Carcinogen Biomarkers Research Articles

Investigation of the Genome-Wide Genetic and Epigenetic Networks for Drug Discovery Based on Systems Biology Approaches in Colorectal Cancer.

Colorectal cancer (CRC) is the third most commonly diagnosed type of cancer worldwide. The mechanisms leading to the progression of CRC are involved in both genetic and epigenetic regulations. In this study, we applied systems biology methods to identify potential biomarkers and conduct drug discovery in a computational approach. Using big database mining, we constructed a candidate protein-protein interaction network and a candidate gene regulatory network, combining them into a genome-wide genetic and epigenetic network (GWGEN). With the assistance of system identification and model selection approaches, we obtain real GWGENs for early-stage, mid-stage, and late-stage CRC. Subsequently, we extracted core GWGENs for each stage of CRC from their real GWGENs through a principal network projection method, and projected them to the Kyoto Encyclopedia of Genes and Genomes pathways for further analysis. Finally, we compared these core pathways resulting in different molecular mechanisms in each stage of CRC and identified carcinogenic biomarkers for the design of multiple-molecule drugs to prevent the progression of CRC. Based on the identified gene expression signatures, we suggested potential compounds combined with known CRC drugs to prevent the progression of CRC with querying Connectivity Map (CMap).

A Streamlined and High-Throughput Error-Corrected Next-Generation Sequencing Method for Low Variant Allele Frequency Quantitation.

Quantifying mutant or variable allele frequencies (VAFs) of ≤10-3 using next-generation sequencing (NGS) has utility in both clinical and nonclinical settings. Two common approaches for quantifying VAFs using NGS are tagged single-strand sequencing and duplex sequencing. While duplex sequencing is reported to have sensitivity up to 10-8 VAF, it is not a quick, easy, or inexpensive method. We report a method for quantifying VAFs that are ≥10-4 that is as easy and quick for processing samples as standard sequencing kits, yet less expensive than the kits. The method was developed using PCR fragment-based VAFs of Kras codon 12 in log10 increments from 10-5 to 10-1, then applied and tested on native genomic DNA. For both sources of DNA, there is a proportional increase in the observed VAF to input VAF from 10-4 to 100% mutant samples. Variability of quantitation was evaluated within experimental replicates and shown to be consistent across sample preparations. The error at each successive base read was evaluated to determine if there is a limit of read length for quantitation of ≥10-4, and it was determined that read lengths up to 70 bases are reliable for quantitation. The method described here is adaptable to various oncogene or tumor suppressor gene targets, with the potential to implement multiplexing at the initial tagging step. While easy to perform manually, it is also suited for robotic handling and batch processing of samples, facilitating detection and quantitation of genetic carcinogenic biomarkers before tumor formation or in normal-appearing tissue.

Toxics source reduction and sewage upgrades eliminated winter flounder liver neoplasia (1984-2017) from Boston Harbor, MA, USA.

Chemical carcinogen biomarkers can validate public investment in environmental remediation. A major factor driving the clean-up of Boston Harbor, MA, USA, induced by the federal Clean Water Act legislation of 1972, was the high prevalence of petroleum and halogenated aromatic hydrocarbon contaminant-associated liver neoplasia in winter flounder Pseudopleuronectes americanus in the harbor in the 1980s. In the present study, we examined the spatial and temporal relationships between the suspended solids and contaminants in the municipal sewage discharge, and liver neoplasia and histopathology in flounder, from 1987 to 2017. Toxics source reduction, sewage treatment, and sludge removal in the 1990s and outfall relocation offshore in 2000 enabled a decreasing prevalence of persistent toxic chemicals in flounder, effluent, and sediment, and consequent disappearance of liver neoplasia and reduction of neoplasm-associated, hydropically vacuolated biliary epithelial cells to background levels. This supports long-term investment in elimination and treatment of anthropogenic waste streams and the value of federal regulatory mandates to maintain and improve regional environmental quality.

A Screening Study of Potential Carcinogen Biomarkers After Surgical Treatment of Obesity.

Obesity has been associated with the development of various types of cancer. Biomarker studies may provide molecular level knowledge of the factors involved in this association, improving clinical practice through new methods of prevention and treatment. The present study aimed to analyze proteins found in the plasma of obese patients prior to and 6months after bariatric surgery, using body mass index (BMI) and percentage total weight loss (%TWL) to evaluate, in a prospective manner, the effects of weight loss on the regulation of proteins related to the appearance of tumors. This was a cohort study designed to compare parameters before and after intervention. A total of 40 patients were divided into two groups: control (n = 10) and obese (n = 30). The latter group was stratified according to surgical technique used (Roux-en-Y gastric bypass (RYGB) n = 11 and sleeve gastrectomy (SG) n = 19) to remove confounding variables. Blood samples were collected for plasma protein studies using two-dimensional electrophoresis. Six proteins related to carcinogenesis were hyperexpressed in the obese patients but were absent in the control group and following surgery. These proteins were the beta-receptor of derived growth factor platelet, the receptor of apolipoprotein B, thrombospondin-2, the low-density lipoprotein receptor, transthyretin, and podoplanin. The current preliminary study thus identified potentially carcinogenic proteins in obese patients. Surgical weight loss resulted in the not detection of these proteins.

Poly‐(pyrrole‐3‐carboxylic acid) Based Nanostructured Platform for the Detection of Carcinoembryonic Antigen

AbstractA nanostructured platform based on pyrrole 3‐carboxylic acid was developed and it was used for the detection of carcinoembryonic antigen, a biomarker for gastro‐intestinal tract cancers. The conductive polymer offers possibilities for functionalization due to the presence of the carboxylic group. The platform was tailored by polymerizing pyrrole 3‐carboxylic acid at the surface of a graphite‐based screen‐printed electrode by using multiple pulse amperometry and cyclic voltammetry. The carboxylic groups were activated, facilitating the formation of amide covalent bonds with the terminal amine groups from the antibody. Each step's optimization was tracked by performing electrochemical impedance spectroscopy. The immunosensor was successfully applied for the detection of carcinoembryonic antigen in synthetic samples. By comparing pyrrole 3‐carboxylic acid with pyrrole, significantly greater increases of the charge transfer resistance were achieved by using the functionalized monomer for the immobilization step. The binding of the anti‐CEA was reproducible and directly proportional with its concentration. The increase of the charge transfer resistance after the incubation with carcinoembryonic antigen was linearly correlated with the concentration of the sample and the LOD was 33.33 pg mL−1. A simple nanostructured platform for the label‐free detection of a carcinogenic biomarker molecule was developed, thus holding great opportunities for clinical diagnostics and other biosensor applications.

Chairside molecular imaging of aberrant glycosylation in subjects with suspicious oral lesions using fluorescently labeled wheat germ agglutinin.

Aberrant sialylation is accepted as a carcinogenic biomarker. In previous work, fluorescently labeled wheat germ agglutinin (WGA) distinguished between cancerous and normal oral biopsies. The purpose of this study was to investigate WGA-fluorescein isothiocyanate (FITC) as a point-of-care tool for detecting oral malignant and dysplastic lesions in vivo. Subject recruitment was divided into two groups: (1) the clinically normal oral mucosa group; or (2) the presence of clinically suspicious oral lesion(s) group. A WGA-FITC solution was topically applied to observable lesions or to half the subject's mouth (sagittal plane) if lesions were absent. Fluorescent molecular imaging was used to evaluate WGA-FITC localization. Fluorescent imaging in 55 subjects demonstrated that WGA-FITC could detect histopathologically-confirmed cancerous and dysplastic lesions with high sensitivity (100% and 81%, respectively) and specificity (82%). This study supports in vivo fluorescent molecular imaging of WGA-FITC to visualize aberrant sialic acid expression associated with carcinogenesis. This technique resulted in the immediate chairside detection of oral cancerous and dysplastic lesions.

Biomarkers of Secondhand Smoke Exposure in Waterpipe Tobacco Venue Employees in Istanbul, Moscow, and Cairo.

Most smoke-free legislation to reduce secondhand smoke (SHS) exposure exempts waterpipe (hookah) smoking venues. Few studies have examined SHS exposure in waterpipe venues and their employees. We surveyed 276 employees of 46 waterpipe tobacco venues in Istanbul, Moscow, and Cairo. We interviewed venue managers and employees and collected biological samples from employees to measure exhaled carbon monoxide (CO), hair nicotine, saliva cotinine, urine cotinine, urine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and urine 1-hydroxypyrene glucuronide (1-OHPG). We estimated adjusted geometric mean ratios (GMR) of each SHS biomarker by employee characteristics and indoor air SHS measures. There were 73 nonsmoking employees and 203 current smokers of cigarettes or waterpipe. In nonsmokers, the median (interquartile) range concentrations of SHS biomarkers were 1.1 (0.2, 40.9) µg/g creatinine urine cotinine, 5.5 (2, 15) ng/mL saliva cotinine, 0.95 (0.36, 5.02) ng/mg hair nicotine, 1.48 (0.98, 3.97) pg/mg creatinine urine NNAL, 0.54 (0.25, 0.97) pmol/mg creatinine urine 1-OHPG, and 1.67 (1.33, 2.33) ppm exhaled CO. An 8-hour increase in work hours was associated with higher urine cotinine (GMR: 1.68, 95% CI: 1.20, 2.37) and hair nicotine (GMR: 1.22, 95% CI: 1.05, 1.43). Lighting waterpipes was associated with higher saliva cotinine (GMR: 2.83, 95% CI: 1.05, 7.62). Nonsmoking employees of waterpipe tobacco venues were exposed to high levels of SHS, including measurable levels of carcinogenic biomarkers (tobacco-specific nitrosamines and PAHs). Smoke-free regulation should be extended to waterpipe venues to protect nonsmoking employees and patrons from the adverse health effects of SHS.

Metabolomic profling in gastric cancer tissues using time-of-flight mass spectrometry.

66 Background: To explore carcinogenic and prognostic biomarkers for gastric cancer, genomics, transcriptomics and proteomics approaches have been extensively applied;however, little has been investigated regarding the role of metabolomics profiles on progression and prognosis of gastric cancer. Therefore, in order to elucidate the role of metabolome on prognosis of gastric cancer, we investigated the metabolic profiles of gastric cancer tissue using time-of-flight mass spectrometry (TOFMS) Methods: A total of 162 patients with gastric cancer underwent gastrectomy from February 2010 to March 2013 were enrolled in this study. Cancer tissues (CA) and adjacent non-cancerous tissues (NC) were obtained from surgically resected sample and were snap-frozen in liquid nitrogen. The samples were then homogenized and then applied to capillary electrophoresis TOFMS (CE-TOFMS) The metabolomics dates were analyzed using principal component analysis (PCA) and hierachical clustering analysis(HCA) in order to compare the metabolic profiles of NC and CA. Metabolites date were further assessed according by the non-parametric Mann–Whitney U-test on the presence or absence of recurrence . Results: A total 96 metabolites were detected and quantified. PCA of the date well-distinguished CA from NC. In CA, lactate / pyruvate ratio was significantly higher, while adenylate energy charge was significantly lower than NC, which reaffirms the Warburug effect of cancer. Total glutathione and reducted glutathione/oxidized glutathione ratio in CA were significantly higher than in NC which possibly contributes to the homeostasis of redox status in CA. Intriguingly, in patients with recurrence, tumor concentrations of β-Ala, Asp, GDP and Gly were significantly lower than in those without recurrence. Conclusions: Metabolomic profiling clearly differentiated CA from NC. Considerably high lactate and amino acids levels expectedly highlighted the metabolome of tumors. Certain metabolite will be a candidate for biomarker in gastric cancer.

Adverse Health Effects of Betel Quid and the Risk of Oral and Pharyngeal Cancers.

Global reports estimate 600 million betel quid (BQ) chewers. BQ chewing has been demonstrated not only to be a risk factor for cancers of the oral cavity and pharynx and oral potentially malignant disorders (OPMD) but also to cause other cancers and adverse health effects. Herein, we summarized the international comparison data to aid in the understanding of the close relationship between the prevalence of BQ chewing, the occurrence of oral and pharyngeal cancers, and adverse health effects. Potential biomarkers of BQ carcinogens, such as areca nut, alkaloids, and 3-methylnitrosaminopropionitrile (MNPN), are closely associated with human health toxicology. Molecular mechanisms or pathways involving autophagy, hypoxia, COX-2, NF-κB activity, and stemness are known to be induced by BQ ingredients and are very closely related to the carcinogenesis of cancers of oral and pharynx. BQ abuse-related monoamine oxidase (MAO) gene was associated with the occurrence and progress of oral and pharyngeal cancers. In summary, our review article provides important insights into the potential roles of environmental BQ (specific alkaloid biomarkers and nitrosamine products MNPN) and genetic factors (MAO) and offers a basis for studies aiming to reduce or eliminate BQ-related OPMD and oral/pharyngeal cancer incidences in the future.

Tobacco smoke toxicant and carcinogen biomarkers and lung cancer susceptibility in smokers

Stephen S. Hecht, Steven G. Carmella, Sharon E. Murphy, Irina Stepanov, Silvia Balbo, Dorothy K. Hatsukami, Jian-Min Yuan, Sungship L. Park, Daniel O. Stram, Christopher Haiman, Yesha Patel, Loic Le Marchand Masonic Cancer Center, University of Minnesota, Minneapolis, MN, University of Pittsburgh Cancer Institute, Pittsburgh, PA, University of Southern California Norris Comprehensive Cancer Center, Los Angeles, CA, University of Hawaii Cancer Center, Honolulu, HI

Activating transcription factor 2 expression mediates cell proliferation and is associated with poor prognosis in human non-small cell lung carcinoma

Activating transcription factor 2 (ATF2) is a member of the cAMP response element binding protein family that heterodimerizes and activates other transcription factors involved in stress and DNA damage responses, growth, differentiation and apoptosis. ATF2 has been investigated as a potential carcinogenic biomarker in certain types of cancer, such as melanoma. However, its function and clinical significance in non-small cell lung cancer (NSCLC) has not been well studied. Therefore, the present study aimed to analyze the association between ATF2/phosphorylated (p)-ATF2 expression and NSCLC malignant behavior, and discuss its clinical significance. Reverse transcription-quantitative polymerase chain reaction and western blotting were used to detect the expression of ATF2 in NSCLC cell lines and fresh NSCLC tissue samples. In addition, immunohistochemistry (IHC) was performed to identify the location and expression of ATF2 and p-ATF2 (threonine 71) in paraffin-embedded sections of NSCLC and adjacent normal tissue. The results demonstrated that ATF2 was markedly overexpressed in the NSCLC cells and significantly overexpressed in the fresh NSCLC tissues compared with the control cells and samples (86 paraffin-embedded tissue sections), respectively (P<0.01). Further data demonstrated that ATF2 expression levels were significantly increased in tumor tissues compared to normal tissues and ATF2 was located in the cytoplasm and nucleus. ATF2 expression was closely associated with adverse clinical characteristics such as TNM stage (P=0.002), tumor size (P=0.018) and metastasis (P=0.027). In addition, nuclear p-ATF2 staining was positive in 65/86 samples of NSCLC. Furthermore, the Kaplan-Meier analysis indicated that patients with high levels of ATF2 and p-ATF2 expression had a significantly shorter overall survival compared with patients exhibiting a low expression (P<0.01 and P<0.05, respectively). Subsequent in vitro experiments revealed that cell growth decreased following knockdown of ATF2 expression using RNA interference, indicating that ATF2 may suppress cell proliferation. Taken together, the results of the present study demonstrated that ATF2 and p-ATF2 were significantly overexpressed in NSCLC tissues, and ATF2 and p-ATF2 overexpression predicted significantly worse outcomes for patients with NSCLC.

Identification of specific mRNA signatures as fingerprints for carcinogenesis in mice induced by genotoxic and nongenotoxic hepatocarcinogens.

Long-term rodent carcinogenicity studies for evaluation of chemicals and pharmaceuticals concerning their carcinogenic potential to humans are currently receiving critical revision. Additional data from mechanistic studies can support cancer risk assessment by clarifying the underlying mode of action. In the course of the IMI MARCAR project, a European consortium of EFPIA partners and academics, which aims to identify biomarkers for nongenotoxic carcinogenesis, a toxicogenomic mouse liver database was generated. CD-1 mice were orally treated for 3 and 14 days with 3 known genotoxic hepatocarcinogens: C.I. Direct Black 38, Dimethylnitrosamine and 4,4'-Methylenedianiline; 3 nongenotoxic hepatocarcinogens: 1,4-Dichlorobenzene, Phenobarbital sodium and Piperonyl butoxide; 4 nonhepatocarcinogens: Cefuroxime sodium, Nifedipine, Prazosin hydrochloride and Propranolol hydrochloride; and 3 compounds that show ambiguous results in genotoxicity testing: Cyproterone acetate, Thioacetamide and Wy-14643. By liver mRNA expression analysis using individual animal data, we identified 64 specific biomarker candidates for genotoxic carcinogens and 69 for nongenotoxic carcinogens for male mice at day 15. The majority of genotoxic carcinogen biomarker candidates possess functions in DNA damage response (eg, apoptosis, cell cycle progression, DNA repair). Most of the identified nongenotoxic carcinogen biomarker candidates are involved in regulation of cell cycle progression and apoptosis. The derived biomarker lists were characterized with respect to their dependency on study duration and gender and were successfully used to characterize carcinogens with ambiguous genotoxicity test results, such as Wy-14643. The identified biomarker candidates improve the mechanistic understanding of drug-induced effects on the mouse liver that result in hepatocellular adenomas and/or carcinomas in 2-year mouse carcinogenicity studies.

Cigarette smoke toxins deposited on surfaces: implications for human health.

Cigarette smoking remains a significant health threat for smokers and nonsmokers alike. Secondhand smoke (SHS) is intrinsically more toxic than directly inhaled smoke. Recently, a new threat has been discovered – Thirdhand smoke (THS) – the accumulation of SHS on surfaces that ages with time, becoming progressively more toxic. THS is a potential health threat to children, spouses of smokers and workers in environments where smoking is or has been allowed. The goal of this study is to investigate the effects of THS on liver, lung, skin healing, and behavior, using an animal model exposed to THS under conditions that mimic exposure of humans. THS-exposed mice show alterations in multiple organ systems and excrete levels of NNAL (a tobacco-specific carcinogen biomarker) similar to those found in children exposed to SHS (and consequently to THS). In liver, THS leads to increased lipid levels and non-alcoholic fatty liver disease, a precursor to cirrhosis and cancer and a potential contributor to cardiovascular disease. In lung, THS stimulates excess collagen production and high levels of inflammatory cytokines, suggesting propensity for fibrosis with implications for inflammation-induced diseases such as chronic obstructive pulmonary disease and asthma. In wounded skin, healing in THS-exposed mice has many characteristics of the poor healing of surgical incisions observed in human smokers. Lastly, behavioral tests show that THS-exposed mice become hyperactive. The latter data, combined with emerging associated behavioral problems in children exposed to SHS/THS, suggest that, with prolonged exposure, they may be at significant risk for developing more severe neurological disorders. These results provide a basis for studies on the toxic effects of THS in humans and inform potential regulatory policies to prevent involuntary exposure to THS.

Urinary 1-hydroxypyrene as a comprehensive carcinogenic biomarker of exposure to polycyclic aromatic hydrocarbons: a cross-sectional study of coke oven workers in China

Polycyclic aromatic hydrocarbons (PAHs) are multiple compounds that include many carcinogens. We conducted a cross-sectional study in steel plant workers in Anshan, China, to identify biomarkers that reflect the carcinogenicity of PAHs. Subjects were 57 workers and 20 controls. Level of personal exposure to PAHs was measured using GC-MS. In accordance with the assessment methods defined by the United States Environmental Protection Agency (US EPA), 15 PAHs were selected for the analysis. For the measurement of urinary metabolites, urine samples were treated with β-glucuronidase and analyzed using HPLC with a fluorescence detector. The mean range of personal exposure to 15 PAHs (total PAHs) was 178.85, 47.08-1,329.45 (geometric mean, 5th and 95th percentile) μg/m(3). Ten known urinary metabolites (1-hydroxynaphthalene, 2-hydroxynaphthalene, 2-hydroxyfluorene, 1-hydroxyphenanthrene, 3-hydroxyphenanthrene, 9-hydroxyphenanthrene, 1-hydroxypyrene, 3-hydroxybenz[a]anthracene, 6-hydroxychrysene, and 3-hydroxybenzo[a]pyrene) and four unknown peaks were detected. The highest correlation was between total PAHs and urinary 2-hydroxynaphthalene (Spearman r = 0.716, P < 0.01). Among the detected urinary metabolites, 2-hydroxyfluorene, 1-hydroxyphenanthrene, 3-hydroxyphenanthrene, and 1-hydroxypyrene were found to correlate significantly with the "Σ carcinogenic potency of PAHs" (sum of seven carcinogenic PAHs calculated from the levels of personal PAHs and relative potency factors), and with the greatest correlation found for 1-hydroxypyrene (Spearman r = 0.630, P < 0.01). The analysis of personal exposure to 15 PAHs and 10 urinary metabolites, and calculation of Σ carcinogenic potency, indicated that urinary 1-hydroxypyrene was the most comprehensive carcinogenic biomarker of exposure to PAHs.

Direct analysis of tobacco-specific nitrosamine NNK and its metabolite NNAL in human urine by LC–MS/MS: evidence of linkage to methylated DNA lesions

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its urinary metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), are the most investigated carcinogenic biomarkers of tobacco-specific nitrosamines. Here, we report the development of a sensitive and selective assay based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to simultaneously measure urinary NNK and NNAL. With the use of isotope internal standards and online solid-phase extraction, urine samples were directly analyzed without prior sample purification. The detection limits of this method were 0.13 and 0.19 pg on column for NNK and NNAL, respectively. Inter- and intra-day imprecision was <10 %. Mean recovery of NNK and NNAL in urine was 99-100 %. This method was applied to measure urinary NNK and NNAL in 101 smokers and 40 nonsmokers to assess tobacco exposure. Urinary nicotine, cotinine, N3-methyladenine (N3-MeA), and N7-methylguanine (N7-MeG) were also measured by isotope-dilution LC-MS/MS methods. The results showed that urinary NNK was not observed in all smokers. Urinary free NNAL (0.10 ± 0.09 ng/mg creatinine) and total NNAL (0.17 ± 0.14 ng/mg creatinine) were detected in all smokers. Urinary concentrations of NNAL were significantly correlated with nicotine, cotinine, N3-MeA, and N7-MeG in smokers (P < 0.001). This method enables the direct and simultaneous measurement of NNK and NNAL in urine using only 50 μL of urine. This study first demonstrated in human that urinary tobacco-specific nitrosamines metabolite (NNAL) are highly correlated with their resulting methylated DNA lesions in urine, which may help to substantiate an increased cancer risk associated with tobacco smoke exposure.

Abstract 1502: Urinary biomarkers of polycyclic aromatic hydrocarbon and volatile organic carcinogens in relation to lung cancer development in lifelong never smokers - The Shanghai Cohort Study.

Abstract Background: In the United States, 10-15% of lung cancer victims are lifelong never smokers. Non-smoking related lung cancer alone ranks as the 6th most frequently diagnosed cancer. The etiological factors for non-smoking lung cancer are unclear. Although strong evidence supports a major role for polycyclic aromatic hydrocarbon (PAH) as causes of lung cancer in smokers, epidemiological studies prospectively investigating biomarkers of PAH and volatile organic carcinogens in relation to lung cancer risk among never smokers are lacking. Methods: A nested case-control study of lung cancer among never smokers was conducted within the Shanghai Cohort Study, a prospective cohort of 18,244 Chinese men aged 45-64 years at enrollment during 1986-1989. After more than 20 years of follow-up, we identified 96 incident lung cancer cases among never smokers. For each case, we randomly chose one control among cohort participants who were never smokers. The control was matched to the index case by age (±2 years), date of urine collection (±1 month) and neighborhood of residence. We quantified urinary free cotinine, 3-hydroxyphenanthrene (3-OH-Phe), total hydroxyphenanthrenes (the sum of 1-, 2-, 3-, and 4-OH-Phe) (total OH-Phe), r-1,t-2,3,c-4-tetrahdroxy-1,2,3,4-tetrahydrophenanthrene (PheT), mercapturic acid metabolites of acrolein [3-hydroxypropyl mercapturic acid (HPMA)], benzene [S-phenyl mercapturic acid (SPMA)], crotonaldehyde [4-hydroxybut-2-yl mercapturic acid (HBMA)], and ethylene oxide [2-hydroxyethyl mercapturic acid (HEMA)]. Self-reported never smokers whose urinary free cotinine was greater than 20 ng/ml were excluded from the present analysis for their potential use of tobacco. The odds ratio (OR) and 95% confidence interval (CI) for lung cancer for each urinary biomarker was estimated using an unconditional logistic regression method. Results: Among never smokers, urinary levels of all 3 measured PAH biomarkers were statistically significantly higher in cases than controls. The geometric means (pmol/mg creatinine) of total OH-Phe, 3-OH-Phe and PheT were 19.10 (in cases) vs 16.08 (in controls) (P = 0.017); 6.64 vs 5.44 (P = 0.011); and 19.84 vs 16.10 (P = 0.035), respectively. Compared with the lowest quartile, ORs (95% CIs) for lung cancer for the highest quartile were 2.59 (1.01-6.65) for total OH-Phe, 3.10 (1.12-7.75) for 3-OH-Phe, and 2.98 (1.13-7.87) for PheT (all Ptrend &amp;lt;0.05). There was no statistically significant difference in urinary levels of HPMA, SPMA, HBMA and HEMA between cases and controls. Conclusion: This is the first prospective study that demonstrates a positive association between urinary biomarkers of PAH and lung cancer risk among never smokers. Given its ubiquitous presence in the environment, PAH could account for large proportion of non-smoking related lung cancer. 1 Citation Format: Jian-Min Yuan, Yu-Tang Gao, Sharon Murphy, Steven G. Carmella, Renwei Wang, Heather H. Nelson, Stephen S. Hecht. Urinary biomarkers of polycyclic aromatic hydrocarbon and volatile organic carcinogens in relation to lung cancer development in lifelong never smokers - The Shanghai Cohort Study. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1502. doi:10.1158/1538-7445.AM2013-1502

Abstract 667: Evaluation of toxicant exposure in U.S. smokers after short-term switching to commercial low-nitrosamine cigarettes

Abstract Mandated reduction of carcinogens and toxicants in cigarette smoke can be required by the U.S. Food and Drug Administration (FDA). Among the initial smoke constituents proposed for regulation are the carcinogenic tobacco-specific nitrosamines (TSNA) 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and Nα-nitrosonornicotine (NNN), which are believed to play an important role in the development of cancers of the lung, pancreas, oral cavity, and esophagus in smokers. Smoking-machine based measurements demonstrate significant variations in TSNA levels both internationally and among currently marketed U.S. brands. However, human smoking patterns are highly complex, and the machine smoking approach does not provide accurate information on actual TSNA intake by individual smokers. Identification of the machine-measured TSNA levels that can result in significant reduction of their current average intake, as determined by changes in biomarker levels in smokers, is critical in informing future FDA policies. In this study, 18 smokers of cigarettes with relatively high machine-measured smoke concentrations of NNK and NNN were asked to switch to Marlboro Virginia Blend (MVB) - cigarettes with the lowest known TSNA levels on the U.S. market. Urinary levels of cotinine, total NNAL, total NNN, and 1-hydroxypyrene (1-HOP) - biomarkers of exposure to nicotine, NNK, NNN, and polycyclic aromatic hydrocarbons (PAH), respectively - were measured at baseline and after one week of smoking MVB. Average(±SD) levels of carcinogen biomarkers at baseline were: total NNAL, 1.0(±0.7) pmol/mg creatinine; total NNN, 0.034(±0.03) pmol/mg creatinine; and 1-HOP, 0.78(±0.6) pmol/mg creatinine. Corresponding levels after switching to MVB were 0.71(±0.5), 0.039(±0.04), and 1.04(±0.7) pmol/mg creatinine, respectively, with the changes in the levels relative to baseline not being statistically significant. Adjustment for nicotine intake revealed a decrease in urinary total NNAL, total NNN, and 1-HOP by 56%, 31%, and 19%, respectively, after switching to MVB compared to baseline. The only statistically significant change was the decrease in total NNAL levels. The lack of significant decrease in exposure to NNN can be explained by the contribution of endogenously formed NNN to the urinary total NNN measured in smokers. The slightly lower levels of pyrene in the smoke of MVB compared to the baseline cigarette brands could have contributed to the modest change in urinary 1-HOP. In summary, our results demonstrate a significant decrease in NNK intake per mg nicotine after short term switching to commercially available low-nitrosamine cigarettes. However, overall exposure to TSNA was greatly influenced by the individual changes in nicotine intake after switching to MVB. Thus, both carcinogen and nicotine levels may need to be modified in order to reach meaningful reductions in carcinogen intake by smokers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 667. doi:1538-7445.AM2012-667

Metabolome analyses in human gastric cancer tissue using capillary electrophoresis time-of-flight mass spectrometry.

61 Background: In order to explore carcinogenic and prognostic biomarkers for gastric cancer, genomic, transcriptomic and proteomic approach had been extensively investigated. However, little information is possible for metabolomics profiles in gastric cancer. As cancer cells demonstrates distinguishable growth pattern from normal cells, presence of specific metabolic pathway in cancer cells is expected. Therefore, we investigated the metabolme profiles of gastric cancer tissue by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Methods: Twenty seven patients with gastric cancer underwent gastrectomy were enrolled in this study. Cancer tissue and adjacent non-cancerous tissue were obtained from surgically resected sample and immediately frozen. Frozen tissues were homogenized and then applied to CF-TOFMS.The metabolomics data by CE-TOFMS were analyzed using principal component analysis (PCA) in order to compare the metabolic profiling of the non cancerous and the cancer tissues. Correlation between metabolic profile and clinicopathologic factors were also investigated. Results: A total of 245 metabolites were detected and quantified. In PCA, the first component separated the cancer tissue data from the non-cancerous tissue data. Metabolic data from cancer tissues showed severe heterogeneity. PCA based on the difference between cancer tissue and non-cancerous tissue revealed that metabolic profile of gastric cancer was classified into two groups. One cancer group (group 1) accumulated lactate and most amino acids excessively, and another group (group 2) didn’t. In group 1, the adenylate energy charge of the cancer tissues was maintained compared to that of the non-cancerous tissues.In comparison with clinicopathologic factors, only histological type demonstrated significant correlation with metabolic classification. Undifferentiated type was significantly predominant in group 2. Conclusions: Gastric cancer was classified into two groups by quantitative metabolome profiling. As this classification significantly correlated with histological type, existence of specific metabolic pathway related to histological type is suggested.

The presence of aflatoxin B1-FAPY adduct and human papilloma virus in cervical smears from cancer patients in Mexico

The carcinogenic biomarker aflatoxin B1-formamidopyrimidine 2,3-dihydro-2-(N-formyl)-2′,5′,6′-triamino-4′-4′-oxy-N-pyrimidyl-3-hydroxy-AFB1 called AFB1-FAPY adduct, and Human Papilloma Virus (HPV) types 16 and 18 were quantified from DNA cervical scrapes from 40 women with cervical cancer (CC) and 14 healthy women as controls. The relationship between the AFB1-FAPY adduct and HPV types 16 and 18 was determined. Competitive inhibitory indirect ELISA was validated with 94% inhibition to quantify the AFB1-FAPY adducts in picograms per milligram of DNA (limit of detection = 0.1 pg/mg, and limit of quantification = 10 pg/mg), polymerase chain reaction and DNA sequencing to identify HPV types. The average concentration of AFB1-FAPY adducts/mg DNA in the CC cases was 1025 pg, 1420 pg with HPV16 and 630 pg sharing HPV18 (p = 0.03). In comparison, healthy controls had ≤2.6 pg/mg DNA, a statistically significant difference (p = 0.00006). The presence of AFB1-FAPY adduct increased six-fold the risk for CC between cases and controls, the odds ratio was 6.1 (95% CI = 1.4–25.4). There was a close relationship between the AFB1-FAPY adducts and HPV16 in CC samples.

Abstract 4656: Relationship of nicotine and carcinogen biomarkers to lung cancer risk among smokers in the Shanghai cohort study

Abstract Individuals who develop lung cancer are predominantly tobacco users and further risk profiling within this group is important to better understand cancer susceptibility and could be used to target interventions. A nested case-control approach was used to evaluate risk factors for lung cancer in a population of Chinese men from Shanghai, China (317 cases and 317 controls). All participants were current smokers at baseline and controls were matched for age, neighborhood, and date of biospecimen collection. Biomarkers assessed in urine were nicotine and its major metabolites, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, a tobacco carcinogen), and r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT, a biomarker of polyaromatic hydrocarbons). The odds ratio for developing lung cancer were greatest for men with the highest versus lowest quintile of urinary cotinine (cotinine + cotinine glucuronide), OR (95% CI); 8.79 (4.42-17.47) unadjusted, and 5.26 (2.39-11.57) adjusted for years of smoking, cigarettes per day, NNAL, and PheT. Cotinine levels are influenced both by nicotine exposure and metabolism. Nicotine and cotinine are metabolized by cytochrome P450 2A6 which is polymorphic and ∼15% of the Chinese population carry low activity alleles. In contrast, nicotine equivalents is a biomarker of exposure that is less influenced by metabolism as it is the sum of nicotine and its metabolites excreted in urine. Higher odds ratios of lung cancer were also observed for increasing nicotine equivalents but this was a weaker biomarker compared to cotinine in this population, OR (95% CI); 4.86 (2.72-8.69) unadjusted, and 2.58 (1.32-5.03) adjusted for years of smoking, cigarettes per day, NNAL, and PheT. The ratio of trans-3′-hydroxycotinine to cotinine per nicotine equivalents was evaluated as a biomarker of oxidation phenotype. Previously the ratio of trans-3′-hydroxycotinine to cotinine has been used to phenotype individuals for P450 2A6 activity, and to account for variation in nicotine dose during smoking the ratio was expressed per nicotine equivalents. Unexpectedly, the highest quintile of the metabolite ratio was associated with a decreased risk of lung cancer compared to the lowest quintile, OR (95%CI); 0.26 (0.15-0.47). This group would include individuals who have a low nicotine metabolism phenotype and smoke an average-to-high amount. Higher urinary NNAL and PheT were also risk factors for lung cancer albeit the odds ratios were lower than for cotinine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4656. doi:10.1158/1538-7445.AM2011-4656