Neurotoxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in cerebellar granule cells

An environmental pollutant, tetrachloro dibenzo dioxin (TCDD) is known to illicit the cognitive disability and motor dysfunction in the developing brain. TCDD induced effects leading to neurodevelopmental and neurobehavioral deficit may have been defined, however underlying molecular mechanism and possible intracellular targets remain to be elucidated. In this study, we attempted to analyze TCDD-induced neurotoxic effects in the granule cells from cerebellum where certain cognitive abilities and motor function command are known to be excuted. [3H]PDBu, (phorbol 12,13-dibutyrate) binding assay indicated that TCDD induced a dose-dependent increase of total PKC activity and its induction was the aryl hydrocarbon receptor (AhR) dependent and N-methyl-D-aspartate receptor (NMDAR) independent. TCDD also caused the translocation of both PKC-alpha and -epsilon in a dose-dependent manner but associated with different receptors; PKC-alpha via AhR but not PKC-epsilon indicating an isozyme-specific pattern of the induction. Increase of the ROS formation was also observed in the cells treated with TCDD in a dose-dependent and an AhR-dependent manner. The treatment of the cells with the diamino dicyano-bis(2-aminophenylthio) butadiene (U0126, MEK-1/2 inhibitor), dizocilpine maleate (MK-801, non-competitive N-methyl-D-aspartate glutamate receptor antagonist) and vitamin E attenuated the TCDD-induced ROS production indicating that TCDD-induced ROS formation may be associated with activation of ERK-1/2 in the MAP kinase pathway or the NMDA receptor. TCDD also increased [Ca2+]i, which is associated with ROS formation and PKC activation in the cerebellar granule cells. It is suggested that TCDD activates the NMDA receptor, which may induce a sustained increase of [Ca2+]i in neurons followed by the ROS formation. Our findings may contribute to understanding the mechanism of TCDD-related neurotoxicity, thereby improving the health risk assessment of neurotoxic compounds in humans.     

6‐Formylindolo[3,2‐b]carbazole (FICZ) is a Very Minor Photoproduct of Tryptophan at Biologically Relevant Doses of UVB and Simulated Sunlight

Exposure to solar UV is at the origin of numerous photodegradation pathways in biomolecules. Tryptophan is readily modified by UVB radiation into ring‐opened and oxidized photoproducts. One of them, 6‐formylindolo[3,2‐b]carbazole (FICZ), has been extensively studied in the recent years because it very efficiently binds to AhR, a major factor in numerous biologic processes, such as metabolism of xenobiotics. Unfortunately, little information is available on the actual yield of FICZ upon exposure to low and biologically relevant doses of UV radiation. In the present work, we used a sensitive and specific HPLC‐tandem mass spectrometry assay to quantify a series of photoproducts induced by UVB and simulated sunlight (SSL) in solutions of tryptophan. FICZ represented only a minute amount of the photoproducts (0.02 and 0.03%, respectively). Experiments were repeated in culture medium where the yield of FICZ was also found to be very low, even when Trp was added. Last, no FICZ could be detected in cytosolic fractions of cultured cells exposed to SSL. Altogether, the present results show that FICZ is a very minor photoproduct and that it cannot be considered the only endogenous photoproduct responsible for the induction of AhR‐dependent responses in UV‐irradiated cells.     

Signaling pathway for 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced TNF-alpha production in differentiated THP-1 human macrophages

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a prototypic halogenated aromatic hydrocarbon (HAH), is known as one of the most potent toxicants. At least a part of its toxic effects appears to be derived from its ability to induce TNF-alpha production. However, the signaling pathway of TCDD that leads to TNF-alpha expression has not been elucidated. In this study, we investigated the signaling mechanism of TCDD-induced TNF-alpha expression in PMA-differentiated THP-1 macrophages. TCDD induced both mRNA and protein expression of TNF-alpha in a dose- and time-dependent manner. Alpha-naphthoflavone (NF), an aryl hydrocarbon receptor (AhR) inhibitor, prevented the TCDD-induced expression of TNF-alpha at both mRNA and protein levels. Genistein, a protein tyrosine kinase (PTK) inhibitor, and PD153035, an EGFR inhibitor, also blocked the increase of TNF-alpha expression by TCDD, indicating the role of EGFR in TCDD-induced TNF-alpha expression. On the other hand, PP2, a c-Src specific inhibitor, did not affect TCDD-induced TNF-alpha expression. EGFR phosphorylation was detected as early as 5 min after TCDD treatment. TCDD-induced EGFR activation was AhR-dependent since co-treatment with alpha-NF prevented it. ERK was found to be a downstream effector of EGFR activation in the signaling pathway leading to TNF-alpha production after TCDD stimulation. Activation of ERK was observed from 30 min after TCDD treatment. PD98059, an inhibitor of the MEK-ERK pathway, completely prevented the TNF-alpha mRNA and protein expression induced by TCDD, whereas inhibitors of JNK and p38 MAPK had no effect. PD153035, an EGFR inhibitor, as well as alpha-NF significantly reduced ERK phosphorylation, suggesting that ERK activation by TCDD was mediated by both EGFR and AhR. These results indicate that TNF-alpha production by TCDD in differentiated THP-1 macrophages is AhR-dependent and involves activation of EGFR and ERK, but not c-Src, JNK, nor p38 MAPK. A signaling pathway is proposed where TCDD induces sequential activation of AhR, EGFR and ERK, leading to the increased expression of TNF-alpha.     

The Endogenous Tryptophan‐derived Photoproduct 6‐formylindolo[3,2‐b]carbazole (FICZ) is a Nanomolar Photosensitizer that Can be Harnessed for the Photodynamic Elimination of Skin Cancer Cells in Vitro and in Vivo

UV‐chromophores contained in human skin may act as endogenous sensitizers of photooxidative stress and can be employed therapeutically for the photodynamic elimination of malignant cells. Here, we report that 6‐formylindolo[3,2‐b]carbazole (FICZ), a tryptophan‐derived photoproduct and endogenous aryl hydrocarbon receptor agonist, displays activity as a nanomolar sensitizer of photooxidative stress, causing the photodynamic elimination of human melanoma and nonmelanoma skin cancer cells in vitro and in vivo. FICZ is an efficient UVA/Visible photosensitizer having absorbance maximum at 390 nm (ε = 9180 L mol^?1 cm^?1), and fluorescence and singlet oxygen quantum yields of 0.15 and 0.5, respectively, in methanol. In a panel of cultured human squamous cell carcinoma and melanoma skin cancer cells (SCC‐25, HaCaT‐ras II‐4, A375, G361, LOX), photodynamic induction of cell death was elicited by the combined action of solar simulated UVA (6.6 J cm^?2) and FICZ (≥10 nm ), preceded by the induction of oxidative stress as substantiated by MitoSOX Red fluorescence microscopy, comet detection of Fpg‐sensitive oxidative genomic lesions and upregulated stress response gene expression (HMOX1, HSPA1A, HSPA6). In SKH1 “high‐risk” mouse skin, an experimental FICZ/UVA photodynamic treatment regimen blocked the progression of UV‐induced tumorigenesis suggesting feasibility of harnessing FICZ for the photooxidative elimination of malignant cells in vivo.     

Hippocampal metabolomics reveals 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity associated with ageing in Sprague-Dawley rats

Metabolomics, the exponentially developing technique, could provide a systemic mapping in toxicology by directly measuring small molecular metabolites. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was found to be neurotoxic in mammalian animals. In this study, we employed liquid chromatography/quadrupole time-of-flight mass spectrometry for non-targeted analysis of metabolic profiling in hippocampal sample sets of the rats exposed to TCDD. Hippocampal metabolome from different ages of the healthy rats (4-week, 12-week and 20-week) was also deciphered. The relationship between the two tested cases was unlocked to delineate TCDD toxicity associated with ageing. Tandem mass spectrometry fragmentation in conjunction with metabolic database searching and compared to authentic standards was utilized for metabolite identification. As a consequence, the reduced levels of phenylalanine and leucine/isoleucine as well as the up-regulation of inosine and hypoxanthine were highlighted for understanding of TCDD toxicity related to age in rats and the trajectory was depicted by principal components analysis.     

Rosmarinus officinalis extract as eco-friendly corrosion inhibitor for copper in 1 M nitric acid solution: Experimental and theoretical studies

Rosmarinus officinalis extract (ROE) was studied chemically (mass loss, ML), electrochemically impedance spectrometry (EIS), and potentiodynamic polarization (PDP) as a corrosion inhibitor in 1 M nitric acid. According to ML, ROE is effective like a copper preservative in 1 M HNO₃ acid solution at R.T by improving inhibitor concentration up to 77 % at 300 ppm and 25 °C. A study was conducted regarding the effect of temperature on copper adsorption, as well as the calculation of adsorption coefficients. Results indicated that physisorption increases with temperature, indicating a decrease in inhibition efficiency (%IE). Langmuir's adsorption model was consistent with the adsorption mechanism. Using the PDP method, the inhibitor accumulated on the copper surface in mixed forms. Moreover, EIS revealed that the value of double-layer capacitance dropped with an increased dose of ROE, while the charge transfer resistance improved. A different approach was taken to the examination of surfaces. Both theoretical studies and practical results were calculated and compared to demonstrate that the results were valid.     

Recovery efficiency of 2,3,7,8-tetrachlorodibenzo-p-dioxin from active carbon and other particulates

The use of Amoco active carbon (grade PX-21) as a cleanup step for the determination of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in environmental samples was investigated. Benzene/toluene (1:1) removed 95% TCDD from Amoco active carbon dispersed in silica gel. Other solvents, including benzene, carbon disulfide, dichloromethane, acetone, hexane, acetone/hexane (1:1), and diethyl ether, did not remove TCDD from Amoco active carbon. o-Dichlorobenzene is needed to remove TCDD from Pittsburgh active carbon completely. Hot benzene with Soxhlet extraction is adequate for TCDD removal from soil and fly ash particulates. The results derived from this study are consistent with past and present analytical practice for isolating TCDD from different matrices.     

A novel piezoelectric immunosensor for the detection of malarial Plasmodium falciparum histidine rich protein-2 antigen

A novel piezoelectric (PZ) immunosensor for the direct detection of malarial Plasmodium falciparum histidine rich protein-2 (PfHRP-2) antigen was developed. The mixed self-assembled monolayers (SAMs) of thioctic acid and 1-dodecanethiol were formed on gold surface of quartz crystal. Cyclic voltammetry, electrochemical impedance spectroscopy and surface Raman spectroscopy techniques were used to characterize the mixed SAMs. The rabbit anti-PfHRP-2 antibodies were coupled on mixed SAM modified gold surface of quartz crystal via NHS/EDC activation method. The PZ immunosensor was applied to detect PfHRP-2 in the linear range of 15-60 ng/ml with a detection limit of 12 ng/ml. It was also found that even after 14 days of storage, 50% of the activity still remained. Clinical human serum samples were tested with this method, and the results were in agreement with those obtained from commercially available ICT kit (NOW^® Malaria).     

Synthesis and conformational behaviour of lower-rim tetraacetylated thiacalix[4]arenes

The acylation of thiacalix[4]arenes with AcCl or Ac₂O gave the corresponding lower-rim tetraacetoxy derivatives. In contrast to classical calix[4]arenes, tetraacetylated thiacalix[4]arenes are conformationally mobile in solution and represent a thermodynamic equilibrium of three different conformers at room temperature. As proven by a dynamic ¹H NMR study, conformational preferences of acetylated thiacalix[4]arenes considerably depend on the upper-rim substitution. Hence, t-Bu thiacalixarene prefers 1,3-alternate and 1,2-alternate conformations (43% and 38%, respectively), while the upper-rim unsubstituted compound adopts preferably the partial cone conformation (70%).     

Piezoelectric Crystal Liquid Flow Detection Systems Based on Alkyl (C18)‐Benzo‐15‐Crown 5 for Organic Compounds and Metal Ions

A water in soluble long‐chain crown ether alkyl (C18)‐benzo‐15‐crown‐5 was synthesized and applied as a coating material on quartz crystal membranes of a liquid flow piezo electric crystal sensor. The oscillating crown ether‐coated piezo electric (PZ) crystal with a home‐made computer inter face was prepared as a liquid chromato graphic (LC) detector for organic species and metal ions in aqueous solutions. The oscillating frequency of the quartz crystal decreased due to the adsorption of organic molecules or metal ions on crown ether molecules. Effects of functional group, molar mass, steric hindrance, and polarity of organic molecules on frequency responses of the crown ether coated PZ crystal detector were investigated. The frequency responses of the crown ether coated PZ crystal detector for various molecules were in the order: amines > carboxylic acids > alcohols > ketones. The crown ether PZ detector also exhibited good sensitivity for some heavy metal ions and the frequency shifts were in the order: Cr³⁺ » Pb²⁺ > Co²⁺ > Cd²⁺ > Ni²⁺ > Cu²⁺. The crown ether coated piezo electric crystal LC detector demonstrated low detection limits for various polar organic molecules, e.g., 6.0 × 10^?5 M for propylamine, and metal ions, e.g., 2.9 × 10^?5 M (1.8 ppm) for Cu²⁺; the crown ether PZ detector also gave good reproducibility when re used. A quite sensitive electrochemical quartz crystal microbalance (EQCM) detection system was also set‐up for detecting trace heavy metal ions in solutions. The variation in frequency of the PZ crystal and the diffusion current were observed simultaneously after the reduction in heavy metal ions such as Cu²⁺ and Ni²⁺. The EQCM detection system exhibited fairly good sensitivity, e.g., 112 Hz/ppm for Cu²⁺ and a good detection limit, e.g., 0.13 ppm for Cu²⁺ ions. Comparison between EQCM and PZ detection systems was made and discussed.     

Reactive kinetics of carbon dioxide loaded aqueous blend of 2-amino-2-ethyl-1,3-propanediol and piperazine using a pressure drop method

In this study, a new pressure drop method has been used to investigate the kinetics of carbon dioxide reaction with aqueous blend of 2-amino-2-ethyl-1,3-propanediol (AEPD) with piperazine (PZ). The blending of a small amount of PZ with AEPD has a significant effect on the observed rate constant, k_obs. It was observed that k_obs values of the blend increased more than twice than the summation of k_obs values of individual alkanolamines. The reaction kinetics in this study were modeled by assuming a termolecular mechanism. The addition of 0.1 mol/L of PZ to 1 mol/L AEPD exhibited an observed rate constant, k_obs of 8824.1 s⁻¹, which is comparable to other alkanolamine mixtures. Hence, PZ/AEPD mixtures can be potentially used for rapid carbon dioxide capture.     

Determination of γ-hexachlorocyclohexane and its metabolites in biological samples from rat

We have determined γ-hexachlorocyclohexane (lindane) and its metabolites in urine, serum and feces samples from rats using HPLC-UV-Vis and confirmation of mass with matrix assisted laser desorption/ ionization-time of flight (MALDI-TOF) analysis. Samples were collected from rats treated orally with lindane (17.6 mg/kg; 1/5 of LD₅₀) or vehicle for 2 weeks. Lindane and metabolites were extracted from samples with hexane and analyzed. The HPLC–MALDI-TOF is highly sensitive to the point of detecting very low level (5 ppm) of lindane and metabolites. The HPLC-UV-Vis analysis confirmed the presence of lindane in urine (386–1652 ppm), serum (207–371 ppm) and feces (5–74 ppm). Control samples had no peak corresponding to lindane. MALDI-TOF analysis of urine and serum samples showed a major peak at 293 m/z, whereas feces showed a minor peak at 292–293 m/z, which were consistent with the peak obtained for standard lindane (293 m/z). Our data indicates that HPLC-UV-Vis–MALDI-TOF combo method is sensitive for detecting and quantifying lindane and its metabolites in serum, urine and feces. Our results further showed that minor quantities of lindane and metabolites were excreted through feces confirming that the main pathway for excretion of lindane and metabolites is through urine.     

The Effect of the Molecular Weight of Polyvinylpyrrolidone and the Model Drug on Laser-Induced In Situ Amorphization

Laser radiation has been shown to be a promising approach for in situ amorphization, i.e., drug amorphization inside the final dosage form. Upon exposure to laser radiation, elevated temperatures in the compacts are obtained. At temperatures above the glass transition temperature (T_g) of the polymer, the drug dissolves into the mobile polymer. Hence, the dissolution kinetics are dependent on the viscosity of the polymer, indirectly determined by the molecular weight (M_w) of the polymer, the solubility of the drug in the polymer, the particle size of the drug and the molecular size of the drug. Using compacts containing 30 wt% of the drug celecoxib (CCX), 69.25 wt% of three different M_w of polyvinylpyrrolidone (PVP: PVP12, PVP17 or PVP25), 0.25 wt% plasmonic nanoaggregates (PNs) and 0.5 wt% lubricant, the effect of the polymer M_w on the dissolution kinetics upon exposure to laser radiation was investigated. Furthermore, the effect of the model drug on the dissolution kinetics was investigated using compacts containing 30 wt% of three different drugs (CCX, indomethacin (IND) and naproxen (NAP)), 69.25 wt% PVP12, 0.25 wt% PN and 0.5 wt% lubricant. In perfect correlation to the Noyes–Whitney equation, this study showed that the use of PVP with the lowest viscosity, i.e., the lowest M_w (here PVP12), led to the fastest rate of amorphization compared to PVP17 and PVP25. Furthermore, NAP showed the fastest rate of amorphization, followed by IND and CCX in PVP12 due to its high solubility and small molecular size.     

Metabolomic profiling of human urine in hepatocellular carcinoma patients using gas chromatography/mass spectrometry

With the technique of metabolomics, gas chromatography/mass spectrometry (GC/MS), urine or serum metabolites can be assayed to explore disease biomarkers. In this work, we present a metabolomic method to investigate the urinary metabolic difference between hepatocellular carcinoma (HCC, n = 20) male patients and normal male subjects (n = 20). The urinary endogenous metabolome was assayed using chemical derivatization followed by GC/MS. After GC/MS analysis, 103 metabolites were detected, of which 66 were annotated as known compounds. By a two sample t-test statistics with p < 0.05, 18 metabolites were shown to be significantly different between the HCC and control groups. A diagnostic model was constructed with a combination of 18 marker metabolites or together with alphafetoprotein, using principal component analysis and receiver-operator characteristic curves. The multivariate statistics of the diagnostic model yielded a separation between the two groups with an area under the curve value of 0.9275. This non-invasive technique of identifying HCC biomarkers from urine may have clinical utility.     

Effect of certain chemical compounds on secondary metabolites of Penicillium janthinellum and P. duclauxii

The secondary metabolites of Penicillium janthinellum and P. duclauxii were affected by different concentrations of cadmium nitrate, sodium chloride, and sucrose. The fungal culture characteristics including mycelial fresh and dry weight, colony diameter, colony spore mass and reverse color, number of spores, and secondary metabolites were affected as well. Cyclopenin, carlosic acid, erythroskyrin, kojic acid, and patulin were produced by P. janthinellum on cadmium nitrate-free medium. However, cyclopenin, carlosic acid, frequentin, and islandicin were produced at 100 ppm, and cyclopenin at 500 ppm. On the other hand, the secondary metabolites produced by P. duclauxii were 9 on cadmium nitrate-free medium, 7 on medium containing 100 and 500 ppm of cadmium nitrate, and 4 at 1000 ppm cadmium nitrate concentration. Secondary metabolite brevianamide A was produced in the presence and absence of cadmium nitrate. The duclauxin, patulin, terrestic acid, and xanthomegin were produced only on cadmium nitrate-free medium. However, mycophenolic acid was produced only on cadmium nitrate-containing medium. The kojic acid was produced by P. janthinellum at 0.0%, 0.5%, 1.0%, 2.0%, 3.0%, and 5.0% concentrations of sodium chloride. The carlosic acid, erythroskyrin, and patulin were produced only at 0.0% and 1.0%. While carolic acid and islandicin were produced at 1%, 2%, and 3% and frequentin was produced only at 2% and 3%. On the other hand, 8 secondary metabolites were produced by P. duclauxii at 0.0%, 0.5%, 1.0%, and 2.0% concentration of sodium chloride and only 4 were produced at 3%. The secondary metabolites produced by P. janthinellum were 7 at 10% and 20%, 6 at 30%, and 2 only at 40% concentrations of sucrose. However, 8 secondary metabolites were produced by P. duclauxii at 10%, 20%, 30% and 3 at 40% concentrations of sucrose.     

Inhibition of CK2 Activity by TCDD via Binding to ATP-competitive Binding Site of Catalytic Subunit: Insight from Computational Studies

Alternative mechanisms of toxic effects induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD), instead of the binding to aryl hydrocarbon receptor(AhR), have been taken into consideration. It has been recently shown that TCDD reduces rapidly the activity of CK2(casein kinase II) both in vivo and in vitro. It is found that TCDD has high molecular similarities to the known inhibitors of CK2 catalytic subunit(CK2α). This suggests that TCDD could also be an ATP-competitive inhibitor of CK2α. In this work, docking TCDD to CK2 was carried out based on the two structures of CK2α from maize and human, respectively. The binding free energies of the predicted CK2α-TCDD complexes estimated by the molecular mechanics/Poisson-Boltzmann surface area(MM/PBSA) method are from -85.1 kJ/mol to -114.3 kJ/mol for maize and are from -96.1 kJ/mol to -118.2 kJ/mol for human, which are comparable to those estimated for the known inhibitor and also ATP with CK2α. The energetic analysis also reveals that the van der Waals interaction is the dominant contribution to the binding free energy. These results are also useful for designing new drugs for a target of overexpressing CK2 in cancers.     

Analysis of cyathane-type diterpenoids from Cyathus striatus and Hericium erinaceus by high-resolution MALDI MS imaging

Fungal secondary metabolites in both fruiting bodies and pellets from submerged cultures of basidiomycetes were analyzed by atmospheric pressure matrix-assisted laser desorption/ionization-mass spectrometry imaging at a lateral resolution of 15 μm, a mass resolution of 140,000 at m/z 200 and a mass accuracy of better than 2 ppm. The striatals A, B, C, and D, and a number of erinacine type metabolites were detected in the basidiomycetes Cyathus striatus and Hericium erinaceus, respectively. The two fungi were selected as model species, as they are well-known for efficient production of terpenoid secondary metabolites with interesting biological activities, e.g., antibacterial, fungicidal, cytotoxic properties, and stimulating effects on nerve growth factor synthesis. The localization of metabolites revealed a mostly homogeneous distribution of the striatals in the pellets of C. striatus, while a concentration gradient, increasing to the center, was observed in the pellets of H. erinaceus. A mostly homogeneous distribution of metabolites was also found in the fruiting body of H. erinaceus.     

Preparation and Application of Fullerene C60‐Polymers in Piezoelectric Quartz Crystal Sensors for Hemoglobin and Olefins

The C60—polycinnamaldehyde (C₆₀—PCA) and C60—polyphenylacetylene (C60—PPA) polymers were synthesized by the Friedel—Craft reaction and applied as piezoelectric (PZ) quartz crystal coating materials. A C60—polycinnamaldehyde (PCA) coated piezoelectric quartz crystal liquid sensor with a homemade computer interface was prepared and applied as a PZ hemoglobin sensor. The adsorption of hemoglobin onto the C60—PCA coated crystal resulted in a decreased oscillating frequency. The variations in crystal frequency were converted to voltage with a frequency to voltage converter, followed by amplification with OPA and data acquisition with an analog to digital converter. The PZ hemoglobin sensor exhibited good sensitivity of 6530 Hz/(mg/mL) with a detection limit at the ppm level for hemoglobin. Further, a C60—polyphenylacetylene (C60—PPA) coated piezoelectric quartz crystal gas sensor with an Intell‐8255 data processing system for various olefin vapors was also made. The aromatic hydrocarbons such as toluene seem to have greater adsorption onto C60—PPA membrane than alkynes, alkenes, and alkanes. The adsorption of polycyclic aromatic hydrocarbons (PAHs) onto the C60—PPA membrane was also examined. The C₆₀—PPA coated PZ crystal gas sensor showed much better sensitivity for PAHs than for other olefins such as toluene, 1‐hexyne and 1‐hexene, and a much larger frequency shift for naphthalene than other PAHs was also found.