Since their discovery 50 years ago, the aflatoxins have become recognized as ubiquitous contaminants of the human food supply throughout the economically developing world. The adverse toxicological consequences of these compounds in populations are quite varied because of a wide range of exposures leading to acute effects, including rapid death, and chronic outcomes such as hepatocellular carcinoma. Furthermore, emerging studies describe a variety of general adverse health effects associated with aflatoxin, such as impaired growth in children. Aflatoxin exposures have also been demonstrated to multiplicatively increase the risk of liver cancer in people chronically infected with hepatitis B virus (HBV) illustrating the deleterious impact that even low toxin levels in the diet can pose for human health. The public health impact of aflatoxin exposure is pervasive. Aflatoxin biomarkers of internal and biologically effective doses have been integral to the establishment of the etiologic role of this toxin in human disease through better estimates of exposure, expanded knowledge of the mechanisms of disease pathogenesis, and as tools for implementing and evaluating preventive interventions.

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Single-walled carbon nanotubes (SWNT) are particularly attractive for biomedical applications, because they exhibit a fluorescent signal in a spectral region where there is minimal interference from biological media. Although SWNT have been used as highly-sensitive detectors for various molecules, their use as in vivo biosensors requires the simultaneous optimization of various parameters, including biocompatibility, molecular recognition, high fluorescence quantum efficiency and signal transduction. Here we demonstrate that a polyethylene glycol ligated copolymer stabilizes near infrared fluorescent SWNT sensors in solution, enabling intravenous injection into mice and the selective detection of local nitric oxide (NO) concentration with a detection limit of 1 μM. The half-life for liver retention is 4 hours, with sensors clearing the lungs within 2 hours after injection, thus avoiding a dominant route of in vivo nanotoxicology. After localization within the liver, it is possible to follow the transient inflammation using NO as a marker and signalling molecule. To this end, we also report a spatial-spectral imaging algorithm to deconvolute fluorescence intensity and spatial information from measurements. Finally, we show that alginate encapsulated SWNT can function as an implantable inflammation sensor for in vivo NO detection, with no intrinsic immune reactivity or other adverse response, for more than 400 days. These results open new avenues for the use of such nanosensors in vivo for biomedical applications.

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Helicobacter hepaticus -infected Rag 2 -/- mice emulate many aspects of human inflammatory bowel disease, including the development of colitis and colon cancer. To elucidate mechanisms of inflammation-induced carcinogenesis, we undertook a comprehensive analysis of histopathology, molecular damage, and gene expression changes during disease progression in these mice. Infected mice developed severe colitis and hepatitis by 10 wk post-infection, progressing into colon carcinoma by 20 wk post-infection, with pronounced pathology in the cecum and proximal colon marked by infiltration of neutrophils and macrophages. Transcriptional profiling revealed decreased expression of DNA repair and oxidative stress response genes in colon, but not in liver. Mass spectrometric analysis revealed higher levels of DNA and RNA damage products in liver compared to colon and infection-induced increases in 5-chlorocytosine in DNA and RNA and hypoxanthine in DNA. Paradoxically, infection was associated with decreased levels of DNA etheno adducts. Levels of nucleic acid damage from the same chemical class were strongly correlated in both liver and colon. The results support a model of inflammation-mediated carcinogenesis involving infiltration of phagocytes and generation of reactive species that cause local molecular damage leading to cell dysfunction, mutation, and cell death. There are strong correlations among histopathology, phagocyte infiltration, and damage chemistry that suggest a major role for neutrophils in inflammation-associated cancer progression. Further, paradoxical changes in nucleic acid damage were observed in tissue- and chemistry-specific patterns. The results also reveal features of cell stress response that point to microbial pathophysiology and mechanisms of cell senescence as important mechanistic links to cancer.

Structural modifications of cellular macromolecules by chemical carcinogens may represent early and requisite events in neoplastic transformation (1, 2). Through interactions of this nature, qualitative changes could be induced in informational macromolecules such as DNA and RNA, and these lesions could provide a molecular basis for alteration of gene expression in carcinogenesis. Identification of the products of these reactions (herein referred to as adducts) is essential in order to: (i) gain insights into mechanisms of carcinogen activation; (ii) determine the reactive centers in these macromolecules; (iii) follow the kinetics of appearance and disappearance of adducts in the cell; and (iv) relate specific patterns of macromolecule modification with the ultimate development of tumors in target organs of susceptible species.Aflatoxin B1 (AFB1) is a very potent liver carcinogen in several animal species (3), and epidemiologic evidence indicates that it is also an important factor in the etiology of human liver cancer in certain sections of the world (4). AFB1 binds covalently to cellular macromolecules, including DNA, in mvo (5-7) and in vitro after metabolic activation (8-10). The relationship of this type of interaction to its mechanism of action has been emphasized (11). Strong indirect evidence has indicated the production of AFB1-2,3-oxide as a major activated metabolite responsible for macromolecular binding in vitro and in vivo (5-7, 9, 12), but structures of specific adducts formed with nucleic acids or proteins have not been determined. The purpose of the research reported here was to determine the structure of the major adduct formed with DNA by AFB1 activated metabolically in vitro. The results indicate that approximately 90% of the binding in vitro can be attributed to a single adduct, which was isolated in sufficient quantity for structural analysis and identified as 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 (structure I).,H0 Ho (c) H3C (c) Hk I MATERIALS AND METHODS Liver microsomes used for metabolic activation of AFB1 were prepared from phenobarbital-treated male Fischer rats (13) by the procedure of Kinoshita et al. (14). The incubation mixture (400 ml) for the binding of AFB1 to DNA included Tris-HCl (pH 7.5,45 mM), MgCl2 (3 mM), glucose-6-phosphate (5 mM), NADP (0.8 mM, Sigma Chemical Co.), glucose-6-phosphate dehydrogenase (0.4 unit/ml, Sigma Chemical Co.), approximately 1 mg of microsomal protein per ml, calf thymus DNA (20 A260 units/ml or a total of 340 mg; type I, Sigma Chemical Co.), AFB1 [224 ,uM added Abbreviations: AFB1, aflatoxin B1; I, 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin Bj; II, 2,3-dihydro-3-hydroxy-2-(4-nitrobenzoxy)-aflatoxin B1; HPLC, high-pressure liquid chromatography; NMR, nuclear magnetic resonance; FD, field-desorption mass spectrometry; EI, electron-impact mass spectrometry.

Recombinase-activating gene-2-deficient (Rag2 ؊/؊ ) mice lacking functional lymphocytes provide a useful model of chronic inflammatory bowel disease-emulating events in human colon cancer. Infection of Rag2 ؊/؊ mice with Helicobacter hepaticus led to accumulation of macrophages and neutrophils in the colon, a process temporally related to up-regulation of tissue inducible nitric oxide synthase (iNOS) expression at the site of infection and increased nitric oxide (NO) production, as evidenced by urinary excretion of nitrate. Progressive development of increasingly severe inflammation, hyperplasia, dysplasia, and cancer accompanied these changes. Concurrent administration of an iNOS inhibitor prevented NO production and abrogated epithelial pathology and inhibited the onset of cancer. The presence of Gr-1 ؉ neutrophils and elevated tumor necrosis factor-␣ (TNF-␣) expression in colon were required for increased iNOS expression and cancer, whereas interleukin-10 (IL-10) down-regulated TNF-␣ and iNOS expression and suppressed cancer. Anti-inflammatory CD4 ؉ regulatory lymphocytes also down-regulated iNOS and reduced cancer formation. Collectively, these results confirm essential roles for inflammation, increased TNF-␣ expression, and elevated NO production in colon carcinogenesis.colorectal cancer ͉ IBD ͉ innate immunity C hronic Helicobacter pylori infection in humans leads to gastritis and has been established as a causative agent for human gastric cancer (1). Inflammatory bowel diseases (IBDs), such as Crohn's disease and ulcerative colitis, also increase risk for colon cancer (2, 3). Generation of nitric oxide (NO) by inducible NO synthase (iNOS) is a central feature of chronic inflammatory diseases in the gastrointestinal tract (4-9), but precise mechanistic roles for NO in colon cancer development remain undefined.Colon cancer patients exhibit evidence of nitrosative and oxidative stresses that increase cancer risk (10), resulting from mutagenic reactive oxygen and nitrogen species derived from NO generated by immune cells (6,(11)(12)(13)(14)(15). Roles for chronic bacterial infection in IBD and colon cancer have been identified recently in recombinase-activating gene-2-deficient mice (Rag2 Ϫ/Ϫ ), which completely lack functional lymphocytes (16-18). We have exploited this mouse model of chronic IBDassociated cancer for studies of the role of NO and its products because it emulates naturally occurring inflammatory events in humans (16,19,20).Rag2 Ϫ/Ϫ mice have been used to assess functions of lymphocytes by adoptive transfer. Populations of CD4 ϩ T cells with low or high expression of CD45RB (17,21, 22) or CD25 (16,18,23,24) prevent or accelerate colitis in these animals. In wild-type (wt) mice, protection against inflammatory pathology induced by bacterial infection has been attributed to interleukin-10 (IL-10) and IL-10-dependent functions of CD4 ϩ cells (18,20,25,26). Collectively, this evidence forms the rationale for the hypothesis that NO overproduction comprises a linkage between Helicobacter hepaticus-i...

Reactive intermediates such as reactive nitrogen species play essential roles in the cell as signaling molecules but, in excess, constitute a major source of cellular damage. We found that nitrosative stress induced by steady-state nitric oxide (NO) caused rapid activation of an ATM damage-response pathway leading to downstream signaling by this stress kinase to LKB1 and AMPK kinases, and activation of the TSC tumor suppressor. As a result, in an ATM-, LKB1-, TSC-dependent fashion, mTORC1 was repressed, as evidenced by decreased phosphorylation of S6K, 4E-BP1, and ULK1, direct targets of the mTORC1 kinase. Decreased ULK1 phosphorylation by mTORC1 at S757 and activation of AMPK to phosphorylate ULK1 at S317 in response to nitrosative stress resulted in increased autophagy: the LC3-II/LC3-I ratio increased as did GFP-LC3 puncta and acidic vesicles; p62 levels decreased in a lysosomedependent manner, confirming an NO-induced increase in autophagic flux. Induction of autophagy by NO correlated with loss of cell viability, suggesting that, in this setting, autophagy was functioning primarily as a cytotoxic response to excess nitrosative stress. These data identify a nitrosative-stress signaling pathway that engages ATM and the LKB1 and TSC2 tumor suppressors to repress mTORC1 and regulate autophagy. As cancer cells are particularly sensitive to nitrosative stress, these data open another path for therapies capitalizing on the ability of reactive nitrogen species to induce autophagy-mediated cell death. signal transduction | cancer therapyA utophagy is a self-digestion process by which a eukaryotic cell degrades and recycles aggregate-prone proteins, macromolecules, and organelles. During autophagy, cytoplasmic contents are sequestered in double-membrane bound vesicles called autophagosomes and delivered to lysosomes for degradation, thereby allowing cells to eliminate and recycle the contents (1-3). Autophagy participates in both prosurvival (recycling of cellular building blocks) and prodeath (excess catalysis) pathways. A comprehensive understanding of signaling pathways that regulate autophagy holds great promise for new therapeutic opportunities by opening the possibility to compromise prosurvival autophagic pathways that enable tumor cells to evade therapy, or by promoting prodeath autophagic pathways to kill cancer cells.The classical pathway regulating autophagy in mammalian cells involves the serine/threonine kinase, mammalian target of rapamycin (mTOR). Active mTOR kinase in the mTORC1 complex phosphorylates and inhibits ULK1, a key proautophagy adapter involved in nucleation of the autophagophore membrane. Inactivation of mTORC1, either pharmacologically with rapamycin or via activation of the tuberous sclerosis complex (TSC) tumor suppressor, leads to downstream dephosphorylation events, including loss of ULK1 phosphorylation at S757. The TSC1/2 heterodimer is itself regulated by upstream kinases, including the AMP-activated protein kinase (AMPK), which regulates several metabolic processes and activates t...