In Vivo Delivery of Nitric Oxide-Sensing, Single-Walled Carbon Nanotubes

Detection of nitric oxide (NO) in vivo by single-walled carbon nanotubes (SWNT) is based on the fluorescent properties of SWNT and the ability of NO to quench the fluorescence signal. Alterations of the signal can be utilized to detect a small molecule in vivo that has not previously been possible by other assay techniques. The protocols described here explain the techniques used to prepare NO-detecting SWNTs and to administer them to mice by both intravenous and subcutaneous routes. These techniques can also be utilized with other SWNT sensors as well as non-SWNT sensorNational Institutes of Health (T32 Training Grant in Environmental Toxicology ES007020

Infection, inflammation and colon carcinogenesis

The importance of chronic inflammation as a risk factor for major cancers is well documented [1], and the inflammatory state is known to involve contributions of both adaptive and innate immune components. In a recent publication [2] we describe an experimental animal model in which infection, inflammation and cancer are mechanistically linked, and provide evidence that chemical mediators of the innate immune system and bacterial toxins both play key roles in driving colon carcinogenesis. In this model, epithelial injury caused by Helicobacter hepaticus infection enhances access of bacterially-associated products to pattern-recognition receptors located on surfaces of macrophages and dendritic cells. Receptor ligation leads to activation of transcription factors, including NF-kappa B, that regulate production of chemo-attractants for macrophages and neutrophils, recruitment of which is a hallmark of inflammation. These acute inflammatory events are re-enforced by expression of powerful inflammatory mediators such as TNF-α and IL-2, which amplify acute inflammatory gene expression and enhance cell survival. If not properly extinguished, the innate inflammatory response is maintained and further amplified by activation of cell-mediated adaptive immunity

Chemistry meets biology in colitis-associated carcinogenesis

The intestine comprises an exceptional venue for a dynamic and complex interplay of numerous chemical and biological processes. Here, multiple chemical and biological systems, including the intestinal tissue itself, its associated immune system, the gut microbiota, xenobiotics, and metabolites meet and interact to form a sophisticated and tightly regulated state of tissue homoeostasis. Disturbance of this homeostasis can cause inflammatory bowel disease (IBD)—a chronic disease of multifactorial etiology that is strongly associated with increased risk for cancer development. This review addresses recent developments in research into chemical and biological mechanisms underlying the etiology of inflammation-induced colon cancer. Beginning with a general overview of reactive chemical species generated during colonic inflammation, the mechanistic interplay between chemical and biological mediators of inflammation, the role of genetic toxicology, and microbial pathogenesis in disease development are discussed. When possible, we systematically compare evidence from studies utilizing human IBD patients with experimental investigations in mice. The comparison reveals that many strong pathological and mechanistic correlates exist between mouse models of colitis-associated cancer, and the clinically relevant situation in humans. We also summarize several emerging issues in the field, such as the carcinogenic potential of novel inflammation-related DNA adducts and genotoxic microbial factors, the systemic dimension of inflammation-induced genotoxicity, and the complex role of genome maintenance mechanisms during these processes. Taken together, current evidence points to the induction of genetic and epigenetic alterations by chemical and biological inflammatory stimuli ultimately leading to cancer formation.Massachusetts Institute of Technology. Center for Environmental Health Sciences (ES002109)National Institutes of Health (U.S.) (NIH (CA26731)

Monocyclic aromatic amines as potential human carcinogens: old is new again

Alkylanilines are a group of chemicals whose ubiquitous presence in the environment is a result of the multitude of sources from which they originate. Exposure assessments indicate that most individuals experience lifelong exposure to these compounds. Many alkylanilines have biological activity similar to that of the carcinogenic multi-ring aromatic amines. This review provides an overview of human exposure and biological effects. It also describes recent investigations into the biochemical mechanisms of action that lead to the assessment that they are most probably more complex than those of the more extensively investigated multi-ring aromatic amines. Not only is nitrenium ion chemistry implicated in DNA damage by alkylanilines but also reactions involving quinone imines and perhaps reactive oxygen species. Recent results described here indicate that alkylanilines can be potent genotoxins for cultured mammalian cells when activated by exogenous or endogenous phase I and phase II xenobiotic-metabolizing enzymes. The nature of specific DNA damage products responsible for mutagenicity remains to be identified but evidence to date supports mechanisms of activation through obligatory N-hydroxylation as well as subsequent conjugation by sulfation and/or acetylation. A fuller understanding of the mechanisms of alkylaniline genotoxicity is expected to provide important insights into the environmental and genetic origins of one or more human cancers and may reveal a substantial role for this group of compounds as potential human chemical carcinogens.National Institute of Environmental Health Sciences (PO1-ES006052)National Institute of Environmental Health Sciences (P30-ES002109

A system for exposing molecules and cells to biologically relevant and accurately controlled steady-state concentrations of nitric oxide and oxygen

Nitric oxide (NO) plays key roles in cell signaling and physiology, with diverse functions mediated by NO concentrations varying over three orders-of-magnitude. In spite of this critical concentration dependence, current approaches to NO delivery in vitro result in biologically irrelevant and poorly controlled levels, with hyperoxic conditions imposed by ambient air. To solve these problems, we developed a system for controlled delivery of NO and O[subscript 2] over large concentration ranges to mimic biological conditions. Here we describe the fabrication, operation and calibration of the delivery system. We then describe applications for delivery of NO and O[subscript 2] into cell culture media, with a comparison of experimental results and predictions from mass transfer models that predict the steady-state levels of various NO-derived reactive species. We also determined that components of culture media do not affect the steady-state levels of NO or O[subscript 2] in the device. This system provides critical control of NO delivery for in vitro models of NO biology and chemistry.National Cancer Institute (U.S.) (CA026731)National Cancer Institute (U.S.) (CA116318)National Institute of Environmental Health Sciences (ES002109

Quantitative Tissue Spectroscopy of Near Infrared Fluorescent Nanosensor Implants

Implantable, near infrared (nIR) fluorescent nanosensors are advantageous for in vivo monitoring of biological analytes since they can be rendered selective for particular target molecule while utilizing their unique optical properties and the nIR tissue transparency window for information transfer without an internal power source or telemetry. However, basic questions remain regarding the optimal encapsulation platform, geometrical properties, and concentration ranges required for effective signal to noise ratio through biological tissue. In this work, we systematically explore these variables quantitatively to optimize the performance of such optical nanosensors for biomedical applications. We investigate both alginate and polyethylene glycol (PEG) as model hydrogel systems, encapsulating d(GT)[subscript 15] ssDNA-wrapped single walled carbon nanotubes (SWNT) as model fluorescent nanoparticle sensors, responsive to riboflavin. Hydrogel sensors implanted 0.5 mm into thick tissue samples cause 50% reduction of initial fluorescence intensity, allowing an optical detection limit of 5.4 mm and 5.1 mm depth in tissue for alginate and PEG gels, respectively, at a SWNT concentration of 10 mg L−1, and 785 nm laser excitation of 80 mW and 30 s exposure. These findings are supported with in vivo nIR fluorescent imaging of SWNT hydrogels implanted subcutaneously in mice. For the case of SWNT, we find that the alginate system is preferable in terms of emission intensity, sensor response, rheological properties, and shelf life.National Institutes of Health (U.S.) (T32 Training Grant in Environmental Toxicology ES007020)National Cancer Institute (U.S.) (Grant P01 CA26731)National Institute of Environmental Health Sciences (Grant P30 ES002109)Arnold and Mabel Beckman Foundation (Young Investigator Award)National Science Foundation (U.S.) (Presidential Early Career Award for Scientists and Engineers)MIT-Technion FellowshipSamsung Scholarship FoundationSanofi Aventis (Firm) (Biomedical Innovation Grant

Sulforaphane, a cancer chemopreventive agent, induces pathways associated with membrane biosynthesis in response to tissue damage by aflatoxin B1

Aflatoxin B[subscript 1] (AFB[subscript 1]) is one of the major risk factors for liver cancer globally. A recent study showed that sulforaphane (SF), a potent inducer of phase II enzymes that occurs naturally in widely consumed vegetables, effectively induces hepatic glutathione S-transferases (GSTs) and reduces levels of hepatic AFB[subscript 1]-DNA adducts in AFB[subscript 1]-exposed Sprague Dawley rats. The present study characterized the effects of SF pre-treatment on global gene expression in the livers of similarly treated male rats. Combined treatment with AFB[subscript 1] and SF caused reprogramming of a network of genes involved in signal transduction and transcription. Changes in gene regulation were observable 4 h after AFB[subscript 1] administration in SF-pretreated animals and may reflect regeneration of cells in the wake of AFB[subscript 1]-induced hepatotoxicity. At 24 h after AFB[subscript 1] administration, significant induction of genes that play roles in cellular lipid metabolism and acetyl-CoA biosynthesis was detected in SF-pretreated AFB[subscript 1]-dosed rats. Induction of this group of genes may indicate a metabolic shift toward glycolysis and fatty acid synthesis to generate and maintain pools of intermediate molecules required for tissue repair, cell growth and compensatory hepatic cell proliferation. Collectively, gene expression data from this study provide insights into molecular mechanisms underlying the protective effects of SF against AFB[subscript 1] hepatotoxicity and hepatocarcinogenicity, in addition to the chemopreventive activity of this compound as a GST inducer.National Institutes of Health (U.S.) (Grants ES016313, P30-ES002109, P01 ES006052, P30 ES003819, and P30 CA006973

Antioksidansi i spojevi selenija inhibiraju toksično djelovanje 3,5-dimetilaminofenola na epitelne bubrežne stanice u ljudi

Exposure to alkyl anilines may lead to bladder cancer, which is the second most frequent cancer of the urogenital tract. 3,5-dimethylaniline is highly used in industry. Studies on its primary metabolite 3,5-dimethylaminophenol (3,5-DMAP) showed that this compound causes oxidative stress, changes antioxidant enzyme activities, and leads to death of different mammalian cells. However, there is no in vitro study to show the direct effects of 3,5-DMAP on human bladder and urothelial cells. Selenocompounds are suggested to decrease oxidative stress caused by some chemicals, and selenium supplementation was shown to reduce the risk of bladder cancer. The main aim of this study was to investigate whether selenocompounds organic selenomethionine (SM, 10 μmol/L) or inorganic sodium selenite (SS, 30 nmol/L) could reduce oxidative stress, DNA damage, and apoptosis in UROtsa cells exposed to 3,5-DMAP. 3,5-DMAP caused a dose-dependent increase in intracellular generation of reactive oxygen species, and its dose of 50 μmol/L caused lipid peroxidation, protein oxidation, and changes in antioxidant enzyme activities in different cellular fractions. The comet assay also showed single-strand DNA breaks induced by the 3,5-DMAP dose of 50 μmol/L, but no changes in double-strand DNA breaks. Apoptosis was also triggered. Both selenocompounds provided partial protection against the cellular toxicity of 3,5-DMAP. Low selenium status along with exposure to alkyl anilines can be a major factor in the development of bladder cancer. More mechanistic studies are needed to specify the role of selenium in bladder cancer.Izloženost alkilnim anilinima može uzrokovati rak mokraćnoga mjehura, koji je drugi po redu po učestalosti raka mokraćnospolnog sustava. 3,5-dimetilanilin često se rabi u industrijskoj proizvodnji, a istraživanja njegova primarnog metabolita, 3,5-dimetilaminofenola (3,5-DMAP), pokazuju da on uzrokuje oksidacijski stres i promjene u aktivnosti antioksidacijskih enzima te u konačnici dovodi do smrti raznih stanica u sisavaca. Dosad, međutim, nije provedeno nijedno istraživanje njegovih izravnih učinaka na epitelne stanice mokraćnoga mjehura i bubrega u ljudi. Za spojeve selenija smatra se da smanjuju oksidacijski stres različitih kemikalija te da dopuna prehrane selenijem smanjuje rizik od raka mokraćnoga mjehura. Primarni je cilj ovoga istraživanja bio utvrditi može li organski spoj selenija selenometionin (SM, 10 μmol/L), odnosno anorganski spoj natrijev selenit (SS, 30 nmol/L], smanjiti oksidacijski stres, oštećenje DNA i apoptozu u UROtsa stanicama izloženima 3,5-dimetilaminofenolu. Jednosatna izloženost stanica 3,5-DMAP-u dovela je do povećanja razina reaktivnih kisikovih spojeva (ROS), lipidne peroksidacije, oksidacije bjelančevina te do promjena u aktivnosti antioksidacijskih enzima u staničnoj citoplazmi i jezgri, ovisno o primijenjenoj dozi. Osim toga, komet-testom su utvrđeni jednolančani, ali ne i dvolančani lomovi DNA. Također, 3,5-DMAP uzrokovao je apoptozu stanica. Oba su spoja selenija pružila djelomičnu zaštitu od njegova toksičnoga djelovanja. Nedostatak selenija pri izloženosti alkilnim anilinskim spojevima stoga može odigrati značajnu ulogu u nastanku raka mokraćnog mjehura. Potrebna su daljnja istraživanja mehanizama djelovanja selenija u njegovu sprječavanju

Mutational spectra of aflatoxin B

Aflatoxin B₁ (AFB₁) and/or hepatitis B and C viruses are risk factors for human hepatocellular carcinoma (HCC). Available evidence supports the interpretation that formation of AFB₁-DNA adducts in hepatocytes seeds a population of mutations, mainly G:C→T:A, and viral processes synergize to accelerate tumorigenesis, perhaps via inflammation. Responding to a need for early-onset evidence predicting disease development, highly accurate duplex sequencing was used to monitor acquisition of high-resolution mutational spectra (HRMS) during the process of hepatocarcinogenesis. Four-day-old male mice were treated with AFB₁ using a regimen that induced HCC within 72 wk. For analysis, livers were separated into tumor and adjacent cellular fractions. HRMS of cells surrounding the tumors revealed predominantly G:C→T:A mutations characteristic of AFB₁ exposure. Importantly, 25% of all mutations were G→T in one trinucleotide context (CGC; the underlined G is the position of the mutation), which is also a hotspot mutation in human liver tumors whose incidence correlates with AFB₁ exposure. The technology proved sufficiently sensitive that the same distinctive spectrum was detected as early as 10 wk after dosing, well before evidence of neoplasia. Additionally, analysis of tumor tissue revealed a more complex pattern than observed in surrounding hepatocytes; tumor HRMS were a composite of the 10-wk spectrum and a more heterogeneous set of mutations that emerged during tumor outgrowth. We propose that the 10-wk HRMS reflects a short-term mutational response to AFB₁, and, as such, is an early detection metric for AFB₁-induced liver cancer in this mouse model that will be a useful tool to reconstruct the molecular etiology of human hepatocarcinogenesis.National Institutes of Health (U.S.) (Grant R01-ES016313)National Institutes of Health (U.S.) (Grant P30-ES002109)National Institutes of Health (U.S.) (Grant T32-ES007020)National Institutes of Health (U.S.) (Grant R01-CA080024

Chemical and cytokine features of innate immunity characterize serum and tissue profiles in inflammatory bowel disease

Inflammatory bowel disease (IBD) arises from inappropriate activation of the mucosal immune system resulting in a state of chronic inflammation with causal links to colon cancer. Helicobacter hepaticus-infected Rag2[superscript −/−] mice emulate many aspects of human IBD, and our recent work using this experimental model highlights the importance of neutrophils in the pathology of colitis. To define molecular mechanisms linking colitis to the identity of disease biomarkers, we performed a translational comparison of protein expression and protein damage products in tissues of mice and human IBD patients. Analysis in inflamed mouse colons identified the neutrophil- and macrophage-derived damage products 3-chlorotyrosine (Cl-Tyr) and 3-nitrotyrosine, both of which increased with disease duration. Analysis also revealed higher Cl-Tyr levels in colon relative to serum in patients with ulcerative colitis and Crohn disease. The DNA chlorination damage product, 5-chloro-2′-deoxycytidine, was quantified in diseased human colon samples and found to be present at levels similar to those in inflamed mouse colons. Multivariate analysis of these markers, together with serum proteins and cytokines, revealed a general signature of activated innate immunity in human IBD. Signatures in ulcerative colitis sera were strongly suggestive of neutrophil activity, and those in Crohn disease and mouse sera were suggestive of both macrophage and neutrophil activity. These data point to innate immunity as a major determinant of serum and tissue profiles and provide insight into IBD disease processes.National Institutes of Health (U.S.) (Grant CA26731)Massachusetts Institute of Technology. Center for Environmental Health Sciences (Grant ES002109))Massachusetts Institute of Technology (Merck Fellowship)German Academic Exchange Service (Fellowship