Gerald N. Wogan

Urinary aflatoxin biomarkers and risk of hepatocellular carcinoma

Aflatoxins have long been suspected to be human hepatic carcinogens but no direct study was feasible until assays to measure individual aflatoxin exposure became available. We have used assays for urinary aflatoxin B1, its metabolites AFP1 and AFM1, and DNA-adducts (AFB1-N7-Gua) to assess the relation between aflatoxin exposure and liver cancer, as part of an ongoing prospective study of 18,244 middle-aged men in Shanghai, Peoples Republic of China. After 35,299 person-years of follow-up, 22 cases of liver cancer had been identified. For each case, 5 or 10 controls were randomly selected from cohort members without liver cancer on the date the disorder was diagnosed in the case and matched to within 1 year for age, within 1 month for sample collection, and for neighbourhood of residence. Subjects with liver cancer were more likely than were controls to have detectable concentrations of any of the aflatoxin metabolites (relative risk 2.4, 95% confidence interval 1.0-5.9). The highest relative risk was for aflatoxin P1 (6.2, 1.8-21.5). In an analysis adjusting for the effects of hepatitis B surface antigen seropositivity, level of education, cigarette smoking, and alcohol consumption, the relative risk for the presence of aflatoxin metabolites was 3.8 (1.2-12.2). There was a strong interaction between serological markers of chronic hepatitis B infection and aflatoxin exposure in liver-cancer risk. Reduction of aflatoxin exposure may be a useful intermediate goal in prevention of liver cancer, since the benefits of wide-scale hepatitis B vaccination will not be apparent for many years.

Aflatoxin: A 50-Year Odyssey of Mechanistic and Translational Toxicology

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.

Nitric oxide in cancer and chemoprevention.

Nitric oxide (NO) is a key molecule involved in many physiological functions. However, evidence is accumulating that sustained high levels of NO over extended periods of time contribute to carcinogenesis. This article reviews recent data and outlines a dual role of NO in animal carcinogenesis. Following an inhibition of NO production, some studies find a protection, while others find an exacerbation of tumorigenesis. These studies reflect the importance of (i). choosing the appropriate compound for NO inhibition; and (ii). genetic background, target tissue, levels of NO, and surrounding free radicals in the overall affects of NO on the tumor growth. These findings highlight the importance of further study of the use of NO inhibitors to inhibit human carcinogenesis.

The rational design of nitric oxide selectivity in single-walled carbon nanotube near infrared fluorescence sensors for biological detection

A major challenge in the synthesis of nanotube or nanowire sensors is to impart selective analyte binding through means other than covalent linkages, which compromise electronic and optical properties. We synthesized a 3,4-diaminophenyl-functionalized dextran (DAP-dex) wrapping for single-walled carbon nanotubes (SWNTs) that imparts rapid and selective fluorescence detection of nitric oxide (NO), a messenger for biological signalling. The near-infrared (nIR) fluorescence of SWNT(DAP-dex) is immediately and directly bleached by NO, but not by other reactive nitrogen and oxygen species. This bleaching is reversible and shown to be caused by electron transfer from the top of the valence band of the SWNT to the lowest unoccupied molecular orbital of NO. The resulting optical sensor is capable of real-time and spatially resolved detection of NO produced by stimulating NO synthase in macrophage cells. We also demonstrate the potential of the optical sensor for in vivo detection of NO in a mouse model.

Carcinogenic effects of low dietary levels of aflatoxin B1 in rats.

Abstract Aflatoxin B1 was added to a semi-synthetic diet at levels of 1, 5, 15, 50 and 100 μg/kg (ppb). These diets were fed to male Fischer rats, beginning when their body weights were 80 g and continuing for up to 105 wk. A control group received the same diet without aflatoxin. Hepatocellular carcinomas were induced in a high incidence by levels of 50 and 100 ppb, and in a lower incidence by the other dietary levels. Tumours were induced in two of 22 animals fed 1 ppb, and pathological lesions were present in the livers of seven of the remaining survivors. These results suggest that, under these experimental conditions, the sensitivity of the Fischer rat to aflatoxin carcinogenesis is approximately the same as that previously reported for the rainbow trout.

Aflatoxin B1: Binding to DNA in vitro and Alteration of RNA Metabolism in vivo

Aflatoxin B1 binds to both native and denatured DNA, as shown by spectroscopy and equilibrium dialysis. It also strongly inhibits incorporation of cytidine into rat liver nuclear RNA and lowers the RNA content of the nucleus. The extremle toxicity and carcinogenicity of aflatoxin B1 may be direct results of the affinity of this agent for DNA.

Infection-induced colitis in mice causes dynamic and tissue-specific changes in stress response and DNA damage leading to colon cancer

Helicobacter hepaticus-infected Rag2-/- 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.

In vivo biosensing via tissue-localizable near-infrared-fluorescent single-walled carbon nanotubes

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.

Nitric oxide and TNF-alpha trigger colonic inflammation and carcinogenesis in Helicobacter hepaticus-infected, Rag2-deficient mice.

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-α (TNF-α) expression in colon were required for increased iNOS expression and cancer, whereas interleukin-10 (IL-10) down-regulated TNF-α 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-α expression, and elevated NO production in colon carcinogenesis.

Reactive nitrogen species regulate autophagy through ATM-AMPK-TSC2-mediated suppression of mTORC1.

Significance NO exposure triggered an ATM-mediated damage response in breast cancer cells involving activation of the LKB1 and TSC2 tumor suppressors, repression of mTORC1, ULK phosphorylation, and increased autophagic flux. The associated loss of cell viability indicates that autophagy can function as a cytotoxic response to nitrosative stress in tumor cells. Collectively, the data identify a nitrosative-stress signaling pathway that regulates autophagy. A more comprehensive understanding of signaling pathways regulating autophagy holds promise for developing new therapeutic approaches compromising prosurvival autophagic pathways that enable tumor cells to evade therapy, or promoting prodeath autophagic pathways that kill cancer cells. 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 lysosome-dependent 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.