Simultaneous toxicokinetic studies of aristolochic acid I and II and aristolactam I and II using a newly-developed microdialysis liquid chromatography-tandem mass spectrometry.

Aristolochic acids (AAs) are naturally occurring genotoxic carcinogens linked to Balkan endemic nephropathy and aristolochic acid nephropathy. Aristolochic acid I and II (AA-I and AA-II) are the most abundant AAs, and AA-I has been reported to be more genotoxic and nephrotoxic than AA-II. This study aimed to explore metabolic differences underlying the differential toxicity. We developed a novel microdialysis sampling coupled with solid-phase extraction liquid chromatography-tandem mass spectrometry (MD-SPE-LC-MS/MS) to simultaneously study the toxicokinetics (TK) of AA-I and AA-II and their corresponding aristolactams (AL-I and AL-II) in the blood of Sprague Dawley rats co-treated with AA-1 and AA-II. Near real-time monitoring of these analytes in the blood of treated rats revealed that AA-I was absorbed, distributed, and eliminated more rapidly than AA-II. Moreover, the metabolism efficiency of AA-I to AL-I was higher compared to AA-II to AL-II. Only 0.58% of AA-I and 0.084% of AA-II was reduced to AL-I and AL-II, respectively. The findings are consistent with previous studies and support the contention that differences in the in vivo metabolism of AA-I and AA-II may be critical factors for their differential toxicities.

Potential Association of Urinary N7-(2-Carbamoyl-2-hydroxyethyl) Guanine with Dietary Acrylamide Intake of Smokers and Nonsmokers.

Acrylamide (AA), a rodent carcinogen, is widely used in industry and present in cigarette smoke as well as in foods processed at high temperatures. The metabolic activation of AA to glycidamide (GA) could be critical for AA carcinogenicity since GA causes DNA adduct formation in vivo. N7-(2-carbamoyl-2-hydroxyethyl) guanine (N7-GAG), the most abundant DNA adduct of AA, is subjected to spontaneous and enzymatic depurination and excreted through urine. Urinary N7-GAG analysis can confirm AA genotoxicity and identify active species of AA metabolites in humans, thereby serving as a risk-associated biomarker for molecular epidemiology studies. This study aimed to develop an isotope-dilution solid-phase extraction liquid chromatography tandem mass spectrometry method to comparatively analyze urinary N7-GAG levels in nonsmokers and smokers. Urinary N-acetyl-S-(propionamide)-cysteine (AAMA), a metabolite of AA, was also analyzed as a biomarker for current AA exposure. Urinary N7-GAG was quantified by monitoring m/z 239 → 152 for N7-GAG and m/z 242 → 152 for (13)C3-labeled N7-GAG under positive electron spray ionization and multiple reaction mode. The median urinary N7-GAG level was 0.93 µg/g creatinine in nonsmokers (n = 33) and 1.41 µg/g creatinine in smokers (n = 30). Multiple linear regression analysis of data revealed that N7-GAG levels were only significantly associated with AAMA levels. These results demonstrate that urinary N7-GAG of nonsmokers and smokers is significantly associated with a very low level of dietary AA intake, assessed by analyzing urinary AAMA.

Simultaneous quantitation of urinary cotinine and acrylonitrile-derived mercapturic acids with ultraperformance liquid chromatography-tandem mass spectrometry.

Acrylonitrile (AN), a widely used industrial chemical also found in tobacco smoke, has been classified as a possible human carcinogen (group 2B) by the International Agency for Research on Cancer. AN can be detoxified by glutathione S-transferase (GST) to form glutathione (GSH) conjugates in vivo. It can be metabolically activated by cytochrome P450 2E1 to form 2-cyanoethylene oxide, which can also be detoxified by GST to generate GSH conjugates. The GSH conjugates can be further metabolized to mercapturic acids (MAs), namely, N-acetyl-S-(2-cyanoethyl)cysteine (CEMA), N-acetyl-S-(2-hydroxyethyl)cysteine (HEMA), and N-acetyl-S-(1-cyano-2-hydroxyethyl)cysteine (CHEMA). This study developed an ultraperformance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) method to quantitatively profile the major AN urinary metabolites (CEMA, HEMA, and CHEMA) to assess AN exposure, as well as analyze urinary cotinine (COT) as an indicator for tobacco smoke exposure. The limits of quantitation were 0.1, 0.1, 1.0, and 0.05 µg/L for HEMA, CEMA, CHEMA, and COT, respectively. This method was applied to analyze the three AN-derived MAs in 36 volunteers with no prior occupational AN exposure. Data analysis showed significant correlations between the level of COT and the levels of these MAs, suggesting them as biomarkers for exposure to low levels of AN. The results demonstrate that a highly specific and sensitive UPLC-MS/MS method has been successfully developed to quantitatively profile the major urinary metabolites of AN in humans to assess low AN exposure.

Analysis of urinary aristolactams by on-line solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry.

Aristolochic acids (AAs), nephrotoxicants and known human carcinogens, are a mixture of structurally related derivatives of nitrophenanthrene carboxylic acids with the major components being aristolochic acid I and aristolochic acid II. People may ingest small amounts of AAs from its natural presence in medicinal plants and herbs of the family Aristolochiaceae, including the genera Aristolochia and Asarum, which have been used worldwide in folk medicine for centuries. In order to assess AA intake, an on-line solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry (on-line SPE-LC/MS/MS) method was developed to analyze their most abundant corresponding metabolites, aristolactams (ALs), in urine to serve as biomarkers. The limits of quantitation were 0.006 ng for aristolactam I (AL-I), and 0.024 ng for aristolactam II (AL-II) on column. Recovery varied from 98.0% to 99.5%, and matrix effects were within 75.3-75.4%. This method was applied to analyze ALs in the urine samples collected on days 1, 2, 4, and 7 from mice treated with 30 mg/kg or 50mg/kg AAs. Their half lives were estimated to be 3.55 h and 4.00 for AL-I, and 4.04 and 4.83 h for AL-II, depending on AAs doses. These results demonstrated that the first simple on-line SPE-LC/MS/MS method was successfully developed to analyze urinary ALs with excellent sensitivity and specificity to serve as biomarkers to assess current AA intake from AAs-containing Chinese herbs.

Comparative analysis of urinary N7-(2-hydroxyethyl)guanine for ethylene oxide- and non-exposed workers.

Ethylene oxide (EO), a direct alkylating agent and a carcinogen, can attack the nucleophilic sites of DNA bases to form a variety of DNA adducts. The most abundant adduct, N7-(2-hydroxyethyl)guanine (N7-HEG), can be depurinated spontaneously or enzymatically from DNA backbone to form abasic sites. Molecular dosimetry of the excised N7-HEG in urine can serve as an EO exposure and potential risk-associated biomarker. This study was to analyze N7-HEG in urine collected from 89 EO-exposed and 48 nonexposed hospital workers and 20 exposed and 10 nonexposed factory workers by using our newly developed on-line solid-phase extraction isotope-dilution LC-MS/MS method. Statistical analysis of data shows that the exposed factory workers excreted significantly greater concentrations of N7-HEG than both the nonexposed factory workers and hospital workers. Multiple linear regression analysis reveals that the EO-exposed factory workers had a significantly greater post-shift urinary N7-HEG than their nonexposed coworkers and hospital workers. These results demonstrate that analysis of urinary N7-HEG can serve as a biomarker of EO exposure for future molecular epidemiology studies to better understand the role of the EO-induced DNA adduct formation in EO carcinogenicity and certainly for routine surveillance of occupational EO exposure for the study of potential health impacts on workers.

The application of mass spectrometry in molecular dosimetry: ethylene oxide as an example.

Mass spectrometry plays an increasingly important role in the search for and quantification of novel chemically specific biomarkers. The revolutionary advances in mass spectrometry instrumentation and technology empower scientists to specifically analyze DNA and protein adducts, considered as molecular dosimeters, derived from reactions of a carcinogen or its active metabolites with DNA or protein. Analysis of the adducted DNA bases and proteins can elucidate the chemically reactive species of carcinogens in humans and can serve as risk-associated biomarkers for early prediction of cancer risk. In this article, we review and compare the specificity, sensitivity, resolution, and ease-of-use of mass spectrometry methods developed to analyze ethylene oxide (EO)-induced DNA and protein adducts, particularly N7-(2-hydroxyethyl)guanine (N7-HEG) and N-(2-hydroxyethyl)valine (HEV), in human samples and in animal tissues. GC/ECNCI-MS analysis after HPLC cleanup is the most sensitive method for quantification of N7-HEG, but limited by the tedious sample preparation procedures. Excellent sensitivity and specificity in analysis of N7-HEG can be achieved by LC/MS/MS analysis if the mobile phase, the inlet (split or splitless), and the collision energy are properly optimized. GC/ECNCI-HRMS and GC/ECNCI-MS/MS analysis of HEV achieves the best performance as compared with GC/ECNCI-MS and GC/EI-MS. In conclusion, future improvements in high-throughput capabilities, detection sensitivity, and resolution of mass spectrometry will attract more scientists to identify and/or quantify novel molecular dosimeters or profiles of these biomarkers in toxicological and/or epidemiological studies.

Biological monitoring for occupational acrylamide exposure from acrylamide production workers.

OBJECTIVE: We conducted a repeated-measurement study to (1) investigate the correlation between occupational exposure to airborne acrylamide (AA) and the time-dependent behavior of urinary AAMA, GAMA2, and GAMA3 and (2) calculate the estimated biological exposure index at the permissible exposure limit (PEL) level of 30 µg/m(3). METHODS: Forty-four workers were recruited--8 were AA-exposed and 36 were controls. Pre- and post-shift urine samples were collected from the exposed group in parallel with personal sampling for 8 consecutive days and only 1 day for the control group and analyzed using liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI-MS/MS). RESULTS: Post-shift urinary AAMA level was significantly associated with personal AA exposure (p < 0.001), indicating that urinary AAMA was a better AA exposure biomarker. The estimated urinary excretion of AAMA was 3.0 mg/g creatinine for nonsmoking workers exposed to the PEL of 30 µg/m(3). The median GAMA (the sum of GAMA2 and GAMA3)/AAMA ratio for exposed workers was 0.03 (range, 0.005-0.14), relatively lower than that of the nonoccupational group. CONCLUSIONS: Although sample size in this study was small, the repeated-measurement data provide useful reference for future studies related to biological monitoring of occupational exposure to AA.

Development of disposable lipid biosensor for the determination of total cholesterol.

A prototype chronoamperometric biosensor for the determination of total cholesterol was developed that consists of a homemade potentiostat and disposable strips immobilized with Fe(3)O(4), cholesterol oxidase (ChOx), and cholesterol esterase (ChE). The principle of sensing cholesterol is based on the detection of reduction signal of hydrogen peroxide generated in two enzymatic reactions. The co-immobilization of ChE and ChOx allows the sensor to detect both concentrations of esterified and free cholesterol. The effects of biosensor on catalyst, enzymes, applied potential, and buffer pH was investigated, and the operation conditions were optimized. The detection of cholesterol can be accomplished in one step, a 10 microL of sample was dropped onto the area of sensing strip and the reduction signal was obtained at an applied potential of -200 mV (vs. Ag/Ag(+)). The pre-reaction time was set at 15s before applying potential on the strip and the sampling time was 5s. The sensing device displays a linear response over the range of 100-400mg/dL (R(2)=0.999) for cholesteryl oleate. The coefficient variation was determined as 5.06% (N=20) for 100mg/dL cholesteryl oleate and the detection limit is 19.4 mg/dL (S/N=3). The probable interferences in bio-matrix were selected to test the selectivity and no significant response was observed in the biosensor.

Rapid and sensitive on-line liquid chromatographic/tandem mass spectrometric determination of an ethylene oxide-DNA adduct, N7-(2-hydroxyethyl)guanine, in urine of nonsmokers.

Ethylene oxide (EtO) is classified as a known human carcinogen. The formation of EtO-DNA adducts is considered as an important early event in the EtO carcinogenic process. An isotope-dilution on-line solid-phase extraction and liquid chromatography coupled with tandem mass spectrometry method was then developed to analyze one of the EtO-DNA adducts, N7-(2-hydroxyethyl)guanine (N7-HEG), in urine of 46 nonsmokers with excellent accuracy, sensitivity and specificity. The merits of this method include small sample volume (only 120 microL urine required), automated sample cleanup, and short total run time (12 minutes per sample). This method demonstrates its high-throughput capacity for future molecular epidemiology studies on the potential health effects resulting from the low-dose EtO exposure.

Simultaneous analysis of urinary 4,4'-methylenebis(2-chloroaniline) and N-acetyl 4,4'-methylenebis(2-chloroaniline) using solid-phase extraction and liquid chromatography/tandem mass spectrometry.

Analysis of 4,4'-methylenebis(2-cholroaniline) (MOCA) or its metabolites in urine has been considered as the appropriate method to assess MOCA exposures through inhalation and skin absorption. MOCA and its metabolite, N-acetyl 4,4'-methylenebis(2-chloroaniline) (acetyl-MOCA), are analyzed using methods either limited by sensitivity or sample preparation. Therefore, a solid-phase extraction (SPE) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed to simultaneously analyze MOCA and acetyl-MOCA in urine to serve as biomarkers for MOCA exposure. Protein was precipitated by using acetonitrile, and SPE were applied to clean up samples to eliminate the matrix effect and to improve the recovery. The limit of quantitation of this method was at 1.0 ng/mL for MOCA and 0.03 ng/mL for acetyl-MOCA (signal-to-noise (S/N) ratio = 10). Urinary MOCA and acetyl-MOCA levels in MOCA-exposed workers were analyzed and quantitated to be 191.9 +/- 373.2 (mean +/- standard deviation (SD)) and 11.79 +/- 23.8 ng/mL (N = 54) with the median values 38.6 and 1.8 ng/mL, respectively. MOCA concentrations are significantly correlated with their corresponding acetyl-MOCA levels in urine (Spearman correlation coefficient r = 0.916, p < 0.001). These results show that this method has been successfully developed and provides high-throughput potential to analyze MOCA and acetyl-MOCA to serve as exposure biomarkers for future study of the potential health effects associated with MOCA exposures.

Application of expression genomics for predicting treatment response in cancer.

During tumor progression, multiple genetic changes in the genome vastly alter the transcriptomes of cancers. Some of these changes, including the mutations of various growth regulatory genes as well as alterations in the transcription of a large number of genes, may lead to resistance to treatment. Therefore, capturing such genomic information of the tumors would enable a physician to decide on the course of treatment options clinically available. Currently, it is still not feasible to identify all the genetic mutations that have occurred in a patient's cancer genome. However, the advent of DNA microarray coupled with the completion of the human genome sequence and the identification of all its genes, have made possible genome-wide gene expression profiling of the cancer genome. In this review, we will focus on the application of expression genomics for identifying signature gene expression profiles in primary cancers to predict response to either radio- or chemotherapy. We envision that transcription profiling of the cancer genomes ultimately will not only reveal how altered gene expression results in resistance to treatment, but also be exploited for predicting and personalizing cancer therapy.

Overexpression of 5-lipoxygenase in rat and human esophageal adenocarcinoma and inhibitory effects of zileuton and celecoxib on carcinogenesis.

PURPOSE: Aberrant arachidonic acid (AA) metabolism, especially through the cyclooxygenase (Cox) and 5-lipoxygenase (5-Lox) pathways, has been suggested to play an important role in the development of esophageal adenocarcinoma (EAC). The purpose of this study was to investigate the expression of 5-Lox in EAC of a rat model and in human samples as well as the chemopreventive effects of zileuton (a specific 5-Lox inhibitor) and celecoxib (a specific Cox2 inhibitor) in the rat EAC model. EXPERIMENTAL DESIGN: 5-Lox expression in EAC of a rat esophagogastroduodenal anastomosis model and of humans was examined with immunohistochemistry. A chemoprevention study was designed to test whether zileuton and celecoxib could suppress aberrant AA metabolism and esophageal adenocarcinogenesis. RESULTS: With immunohistochemistry, we found that 5-Lox was overexpressed during esophageal adenocarcinogenesis in our rat model and in humans. In the chemoprevention study, EAC incidence was reduced in a dose-dependent manner from 68.8% (11 of 16) to 44.4% (8 of 18; P > 0.05) and 31.3% (5 of 16; P < 0.05) by 500 and 1,000 ppm zileuton, respectively, and to 33.3% (7 of 21; P < 0.05) and 20% (3 of 15; P < 0.05) by 500 and 1,000 ppm celecoxib, respectively. With isobolographic analysis, zileuton and celecoxib, both at a dose of 500 ppm, had an additive effect by reducing the tumor incidence to 16.7% (3 of 18, P < 0.01). Leukotriene B4 and prostaglandin E2 levels in the esophageal tissues were also significantly reduced by zileuton and celecoxib. CONCLUSIONS: This study clearly demonstrated that 5-Lox and Cox2 play important roles in the development of EAC. Both zileuton and celecoxib had inhibitory effects on esophageal adenocarcinogenesis through inhibition on their respective enzymes of AA metabolism.

Extracellular cAMP-dependent protein kinase (ECPKA) in melanoma.

Melanoma is one of the fastest rising malignancies in the United States. When detected early, primary melanomas are curable through surgery. However, despite significant improvements in diagnosis and surgical, local and systemic therapy, mortality rate in metastatic melanoma remains high. Furthermore, genetic alterations associated with the development and stepwise progression of melanoma, are still unclear. Previous reports show that the catalytic kinase subunit of the cAMP-dependent protein kinase is secreted by tumor cells and can be detected in the serum of cancer patients. We examine in this report the clinical significance of this secreted C subunit kinase termed extracellular protein kinase (ECPKA) in melanoma patients. Our results showed the presence of ECPKA activity in the serum of melanoma patients and correlate with the appearance and size of the tumor. Most importantly, surgical removal of melanoma causes a precipitous decrease in ECPKA activity in the sera of patients, suggesting that ECPKA may be a novel predictive marker in melanoma.

Analyzing excessive no changes in clinical trials with clustered data.

This article considers clinical trials in which the efficacy measure is taken from several sites within each patient, such as the alveolar bone height of the tooth sites, or bone mineral densities of the lumbar spine sites. Since usually only a small portion of these sites will exhibit changes, the conventional method using per patient average gives a diluted result due to excessive no changes in the data. Different methods have been proposed for this type of data in the case where the observations are mutually independent. This includes the popular "two-part model" (Lachenbruch, 2001, Statistics in Medicine 20, 1215-1234; 2002, Statistical Methods in Medical Research 11, 297-302), which is related to the "composite approach" for discrete and continuous data in Shih and Quan (1997, Statistics in Medicine16, 1225-1239; 2001, Statistica Sinica 11, 53-62). In this article, we model the data with excessive zeros (no changes) in clustered data using a mixture of distributions, and taking into account possible measurement errors. This mixture model includes the two-part model as a special case when one component of the mixture degenerates.

Gene expression of TPA induced differentiation in HL-60 cells by DNA microarray analysis.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent inducer of differentiation in human promyelocytic leukemia cells. Recently, TPA has been successfully administered to patients with myelocytic leukemia and has produced therapeutic effects that led to temporary remission. These studies demonstrated the potential efficacy of TPA in cancer chemotherapy. We now seek to understand the biological effects and molecular mechanisms of differentiation in response to TPA treatment in leukemia cells by expression profiling using DNA microarray. Our results show distinct temporal and coordinated gene changes that are consistent with differentiation and activation of multiple biochemical pathways in HL-60 cells exposed to TPA. Alterations of gene expression in HL-60 cells include various transcription factors, cytokines and protein markers that are consistent with the induction of differentiation elicited by TPA. These temporal patterns of gene expression were abolished or greatly diminished in an HL-60 derived TPA- resistant variant cell line (HL-525), thus revealing transcriptional and consequential biochemical changes that may be required for TPA-induced differentiation. In addition, certain genes were upregulated by TPA in TPA-resistant HL-525 cells but not in TPA-sensitive HL-60 cells suggesting that these genes may play a role in the resistant phenotype. These patterns of gene expression may be important for predicting response to TPA.

DNA microarray analysis of the expression profiles of luteinized granulosa cells as a function of ovarian reserve.

OBJECTIVE: To examine the expression profiles of luteinized granulosa cells isolated from women with normal or diminished ovarian reserve undergoing in vitro fertilization. DESIGN: Expression profiling by complementary DNA microarray analysis. SETTING: Women undergoing in vitro fertilization-embryo transfer in a university-based fertility clinic. PATIENT(S): Eighteen women with normal or decreased ovarian reserve. INTERVENTION(S): All patients were given gonadotropin stimulation in preparation for IVF with granulosa cells isolated at the time of follicular aspiration. MAIN OUTCOME MEASURE(S): Expression profiles of luteinized granulosa cells isolated from each woman were determined by using DNA microarray analysis. RESULT(S): Changes in patterns of gene expression in granulosa cells were observed between women with normal and diminished ovarian reserve. These genes included several anonymous expressed sequence tags and also expressed sequence tags that correspond to known genes. CONCLUSION(S): Expression profiling of granulosa cells by DNA microarray may yield signature patterns that reflect the status of ovarian reserve and the competence of granulosa cells.