQx exposure, blood and urine were collected for pharmacokinetic and metabolite analysis. MeIQx-DNA adducts levels were quantified by accelerator mass spectrometry from pancreatic tissue excised during surgery from the cancer patient group. Pharmacokinetic analysis of plasma revealed a rapid distribution of MeIQx with a plasma elimination half-life of approximately 3.5 h in 50% of the cancer patients and all of the control volunteers. In 2 of the 4 cancer patients, very low levels of MeIQx were detected in plasma and urine suggesting low absorption from the gut into the plasma. Urinary metabolite analysis revealed five MeIQx metabolites with 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid being the most abundant accounting for 25%-50% of the recovered 14-carbon/mL urine. There was no discernible difference in metabolite levels between the cancer patient volunteers and the control group. MeIQx-DNA adduct analysis of pancreas and duodenum tissue revealed adduct levels indistinguishable from background levels. Although other meat-derived HCA mutagens have been shown to bind DNA in pancreatic tissue, indicating that exposure to HCAs from cooked meat cannot be discounted as a risk factor for pancreatic cancer, the results from this current study show that exposure to a single dietary dose of MeIQx does not readily form measurable DNA adducts under the conditions of the experiment.

Use of microdosing and accelerator mass spectrometry to evaluate the pharmacokinetic linearity of a novel tricyclic GyrB/ParE inhibitor in rats.

Determining the pharmacokinetics (PKs) of drug candidates is essential for understanding their biological fate. The ability to obtain human PK information early in the drug development process can help determine if future development is warranted. Microdosing was developed to assess human PKs, at ultra-low doses, early in the drug development process. Microdosing has also been used in animals to confirm PK linearity across subpharmacological and pharmacological dose ranges. The current study assessed the PKs of a novel antimicrobial preclinical drug candidate (GP-4) in rats as a step toward human microdosing studies. Dose proportionality was determined at 3 proposed therapeutic doses (3, 10, and 30 mg/kg of body weight), and PK linearity between a microdose and a pharmacological dose was assessed in Sprague-Dawley rats. Plasma PKs over the 3 pharmacological doses were proportional. Over the 10-fold dose range, the maximum concentration in plasma and area under the curve (AUC) increased 9.5- and 15.8-fold, respectively. PKs from rats dosed with a (14)C-labeled microdose versus a (14)C-labeled pharmacological dose displayed dose linearity. In the animals receiving a microdose and the therapeutically dosed animals, the AUCs from time zero to infinity were 2.6 ng · h/ml and 1,336 ng · h/ml, respectively, and the terminal half-lives were 5.6 h and 1.4 h, respectively. When the AUC values were normalized to a dose of 1.0 mg/kg, the AUC values were 277.5 ng · h/ml for the microdose and 418.2 ng · h/ml for the pharmacological dose. This 1.5-fold difference in AUC following a 300-fold difference in dose is considered linear across the dose range. On the basis of the results, the PKs from the microdosed animals were considered to be predictive of the PKs from the therapeutically dosed animals.

Directly coupled high-performance liquid chromatography-accelerator mass spectrometry measurement of chemically modified protein and peptides.

Quantitation of low-abundance protein modifications involves significant analytical challenges, especially in biologically important applications, such as studying the role of post-translational modifications in biology and measurement of the effects of reactive drug metabolites. (14)C labeling combined with accelerator mass spectrometry (AMS) provides exquisite sensitivity for such experiments. Here, we demonstrate real-time (14)C quantitation of high-performance liquid chromatography (HPLC) separations by liquid sample accelerator mass spectrometry (LS-AMS). By enabling direct HPLC-AMS coupling, LS-AMS overcomes several major limitations of conventional HPLC-AMS, where individual HPLC fractions must be collected and converted to graphite before measurement. To demonstrate LS-AMS and compare the new technology to traditional solid sample AMS (SS-AMS), reduced and native bovine serum albumin (BSA) was modified by (14)C-iodoacetamide, with and without glutathione present, producing adducts on the order of 1 modification in every 10(6) to 10(8) proteins. (14)C incorporated into modified BSA was measured by solid carbon AMS and LS-AMS. BSA peptides were generated by tryptic digestion. Analysis of HPLC-separated peptides was performed in parallel by LS-AMS, fraction collection combined with SS-AMS, and (for peptide identification) electrospray ionization and tandem mass spectrometry (ESI-MS/MS). LS-AMS enabled (14)C quantitation from ng sample sizes and was 100 times more sensitive to (14)C incorporated in HPLC-separated peptides than SS-AMS, resulting in a lower limit of quantitation of 50 zmol (14)C/peak. Additionally, LS-AMS turnaround times were minutes instead of days, and HPLC trace analyses required 1/6th the AMS instrument time required for analysis of graphite fractions by SS-AMS.

Determining the pharmacokinetics and long-term biodistribution of SiO2 nanoparticles in vivo using accelerator mass spectrometry.

Biodistribution is an important factor in better understanding silica dioxide nanoparticle (SiNP) safety. Currently, comprehensive studies on biodistribution are lacking, most likely due to the lack of suitable analytical methods. Accelerator mass spectrometry was used to investigate the relationship between administered dose, pharmacokinetics (PK), and long-term biodistribution of (14)C-SiNPs in vivo. PK analysis showed that SiNPs were rapidly cleared from the central compartment, were distributed to tissues of the reticuloendothelial system, and persisted in the tissue over the 8 week time course, raising questions about the potential for bioaccumulation and associated long-term effects.

Impact of phenanthrene co-administration on the toxicokinetics of benzo[a]pyrene in humans. UPLC-accelerator mass spectrometry following oral microdosing.

Current risk assessments for environmental carcinogens rely on animal studies utilizing doses orders of magnitude higher than actual human exposures. Epidemiological studies of people with high exposures (e.g., occupational) are of value, but rely on uncertain exposure data. In addition, exposures are typically not to a single chemical but to mixtures, such as polycyclic aromatic hydrocarbons (PAHs). The extremely high sensitivity of accelerator mass spectrometry (AMS) allows for dosing humans with known carcinogens with de minimus risk. In this study UPLC-AMS was used to assess the toxicokinetics of [14C]-benzo[a]pyrene ([14C]-BaP) when dosed alone or in a binary mixture with phenanthrene (Phe). Plasma was collected for 48 h following a dose of [14C]-BaP (50 ng, 5.4 nCi) or the same dose of [14C]-BaP plus Phe (1250 ng). Following the binary mixture, Cmax of [14C]-BaP significantly decreased (4.4-fold) whereas the volume of distribution (Vd) increased (2-fold). Further, the toxicokinetics of twelve [14C]-BaP metabolites provided evidence of little change in the metabolite profile of [14C]-BaP and the pattern was overall reduction consistent with reduced absorption (decrease in Cmax). Although Phe was shown to be a competitive inhibitor of the major hepatic cytochrome P-450 (CYP) responsible for metabolism of [14C]-BaP, CYP1A2, the high inhibition constant (Ki) and lack of any increase in unmetabolized [14C]-BaP in plasma makes this mechanism unlikely to be responsible. Rather, co-administration of Phe reduces the absorption of [14C]-BaP through a mechanism yet to be determined. This is the first study to provide evidence that, at actual environmental levels of exposure, the toxicokinetics of [14C]-BaP in humans is markedly altered by the presence of a second PAH, Phe, a common component of environmental PAH mixtures.

Benzo[a]pyrene (BaP) metabolites predominant in human plasma following escalating oral micro-dosing with [14C]-BaP.

Benzo[a]pyrene (BaP) is formed by incomplete combustion of organic materials (petroleum, coal, tobacco, etc.). BaP is designated by the International Agency for Research on Cancer as a group 1 known human carcinogen; a classification supported by numerous studies in preclinical models and epidemiology studies of exposed populations. Risk assessment relies on toxicokinetic and cancer studies in rodents at doses 5-6 orders of magnitude greater than average human uptake. Using a dose-response design at environmentally relevant concentrations, this study follows uptake, metabolism, and elimination of [14C]-BaP in human plasma by employing UPLC - accelerator mass spectrometry (UPLC-AMS). Volunteers were administered 25, 50, 100, and 250 ng (2.7-27 nCi) of [14C]-BaP (with interceding minimum 3-week washout periods) with quantification of parent [14C]-BaP and metabolites in plasma measured over 48 h. [14C]-BaP median Tmax was 30 min with Cmax and area under the curve (AUC) approximating dose-dependency. Marked inter-individual variability in plasma pharmacokinetics following a 250 ng dose was seen with 7 volunteers as measured by the Cmax (8.99 ± 7.08 ng × mL-1) and AUC0-48hr (68.6 ± 64.0 fg × hr-1 × mL-1). Approximately 3-6% of the [14C] recovered (AUC0-48 hr) was parent compound, demonstrating extensive metabolism following oral dosing. Metabolite profiles showed that, even at the earliest time-point (30 min), a substantial percentage of [14C] in plasma was polar BaP metabolites. The best fit modeling approach identified non-compartmental apparent volume of distribution of BaP as significantly increasing as a function of dose (p = 0.004). Bay region tetrols and dihydrodiols predominated, suggesting not only was there extensive first pass metabolism but also potentially bioactivation. AMS enables the study of environmental carcinogens in humans with de minimus risk, allowing for important testing and validation of physiologically based pharmacokinetic models derived from animal data, risk assessment, and the interpretation of data from high-risk occupationally exposed populations.

A microdosing approach for characterizing formation and repair of carboplatin-DNA monoadducts and chemoresistance.

Formation and repair of platinum (Pt)-induced DNA adducts is a critical step in Pt drug-mediated cytotoxicity. Measurement of Pt-DNA adduct kinetics in tumors may be useful for better understanding chemoresistance and therapeutic response. However, this concept has yet to be rigorously tested because of technical challenges in measuring the adducts at low concentrations and consistent access to sufficient tumor biopsy material. Ultrasensitive accelerator mass spectrometry was used to detect [(14)C]carboplatin-DNA monoadducts at the attomole level, which are the precursors to Pt-DNA crosslink formation, in six cancer cell lines as a proof-of-concept. The most resistant cells had the lowest monoadduct levels at all time points over 24 hr. [(14)C]Carboplatin "microdoses" (1/100th the pharmacologically effective concentration) had nearly identical adduct formation and repair kinetics compared to therapeutically relevant doses, suggesting that the microdosing approach can potentially be used to determine the pharmacological effects of therapeutic treatment. Some of the possible chemoresistance mechanisms were also studied, such as drug uptake/efflux, intracellular inactivation and DNA repair in selected cell lines. Intracellular inactivation and efficient DNA repair each contributed significantly to the suppression of DNA monoadduct formation in the most resistant cell line compared to the most sensitive cell line studied (p < 0.001). Nucleotide excision repair (NER)-deficient and -proficient cells showed substantial differences in carboplatin monoadduct concentrations over 24 hr that likely contributed to chemoresistance. The data support the utility of carboplatin microdosing as a translatable approach for defining carboplatin-DNA monoadduct formation and repair, possibly by NER, which may be useful for characterizing chemoresistance in vivo.

Yeast dynamic metabolic flux measurement in nutrient-rich media by HPLC and accelerator mass spectrometry.

Metabolic flux, the flow of metabolites through networks of enzymes, represents the dynamic productive output of cells. Improved understanding of intracellular metabolic fluxes will enable targeted manipulation of metabolic pathways of medical and industrial importance to a greater degree than is currently possible. Flux balance analysis (FBA) is a constraint-based approach to modeling metabolic fluxes, but its utility is limited by a lack of experimental measurements. Incorporation of experimentally measured fluxes as system constraints will significantly improve the overall accuracy of FBA. We applied a novel, two-tiered approach in the yeast Saccharomyces cerevisiae to measure nutrient consumption rates (extracellular fluxes) and a targeted intracellular flux using a (14)C-labeled precursor with HPLC separation and flux quantitation by accelerator mass spectrometry (AMS). The use of AMS to trace the intracellular fate of (14)C-glutamine allowed the calculation of intracellular metabolic flux through this pathway, with glutathione as the metabolic end point. Measured flux values provided global constraints for the yeast FBA model which reduced model uncertainty by more than 20%, proving the importance of additional constraints in improving the accuracy of model predictions and demonstrating the use of AMS to measure intracellular metabolic fluxes. Our results highlight the need to use intracellular fluxes to constrain the models. We show that inclusion of just one such measurement alone can reduce the average variability of model predicted fluxes by 10%.

Statistical analysis of variation in the human plasma proteome.

Quantifying the variation in the human plasma proteome is an essential prerequisite for disease-specific biomarker detection. We report here on the longitudinal and individual variation in human plasma characterized by two-dimensional difference gel electrophoresis (2-D DIGE) using plasma samples from eleven healthy subjects collected three times over a two week period. Fixed-effects modeling was used to remove dye and gel variability. Mixed-effects modeling was then used to quantitate the sources of proteomic variation. The subject-to-subject variation represented the largest variance component, while the time-within-subject variation was comparable to the experimental variation found in a previous technical variability study where one human plasma sample was processed eight times in parallel and each was then analyzed by 2-D DIGE in triplicate. Here, 21 protein spots had larger than 50% CV, suggesting that these proteins may not be appropriate as biomarkers and should be carefully scrutinized in future studies. Seventy-eight protein spots showing differential protein levels between different individuals or individual collections were identified by mass spectrometry and further characterized using hierarchical clustering. The results present a first step toward understanding the complexity of longitudinal and individual variation in the human plasma proteome, and provide a baseline for improved biomarker discovery.

Towards biomarker-dependent individualized chemotherapy: exploring cell-specific differences in oxaliplatin-DNA adduct distribution using accelerator mass spectrometry.

Oxaliplatin is a third-generation platinum-based anticancer drug that is currently used in the treatment of metastatic colorectal cancer. Oxaliplatin, like other platinum-based anticancer drugs such as cisplatin and carboplatin, is known to induce apoptosis in tumor cells by binding to nuclear DNA, forming monoadducts, and intra- and interstrand diadducts. Previously, we reported an accelerator mass spectrometry (AMS) assay to measure the kinetics of oxaliplatin-induced DNA damage and repair [Hah, S. S.; Sumbad, R. A.; de Vere White, R. W.; Turteltaub, K. W.; Henderson, P. T. Chem. Res. Toxicol.2007, 20, 1745]. Here, we describe another application of AMS to the measurement of oxaliplatin-DNA adduct distribution in cultured platinum-sensitive testicular (833K) and platinum-resistant breast (MDA-MB-231) cancer cells, which resulted in elucidation of cell-dependent differentiation of oxaliplatin-DNA adduct formation, implying that differential adduction and/or accumulation of the drug in cellular DNA may be responsible for the sensitivity of cancer cells to platinum treatment. Ultimately, we hope to use this method to measure the intrinsic platinated DNA adduct repair capacity in cancer patients for use as a biomarker for diagnostics or a predictor of patient outcome.

Oxaliplatin-DNA Adducts as Predictive Biomarkers of FOLFOX Response in Colorectal Cancer: A Potential Treatment Optimization Strategy.

FOLFOX is one of the most effective treatments for advanced colorectal cancer. However, cumulative oxaliplatin neurotoxicity often results in halting the therapy. Oxaliplatin functions predominantly via the formation of toxic covalent drug-DNA adducts. We hypothesize that oxaliplatin-DNA adduct levels formed in vivo in peripheral blood mononuclear cells (PBMC) are proportional to tumor shrinkage caused by FOLFOX therapy. We further hypothesize that adducts induced by subtherapeutic "diagnostic microdoses" are proportional to those induced by therapeutic doses and are also predictive of response to FOLFOX therapy. These hypotheses were tested in colorectal cancer cell lines and a pilot clinical study. Four colorectal cancer cell lines were cultured with therapeutically relevant (100 µmol/L) or diagnostic microdose (1 µmol/L) concentrations of [14C]oxaliplatin. The C-14 label enabled quantification of oxaliplatin-DNA adduct level with accelerator mass spectrometry (AMS). Oxaliplatin-DNA adduct formation was correlated with oxaliplatin cytotoxicity for each cell line as measured by the MTT viability assay. Six colorectal cancer patients received by intravenous route a diagnostic microdose containing [14C]oxaliplatin prior to treatment, as well as a second [14C]oxaliplatin dose during FOLFOX chemotherapy, termed a "therapeutic dose." Oxaliplatin-DNA adduct levels from PBMC correlated significantly to mean tumor volume change of evaluable target lesions (5 of the 6 patients had measurable disease). Oxaliplatin-DNA adduct levels were linearly proportional between microdose and therapeutically relevant concentrations in cell culture experiments and patient samples, as was plasma pharmacokinetics, indicating potential utility of diagnostic microdosing.

Recent advances in biomedical applications of accelerator mass spectrometry.

The use of radioisotopes has a long history in biomedical science, and the technique of accelerator mass spectrometry (AMS), an extremely sensitive nuclear physics technique for detection of very low-abundant, stable and long-lived isotopes, has now revolutionized high-sensitivity isotope detection in biomedical research, because it allows the direct determination of the amount of isotope in a sample rather than measuring its decay, and thus the quantitative analysis of the fate of the radiolabeled probes under the given conditions. Since AMS was first used in the early 90's for the analysis of biological samples containing enriched 14C for toxicology and cancer research, the biomedical applications of AMS to date range from in vitro to in vivo studies, including the studies of 1) toxicant and drug metabolism, 2) neuroscience, 3) pharmacokinetics, and 4) nutrition and metabolism of endogenous molecules such as vitamins. In addition, a new drug development concept that relies on the ultrasensitivity of AMS, known as human microdosing, is being used to obtain early human metabolism information of candidate drugs. These various aspects of AMS are reviewed and a perspective on future applications of AMS to biomedical research is provided.

Tamoxifen forms DNA adducts in human colon after administration of a single [14C]-labeled therapeutic dose.

Tamoxifen is widely prescribed for the treatment of breast cancer and is also licensed in the United States for the prevention of this disease. However, tamoxifen therapy is associated with an increased occurrence of endometrial cancer in women, and there is also evidence that it may elevate the risk of colorectal cancer. The underlying mechanisms responsible for tamoxifen-induced carcinogenesis in women have not yet been elucidated, but much interest has focused on the role of DNA adduct formation. We investigated the propensity of tamoxifen to bind irreversibly to colorectal DNA when given to 10 women as a single [(14)C]-labeled therapeutic (20 mg) dose, approximately 18 h before undergoing colon resections. Using the sensitive technique of accelerator mass spectrometry, coupled with high-performance liquid chromatography separation of enzymatically digested DNA, a peak corresponding to authentic dG-N(2)-tamoxifen adduct was detected in samples from three patients, at levels ranging from 1 to 7 adducts/10(9) nucleotides. No [(14)C]-radiolabel associated with tamoxifen or its major metabolites was detected. The presence of detectable CYP3A4 protein in all colon samples suggests that this tissue has the potential to activate tamoxifen to alpha-hydroxytamoxifen, in addition to that occurring in the systemic circulation, and direct interaction of this metabolite with DNA could account for the binding observed. Although the level of tamoxifen-induced damage displayed a degree of interindividual variability, when present, it was approximately 10 to 100 times higher than that reported for other suspect human colon carcinogens such as 2-amino-1-methyl-6-phenyimidazo[4,5-b]pyridine. These findings provide a mechanistic basis through which tamoxifen could increase the incidence of colon cancers in women.