Deuterium Magnetic Resonance Imaging Using Deuterated Water-Induced 2H-Tissue Labeling Allows Monitoring Cancer Treatment at Clinical Field Strength.

PURPOSE: An accurate and noninvasive assessment of tumor response following treatment other than traditional anatomical imaging techniques is essential. Deuterium magnetic resonance spectroscopic (MRS) imaging has been demonstrated as an alternative for cancer metabolic imaging by high-field MRI using deuterium-labeled molecules. The study aim was to use 2H tissue labeling and deuterium MRI at clinical field strength for tumor visualization and assessment of three anticancer therapies in pancreatic cancer model mice. EXPERIMENTAL DESIGN: MIA PaCa-2 pancreatic carcinoma and C26 colorectal carcinoma models of BALB/c-nu mice was prepared, and repeated deuterium MRI was performed during the first 10 days of free drinking of 30% D2O to track 2H distribution in tissues. 2H accumulation in the tumor after irradiation, bevacizumab administration, or gemcitabine administration was also measured in MIA PaCa-2-bearing mice. Confirmatory proton MRI, ex vivo metabolic hyperpolarization 13C-MRS, and histopathology were performed. RESULTS: The mouse's whole-body distribution of 2H was visible 1 day after drinking, and the signal intensity increased daily. Although the tumor size did not change 1 and 3 days after irradiation, the amount of 2H decreased significantly. The 2H image intensity of the tumor also significantly decreased after the administration of bevacizumab or gemcitabine. Metabolic hyperpolarization 13C-MRS, proton MRI, and 2H-NMR spectroscopy confirmed the efficacy of the anticancer treatments. CONCLUSIONS: Deuterium MRI at 1.5T proved feasible to track 2H distribution throughout mouse tissues during D2O administration and revealed a higher 2H accumulation in the tumor xenografts. This research demonstrated a promising successful method for preliminary assessment of radiotherapy and chemotherapy of cancer.

Spatiotemporal imaging of redox status using in vivo dynamic nuclear polarization magnetic resonance imaging system for early monitoring of response to radiation treatment of tumor.

In general, the effectiveness of radiation treatment is evaluated through the observation of morphological changes with computed tomography (CT) or magnetic resonance imaging (MRI) images after treatment. However, the evaluation of the treatment effects can be very time consuming, and thus can delay the verification of patient cases where treatment has not been fully effective. It is known that the treatment efficacy depends on redox modulation in tumor tissues, which is an indirect effect of oxidizing redox molecules such as hydroxyl radicals and of reactive oxygen species generated by radiation treatment. In vivo dynamic nuclear polarization-MRI (DNP-MRI) using carbamoyl-PROXYL (CmP) as a redox sensitive DNP probe enables the accurate monitoring of the anatomical distribution of free radicals based on interactions of electrons and nuclear spin, known as Overhauser effect. However, spatiotemporal response of the redox status in tumor tissues post-irradiation remains unknown. In this study, we demonstrate the usefulness of spatiotemporal redox status as an early imaging biomarker of tumor response after irradiation using in vivo DNP-MRI. Our results highlight that in vivo DNP-MRI/CmP allowed us to visualize the tumor redox status responses significantly faster and earlier compared to the verification of morphological changes observed with 1.5 T MRI and cancer metabolism (Warburg effect) obtained by hyperpolarized 13C pyruvate MRS. Our findings suggest that the early assessment of redox status alterations with in vivo DNP-MRI/CmP probe may provide very efficient information regarding the effectiveness of the subsequent radiation treatment.

In Vivo Dynamic Nuclear Polarization Magnetic Resonance Imaging for the Evaluation of Redox-Related Diseases and Theranostics.

Significance:In vivo molecular and metabolic imaging is an emerging field in biomedical research that aims to perform noninvasive detection of tissue metabolism in disease states and responses to therapeutic agents. The imbalance in tissue oxidation/reduction (Redox) states is related to the onset and progression of several diseases. Tissue redox metabolism provides biomarkers for early diagnosis and drug treatments. Thus, noninvasive imaging of redox metabolism could be a useful, novel diagnostic tool for diagnosis of redox-related disease and drug discovery. Recent Advances:In vivo dynamic nuclear polarization magnetic resonance imaging (DNP-MRI) is a technique that enables the imaging of free radicals in living animals. DNP enhances the MRI signal by irradiating the target tissue or solution with the free radical molecule's electron paramagnetic resonance frequency before executing pulse sequence of the MRI. In vivo DNP-MRI with redox-sensitive nitroxyl radicals as the DNP redox contrast agent enables the imaging of the redox metabolism on various diseases. Moreover, nitroxyl radicals show antioxidant effects that suppress oxidative stress. Critical Issues: To date, considerable progress has been documented preclinically in the development of animal imaging systems. Here, we review redox imaging of in vivo DNP-MRI with a focus on the recent progress of this system and its uses in patients with redox-related diseases. Future Directions: This technique could have broad applications in the study of other redox-related diseases, such as cancer, inflammation, and neurological disorders, and facilitate the evaluation of treatment response as a theranostic tool. Antioxid. Redox Signal. 36, 172-184.

Formation of biogenic amines in fish: Dietary intakes and health risk assessment.

Biogenic amines (BAs) are toxicants that are produced during the proteolytic activities of some microorganisms, or naturally during the metabolism of their precursor amino acids. The objective of this study was to estimate the formed BAs in six types of fish retailed in Egypt including tilapia, mullet, mackerel, sardine, herring, and tuna. In addition, total mesophilic (TMC) and total psychrophilic (TPsC) bacterial counts were investigated. Furthermore, the estimated daily intakes (EDI) of BAs via the ingestion of various types of fish in Egypt were calculated, and their potential health risks were discussed. The achieved results indicated the formation of histamine (HIS), tyramine (TYR), cadaverine (CAD), putrescine, spermine, and spermidine at different concentrations. Tilapia had the lowest concentration levels for the different BAs. In contrast, mackerel and tuna had the highest concentrations of BAs. Total biogenic amines (TBAs) showed significant positive correlations with TMC in the examined fish species. The recorded EDI values of the different BAs in the current study would not have adverse effects, except for mackerel and tuna. Excessive consumption of fish contaminated with BA might have serious health hazards such as symptoms of histamine poisoning, including rashes, flushing, palpitations, and asthma. Therefore, the adoption of strict hygienic measures during the processing, storage, and distribution of fish is highly recommended to reduce the formation of BAs in fish.

Development of 20 cm sample bore size dynamic nuclear polarization (DNP)-MRI at 16 mT and redox metabolic imaging of acute hepatitis rat model.

Tissue redox metabolism is involved in various diseases, and an understanding of the spatio-temporal dynamics of tissue redox metabolism could be useful for diagnosis of progression and treatment. In in vivo dynamic nuclear polarization (DNP)-MRI, electron paramagnetic resonance (EPR) irradiation at the resonance frequency of nitroxyl radicals administered as a redox probe for induction of DNP, increases the intensity of MRI signals. For electron spin, it is necessary to apply a resonant frequency 658 times higher than that required for nuclear spin because of the higher magnetic moment of unpaired electrons. Previous studies using a disease model of small animals and in vivo DNP-MRI have revealed that an abnormal redox status is involved in many diseases, and that it could be used to visualize the dynamics of alterations in redox metabolism. To use the current methods in clinical practice, the development of a prototype DNP-MRI system for preclinical examinations of large animals is indispensable for clarifying the problems peculiar to the increase in size of the DNP-MRI device. Therefore, we developed a in vivo DNP-MRI system with a sample bore size of 20 cm and a 16-mT magnetic field using a U-shaped permanent magnet. Because the NMR frequency is very low, we adopted a digital radiofrequency transmission/reception system with excellent filter and dynamic range characteristics and equipped with a digital eddy current compensation system to suppress large eddy currents. The pulse sequence was based on the fast spin-echo sequence, which was improved for low frequency and large-eddy current equipment. The in vivo DNP-MRI system developed was used to non-invasively image the redox reaction of a carbamoyl-PROXYL probe in the livers of large rats weighing 800 g. Furthermore, DNP-MRI analysis was able to capture significant changes in redox metabolism in hepatitis-model rats.

Evaluation of enzymatic and magnetic properties of γ-glutamyl-[1-13C]glycine and its deuteration toward longer retention of the hyperpolarized state.

Dynamic nuclear polarization (DNP) is an emerging cutting-edge method of acquiring metabolic and physiological information in vivo. We recently developed γ-glutamyl-[1-13C]glycine (γ-Glu-[1-13C]Gly) as a DNP nuclear magnetic resonance (NMR) molecular probe to detect γ-glutamyl transpeptidase (GGT) activity in vivo. However, the detailed enzymatic and magnetic properties of this probe remain unknown. Here, we evaluate a γ-Glu-Gly scaffold and develop a deuterated probe, γ-Glu-[1-13C]Gly-d 2, that can realize a longer lifetime of the hyperpolarized signal. We initially evaluated the GGT-mediated enzymatic conversion of γ-Glu-Gly and the magnetic properties of 13C-enriched γ-Glu-Gly (γ-Glu-[1-13C]Gly and γ-[5-13C]Glu-Gly) to support the validity of γ-Glu-[1-13C]Gly as a DNP NMR molecular probe for GGT. We then examined the spin-lattice relaxation time (T 1) of γ-Glu-[1-13C]Gly and γ-Glu-[1-13C]Gly-d 2 under various conditions (D2O, PBS, and serum) and confirmed that the T 1 of γ-Glu-[1-13C]Gly and γ-Glu-[1-13C]Gly-d 2 was maintained for 30 s (9.4 T) and 41 s (9.4 T), respectively, even in serum. Relaxation analysis of γ-Glu-[1-13C]Gly revealed a significant contribution of the dipole-dipole interaction and the chemical shift anisotropy relaxation pathway (71% of the total relaxation rate at 9.4 T), indicating the potential of deuteration and the use of a lower magnetic field for realizing a longer T 1. In fact, by using γ-Glu-[1-13C]Gly-d 2 as a DNP probe, we achieved longer retention of the hyperpolarized signal at 1.4 T.

Free radical imaging of endogenous redox molecules using dynamic nuclear polarization magnetic resonance imaging.

Redox reactions accompanied by the oxidation-reduction of endogenous molecules play important roles in maintaining homeostasis in living organisms. In humans, numerous endogenous molecules that contribute toward maintaining physiological conditions form free radicals via electron transfer. A typical example of this is the mitochondrial electron transport chain, which is involved in energy production. If free radicals derived from endogenous molecules could be visualized and exploited as biological and functional probes, redox reactions mediated by endogenous molecules could be detected non-invasively. We succeeded in visualizing the free radicals derived from endogenous molecules using an in vivo dynamic nuclear polarization (DNP) magnetic resonance imaging (MRI) system. In this review, we describe the visualization of endogenous redox molecules, such as flavins and ubiquinones, which are mitochondrial electron carriers, as well as vitamin E and vitamin C (ascorbate). In addition, we describe the application of melanin free radicals for the in vivo visualization of metabola without using probes via in vivo DNP-MRI.

Effects of Pyrene on Human Liver HepG2 Cells: Cytotoxicity, Oxidative Stress, and Transcriptomic Changes in Xenobiotic Metabolizing Enzymes and Inflammatory Markers with Protection Trial Using Lycopene.

Pyrene is one of the major polycyclic aromatic hydrocarbons formed during heat treatment of meat and in car exhausts; however, few studies have investigated pyrene-induced adverse effects on human cell lines. This study aimed at the investigation of pyrene-induced cytotoxicity and oxidative damage in human liver HepG2 cells at environmentally relevant concentrations. Pyrene-induced changes in mRNA expression of xenobiotic metabolizing enzymes (XMEs), xenobiotic transporters, antioxidant enzymes, and inflammatory markers were investigated using real-time PCR. As a protection trial, the ameliorative effects of lycopene, a carotenoid abundantly found in tomato, were investigated. The possible mechanisms behind such effects were examined via studying the co exposure effects of pyrene and lycopene on regulatory elements including the aryl hydrocarbon receptor (Air) and elytroid 2-related factor 2 (RF). The achieved results indicated that pyrene caused significant cytotoxicity at 50 n, with a clear production of reactive oxygen species (ROS) in a dose-dependent manner. Pyrene upregulated mRNA expression of phase I enzymes including CYP1A1, 1A2, and CYP1B1 and inflammatory markers including TNFα and Cox2. However, pyrene significantly downregulated phase II enzymes, xenobiotic transporters, and antioxidant enzymes. Interestingly, lycopene significantly reduced pyrene-induced cytotoxicity and ROS production. Moreover, lycopene upregulated detoxification and antioxidant enzymes, probably via its regulatory effects on Air- and RF-dependent pathways.

A designer molecular chaperone against transmissible spongiform encephalopathy slows disease progression in mice and macaques.

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases that lack therapeutic solutions. Here, we show that the molecular chaperone (N,N'-([cyclohexylmethylene]di-4,1-phenylene)bis(2-[1-pyrrolidinyl]acetamide)), designed via docking simulations, molecular dynamics simulations and quantum chemical calculations, slows down the progress of TSEs. In vitro, the designer molecular chaperone stabilizes the normal cellular prion protein, eradicates prions in infected cells, prevents the formation of drug-resistant strains and directly inhibits the interaction between prions and abnormal aggregates, as shown via real-time quaking-induced conversion and in vitro conversion NMR. Weekly intraperitoneal injection of the chaperone in prion-infected mice prolonged their survival, and weekly intravenous administration of the compound in macaques infected with bovine TSE slowed down the development of neurological and psychological symptoms and reduced the concentration of disease-associated biomarkers in the animals' cerebrospinal fluid. The de novo rational design of chaperone compounds could lead to therapeutics that can bind to different prion protein strains to ameliorate the pathology of TSEs.

Estimation of cadmium content in Egyptian foodstuffs: health risk assessment, biological responses of human HepG2 cells to food-relevant concentrations of cadmium, and protection trials using rosmarinic and ascorbic acids.

Cadmium (Cd) is an environmental pollutant that can get entry into human body via ingestion of contaminated foods causing multiple organ damage. This study aimed at monitoring Cd residues in 20 foodstuffs of animal origin that are commonly consumed in Egypt. Health risk assessment was conducted via calculation of Cd dietary intakes and non-carcinogenic target hazard quotient. An in vitro approach was performed to investigate the constitutive effects of Cd on human hepatoma (HepG2) cells under food-relevant concentrations. Trials to reduce Cd-induced adverse effects on HepG2 cells were done using rosmarinic (RMA) and ascorbic acids (ASA). The achieved results indicated contamination of the tested foodstuffs with Cd at high levels with potential human health hazards. Cd at food-relevant concentrations caused significant cytotoxicity to HepG2 cells. This may be attributed to induction of oxidative stress and inflammation, as indicated by the overexpression of stress and inflammatory markers. At the same time, Cd downregulated xenobiotic transporters and upregulated the proliferation factors. Co-exposure of HepG2 cells to Cd and micronutrients such as RMA and ASA led to recovery of cells from the oxidative damage, and subsequently cell viability was strongly improved. RMA and ASA ameliorated the biological responses of HepG2 cells to Cd exposure.

Determination of polycyclic aromatic hydrocarbon content in heat-treated meat retailed in Egypt: Health risk assessment, benzo[a]pyrene induced mutagenicity and oxidative stress in human colon (CaCo-2) cells and protection using rosmarinic and ascorbic acids.

This study was undertaken to estimate the concentrations of the formed polycyclic aromatic hydrocarbons (PAHs) in heat-treated (boiled, pan-fried and grilled) meats collected from Egypt. Dietary intakes and cancer risks of PAHs among Egyptian adults were calculated. Benzo[a]pyrene (B[a]P)-induced mutagenicity and oxidative stress in human colon (CaCo-2) cell line and mechanisms behind such effects were also investigated. Finally, protection trials using rosmarinic (RMA) and ascorbic acids (ASA) were carried out. The results indicated formation of PAHs at high levels in the heat-treated meats. Calculated incremental life time cancer risk among Egyptian adults were 7.05179E-07, 7.00604 E-06 and 1.86069 E-05 due to ingestion of boiled, pan-fried and grilled meats, respectively. B[a]P-exposed CaCo-2 cells had high abilities for mutagenicity (490.05 ±â€¯21.37 His + revertants) and production of reactive oxygen species. RMA and ASA protected CaCo-2 cells via reduction of B[a]P-induced mutagenicity and oxidative stress and upregulation of phase II detoxification enzymes and xenobiotic transporters.

Poly-L-histidine inhibits prion propagation in a prion-infected cell line.

Transmissible spongiform encephalopathies (TSEs) are a group of lethal neurodegenerative diseases involving the structural conversion of cellular prion protein (PrPC) into the pathogenic isoform (PrPSc) for which no effective treatment is currently available. Previous studies have implicated that a polymeric molecule with a repeating unit, such as pentosane polysulfate and polyamidoamide dendrimers, exhibits a potent anti-prion activity, suggesting that poly-(amino acid)s could be a candidate molecule for inhibiting prion propagation. Here, by screening a series of poly-(amino acid)s in a prion-infected neuroblastoma cell line (GTFK), we identified poly-L-His as a novel anti-prion compound with an IC50 value of 1.8 µg/mL (0.18 µM). This potent anti-prion activity was specific to a high-molecular-weight poly-L-His and absent in monomeric histidine or low-molecular-weight poly-L-His. Solution NMR data indicated that poly-L-His directly binds to the loop region connecting Helix 2 and Helix 3 of PrPC and sterically blocks the structural conversion toward PrPSc. Poly-L-His, however, did not inhibit prion propagation in a prion-infected mouse when administered intraperitoneally, suggesting that the penetration of blood-brain barrier and/or the chemical stability of this polypeptide must be addressed before its application in vivo. Taken together, this study revealed the potential use of poly-L-His as a novel treatment against TSEs. (203 words).

Acceleration of nucleation of prion protein during continuous ultrasonication.

Although pulsatile irradiation of ultrasonication is frequently used for generating amyloid fibrils in vitro, the potential for inducing amyloid fibrillation of proteins during continuous ultrasonication is unknown. In this study, we implemented a continuous irradiation system and measured far-ultraviolet circular dichroism in a real-time manner. During the continuous ultrasonication, the conformation of full-length mouse prion protein (mPrP) was rapidly altered without a lag time and electron microscopy revealed that distorted fibrils, ß-oligomers and amorphous aggregates were formed at pH 2.2, 4.0 and 9.1, respectively. Similarly, hen egg white lysozyme formed distorted fibrils and small and large amorphous aggregates at pH 2.2 and 7.1 and 11.9, respectively, without a lag time. The concentration dependencies of the initial rates were different between the two systems. The aggregate formation of mPrP followed a first-order reaction, whereas that of lysozyme followed the zeroth-order reaction. Importantly, the reactions were immediately stopped by switching off ultrasonication, and restarted instantaneously when ultrasonication was restarted. Thus, the continuous ultrasonication significantly accelerates the nucleations of mPrP and lysozyme aggregates by the interaction between monomer and cavitation bubble. These cavitation bubbles may act as catalysts that decrease the activation free energy for nucleation, which is low in mPrP and high in lysozyme.

Logical design of an anti-cancer agent targeting the plant homeodomain in Pygopus2.

Pygopus2 (Pygo2) is a component of the Wnt signaling pathway, which is required for ß-catenin mediated transcription. Plant homeodomain (PHD) finger in Pygo2 intercalates the methylated histone 3 (H3K4me) tail and HD1 domain of BCL9 that binds to ß-catenin. Thus, PHD finger may be a potential target for the logical design of an anti-cancer drug. Here, we found that Spiro[2H-naphthol[1,2-b]pyran-2,4'-piperidine]-1'ethanol,3,4-dihydro-4-hydroxy-α-(6-methyl-1H-indol-3-yl)) termed JBC117 interacts with D339, A348, R356, V376 and A378 in PHD corresponding to the binding sites with H3K4me and/or HD1, and has strong anti-cancer effects. For colon (HCT116) and lung (A549) cancer cell lines, IC50 values were 2.6 ± 0.16 and 3.3 ± 0.14 µM, respectively, while 33.80 ± 0.15 µM for the normal human fibroblast cells. JBC117 potently antagonized the cellular effects of ß-catenin-dependent activity and also inhibited the migration and invasion of cancer cells. In vivo studies showed that the survival time of mice was significantly prolonged by the subcutaneous injection of JBC117 (10 mg/kg/day). In conclusion, JBC117 is a novel anti-cancer lead compound targeting the PHD finger of Pygo2 and has a therapeutic effect against colon and lung cancer.

Characterization of early transient accumulation of PrP(Sc) in immune cells.

PrP(Sc) is known to elicit no specific immune response and the immune cells are suspected to support its accumulation. In the present study, we investigated the response of some immune cell types to PrP(Sc) to characterize an observed early transient accumulation of PrP(Sc). After cells were treated with PrP(Sc)-brain homogenate, PrP(Sc) was transiently accumulated for the first 8-12h post-exposure then completely cleared by the 5th day of the experiment. The accumulated PrP(Sc) was not a de novo product of the cell PrP(C). Further investigation of this phenomenon revealed some potential factors influencing it. These factors included cholesterol homeostasis, temperature, the degradation power of the cell and the availability of sufficient PrP(C). Our in vitro results suggest that immune cells, especially macrophages are potential risk factors for the accumulation and intercellular spread of PrP(Sc) if the complete clearance of PrP(Sc) were not fulfilled.

Virulence characteristics of Yersinia pseudotuberculosis isolated from breeding monkeys in Japan.

Between April 2001 and 2007, 18 Yersinia pseudotuberculosis outbreaks occurred in breeding monkeys at 12 zoological gardens in Japan, and 28 monkeys of 8 species died. A total of 18 Y. pseudotuberculosis strains from the dead monkeys, comprising one strain per outbreak, were examined for serotype and the presence of the virulence genes virF, inv, ypm (ypmA, ypmB and ypmC) and irp2. Of the 18 Y. pseudotuberculosis strains, 7 (38.9%) were serotype 4b, 7 (38.9%) were serotype 1b, and there was one each of serotypes 2b, 3, 6 and 7. All the 18 strains examined harbored virF and inv. Sixteen (88.9%) strains, including the strain of serotype 7, harbored ypmA. However, no strain harbored ypmB, ypmC and irp2. This study demonstrated that among other pathogenic factors, almost all the Y. pseudotuberculosis isolated from the outbreaks had the ypm gene encoding the superantigenic toxin, YPM. As most of the monkeys who died in those outbreaks originated from South America and other regions, where the presence of the ypm gene have not been reported, YPM might be the cause, or at least the most important factor for, the high mortality of the breeding monkeys infected by Y. pseudotuberculosis in Japan. This is also the first report of a fatal case due to Y. pseudotuberculosis serotype 7 infection in the world.