Cytotoxicity evaluation of three pairs of hexabromocyclododecane (HBCD) enantiomers on Hep G2 cell

Hexabromocyclododecanes (HBCDs) are additive brominated flame retardants mainly used in plastics and textiles. At the present time, these compounds are found in almost all environmental and human samples. In order to evaluate the environmental safety and health risk of HBCDs, the enantiomerically pure alpha-, beta-, and gamma-HBCD were prepared using high performance liquid chromatography (HPLC) on a PM-P-CD column and the cytotoxicities of their enantiomers were evaluated in Hep G2 cells. Results from the 3-(4,5-dimethylthioazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), resazurin reduction and lactate dehydrogenase (LDH) release assays showed a good agreement that the order of cytotoxicity was gamma-HBCD >= beta-HBCD > alpha-HBCD, and that significantly lower cell viability and higher LDH release were observed in all (+)-enantiomers ((+) alpha-, (+) beta- and (+) gamma-HBCD) than the corresponding (-)-forms ((-) alpha-, (-) beta- and (-) gamma-HBCD). Additionally, the formation of reactive oxygen species (ROS) induced by these HBCD enantiomers were detected. The positive correlation between the LDH release and ROS formation demonstrated that the toxic mechanism might be mediated by oxidative damage. These results suggest that environmental and human health risks of HBCDs must be evaluated at the level of individual enantiomers. (C) 2008 Published by Elsevier Ltd.

DE-71-induced apoptosis involving intracellular calcium and the Bax-mitochondria-caspase protease pathway in human neuroblastoma cells In vitro

Polybrominated diphenyl ethers (PBDEs) are used extensively as flame-retardants and are ubiquitous in the environment and in wildlife and human tissue. Recent studies have shown that PBDEs induce neurotoxic effects in vivo and apoptosis in vitro. However, the signaling mechanisms responsible for these events are still unclear. In this study, we investigated the action of a commercial mixture of PBDEs (pentabrominated diphenyl ether, DE-71) on a human neuroblastoma cell line, SK-N-SH. A cell viability test showed a dose-dependent increase in lactate dehydrogenase leakage and 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyl-tetrazolium bromide reduction. Cell apoptosis was observed through morphological examination, and DNA degradation in the cell cycle and cell apoptosis were demonstrated using flow cytometry and DNA laddering. The formation of reactive oxygen species was not observed, but DE-71 was found to significantly induce caspase-3, -8, and -9 activity, which suggests that apoptosis is not induced by oxidative stress but via a caspase-dependent pathway. We further investigated the intracellular calcium ([Ca2+](i)) levels using flow cytometry and observed an increase in the intracellular Ca2+ concentration with a time-dependent trend. We also found that the N-methyl d-aspartate (NMDA) receptor antagonist MK801 (3 mu M) significantly reduced DE-71-induced cell apoptosis. The results of a Western blotting test demonstrated that DE-71 treatment increases the level of Bax translocation to the mitochondria in a dose-dependent fashion and stimulates the release of cytochrome c (Cyt c) from the mitochondria into the cytoplasm. Overall, our results indicate that DE-71 induces the apoptosis of ([Ca2+](i)) in SK-N-SH cells via Bax insertion, Cyt c release in the mitochondria, and the caspase activation pathway.

Apoptosis induction on human hepatoma cells Hep G2 of decabrominated diphenyl ether (PBDE-209)

Polybrominated diphenyl ethers (PBDEs) are an important class of halogenated organic brominated flame retardants. Because of their presence in abiotic and biotic environments widely and their structural similarity to polychlorinated biphenyls (PCBs), concern has been raised on their possible adverse health effects to humans. This study was designed to determine the anti-proliferative, apoptotic properties of decabrominated diphenyl ether (PBDE-209), using a human hepatoma Hep G2 line as a model system. Hep G2 cells were cultured in the presence of PBDE-209 at various concentrations (1.0-100.0 mu mol/L) for 72 h and the percentage of cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The results showed that PBDE-209 inhibited the cells viability in time and concentration-dependent characteristics at concentrations (10.0-100.0 mu mol/L). We found that anti-proliferative effect of PBDE-209 was associated with apoptosis on Hep G2 cells by determinations of morphological changes, cell cycle and apoptosis. Mechanism study showed that PBDE-209 could increase the generation of intracellular reactive oxygen species (ROS) concentration-dependently. Antioxidant N-acetylcyteine partially inhibited the increase of ROS. The mechanism for its hepatoma-inhibitory effects was the induction of cellular apoptosis through ROS generation. In addition, activity of lactate dehydrogenase (LDH) release increased when the cells incubated with PBDE-209 at various concentrations and times. These results suggested that PBDE-209 had the toxicity activity of anti-proliferation and induction of apoptosis in tumor cells in vitro. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

Sequential ultrastructural and biochemical changes induced in vivo by the hepatotoxic microcystins in liver of the phytoplanktivorous silver carp Hypophthalmichthys molitrix

Up to now, in vivo studies on the toxic effects of microcystins (MCs) on the ultrastructures of fish liver have been very limited. The phytoplanktivorous silver carp was injected i.p. with extracted hepatotoxic microcystins (mainly MC-RR and -LR) at a dose of 1000 mu g MC-LReq. kg(-1) body weight, showing a time-dependent ultrastructural change in liver as well as significant increases in enzyme activity of plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH). We observed for the first time the occurrence of a large amount of activated secondary lysosomes, which might be an adaptive mechanism to eliminate or lessen cell damage caused by MCs through lysosome activation. Quantitative and qualitative determinations of MCs in the liver were conducted by HPLC and LC-MS2, respectively. MCs concentration in the liver reached the maximum (114.20 mu g g(-1) dry weight) after 3 h post-injection, and then rapidly dropped to 7.57 mu g g(-1) dry weight at 48 h, indicating a deputation of 99% accumulated MC-LReq. On the other hand, a decrease trend in glutathione (GSH) concentration was observed in the liver of silver carp while the activity of glutathione S-transferase (GST) increased significantly after injection. The high tolerance of silver carp to MCs might be due to the high basic GSH level in their liver, and/or an increased GSH synthesis. (C) 2007 Elsevier Inc. All rights reserved.

First report of aphantoxins in China - waterblooms of toxigenic Aphanizomenon flos-aquae in Lake Dianchi

The oligohaline cyanobacterium Aphanizomenon flos-aquae (L.) Ralfs (A. flos-aquae) has been reported in several countries to produce paralytic shellfish poisons (PSPs) or protracted toxic effects. In the past years, A. flos-aquae blooms have occurred annually in the eutrophic Lake Dianchi (300 km(2) in area, located in southwestern China). Material from natural blooms dominated by A. flosaquae was collected and lyophilized. Acute toxicity testing was performed by mouse bioassay using extracts from the lyophilized material. Clear symptoms of PSPs, intoxications were observed. To confirm the production of PSPs, a strain of A. flos-aquae (DC-1) was isolated and maintained in culture. Histopathological effects were studied by examining the organ damages using transmission electron microscopy (TEM). Slight hepatocytic damage with swollen mitochondria was found. The ultrastructural pulmonary lesions were characterized by distortied nuclei and indenting of karyotheca, together with degeneration and tumefaction of mitochondria and endoplasmic reticulum. Control animals injected with acetic acid did not exhibit histopathological damage in any organ. Toxic effects of cultured algal cells on enzymatic systems in the mouse were studied using sublethal doses of extracts. Significant glutathione-S-transferase (GST) and lactate dehydrogenase (LDH) increases, together with decrease of the glutathione (GSH) level, were measured. These results indicated a potential role of PSPs intoxicating and metabolizing in the test animals. HPLC-FLD and LC/MS analysis of extracts from cultured material demonstrated the PSP toxins produced by A. flos-aquae bloom. To the best of our knowledge, this is the first study reporting chemically and toxicologically confirmed PSP toxins related to A. flosaquae in China. (c) 2005 Elsevier Inc. All rights reserved.

EROD activities in a primary cell culture of grass carp (Ctenopharyngodon idellus) hepatocytes exposed to polychlorinated aromatic hydrocarbonas

Aryl hydrocarbon (Ah) receptor (Ah-agonist) effects of environmental samples containing polychlorinated aromatic hydrocarbons were evaluated using a 7-ethoxyresorufin-O-deethylase (FROD) assay of a primary hepatocyte culture from grass carp (Ctenopharyngodon idellus). The results were compared with those obtained from the assay using the rat hepatoma cell line H4IIE and chemical analysis using high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS). A dose-response relationship was observed between the EROD activities, either from primary hepatocyte culture assay or from H4IIE assay, and concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The results showed that the assay based on the H4IIE cell line (EC50 = 0.83 mug/mL) is more sensitive to TCDD than the assay based on primary hepatocyte Culture (EC50 = 9.7 pg/mL). In tests of environmental samples, the results from the assay using primary hepatocyte culture were comparable to those from the assay using the H4IIE cell line and chemical analysis of concentrations of mixtures of polychlorinated dibenzo-p-dioxin and dibenzofuran (PCDD/PCDF). The lack of a change in the activities of glutathione-S-transferase (GST) and lactate dehydrogenase (LDH) in cell culture upon exposure to TCDD indirectly indicates that the compound is persistent to biodegradation in the cell culture system. (C) 2004 Elsevier Inc. All rights reserved.

Polymerization of 2,2 '-Dimethyltrimethylene Carbonate by Lutetium Complexes Bearing Amino-Phosphine Ligands

A series of lutetium alkyl, amino, and guanidinato complexes based upon an amino-phosphine ligand framework had been prepared. These complexes were applied to initiate ring-opening polymerization of 2,2'-dimethyltrimethylene carbonate (DTC). The type of the initiator significantly influenced the catalytic activity of these complexes in a trend as follows: alkyl approximate to guanidinate > amide, whereas the complexes with flexible backbone between P and N atoms within the ligand exhibited higher activity than those with rigid backbone. The isolated PDTC had bimodal-mode molecular weight distribution. The molecular weights of each fraction increased linearly with the conversion, indicating that there might be two active species. This had been confirmed by analyses of oligomeric DTC living species and oligomer with NMR technique as the metal-alkoxide and the four-membered metallocyclic lactate. Kinetic investigation displayed that the polymerization rate was the first order with the monomer concentration.

Biochemical effects of gadolinium chloride in rats liver and kidney studied by H-1 NMR metabolomics

The biochemical effects of gadolinium chloride were studied using high-resolution H-1 nuclear magnetic resonance (NMR) spectroscopy to investigate the biochemical composition of tissue (liver and kidney) aqueous extracts obtained from control and gadolinium chloride (GdCl3) (10 and 50 mg/kg body weight, intraperitoneal injection. i.p.) treated rats. Tissue samples were collected at 48, 96 and 168 h p.d. after exposure to GdCl3, and extracted using methanol/chloroform solvent system. H-1 NMR spectra of tissue extracts were analyzed by pattern recognition using principal components analysis. The liver damages caused by GdCl3 were characterized by increased succinate and decreased glycogen level and elevated lactate, alanine and betaine concentration in liver. Furthermore, the increase of creatine and lactate, and decrease of glutamate, alanine, phosphocholine, glycophosphocholine (GPC), betaine, myo-inositol and trimethylamine N-oxide (TMAO) levels in kidney illustrated kidney disturbance induced by GdCl3.

Metabolic profiling of serum from gadolinium chloride-treated rats by H-1 NMR spectroscopy

Metabolic profiling of serum from gadolinium chloride (GdCl3, 10 and 50 mg/kg body weight, intraperitoneal [i.p.])-treated rats was investigated by the NMR spectroscopic-based metabonomic strategy. Serum samples were collected at 48, 96, and 168 h postdose (p.d.) after exposure to GdCl3. H-1 NMR spectra of serum were analyzed by pattern recognition using principal components analysis. The studies showed that there was a dose-related biochemical effect of GdCl3 treatment on the levels of a range of low-molecular weight compounds in serum. The liver damage induced by GdCl3 was characterized by the elevation of lactate, pyruvate, and creatine as well as the decrease of branched-chain amino acids (valine and isoleucine), alanine, glucose, and trimethylamine-N-oxide concentration in serum samples. The biochemical effects of GdCl3 in rats could be consulted when evaluating the biochemical profile of gadolinium-containing compounds that are being developed for nuclear magnetic resonance imaging.

Layer-by-layer electrodeposition of redox polymers and enzymes on screen-printed carbon electrodes for the preparation of reagentless biosensors

Layer-by-layer electrodeposition of redox polymer/enzyme composition films on screen-printed carbon electrodes for fabrication of reagentless enzyme biosensors has been proposed and the resulting films were found to be very stable and rigid.

Medicated wound dressings based on poly(vinyl alcohol)/poly(N-vinyl pyrrolidone)/chitosan hydrogels

Poly(vinyl alcohol) /poly(N-vinyl pyrrolidone) (PVP)/chitosan hydrogels were prepared by a low-temperature treatment and subsequent Co-60 -gamma-ray irradiation and then were medicated with ciprofloxacin lactate (an antibiotic) and chitosan oligomer (molecular weight = 3000 g/mol). The gel content, swelling ratio, tensile strength, and crystallinity of the hydrogels were determined. The effects of the chitosan molecular weight, the low-temperature treatment procedure, and the radiation dosage on the hydrogel properties were examined. The molecular weight of chitosan was lowered by the irradiation, but its basic polysaccharide structure was not destroyed. Repeating the low-temperature treatment and gamma-ray irradiation caused effective physical crosslinking and chemical crosslinking, respectively, and contributed to the mechanical strength of the final hydrogels. The incorporation of PVP and chitosan resulted in a significant improvement in the equilibrium swelling ratio. and elongation ratio of the prepared hydrogels. The ciprofloxacin lactate and chitosan oligomer were soaked into the hydrogels. Their in vitro release behaviors were examined, and they were found to follow diffusion-controlled kinetics.

Investigation on acute biochemical effects of Ce(NO3)(3) on liver and kidney tissues by MAS H-1 NMR spectroscopic-based metabonontic approach

High resolution magic angle spinning (MAS)-H-1 nuclear magnetic resonance (NMR) spectroscopic-based metabonomic approach was applied to the investigation on the acute biochemical effects of Ce(No-3)(3). Male Wistar rats were administrated with various doses of Ce (NO3)(3)(2, 10, and 50 mg(.)kg(-1) body weight), and MAS H-1 NMR spectra of intact liver and kidney tissues were analyzed using principal component analysis to extract toxicity information. The biochemical effects of Ce (NO3)(3) were characterized by the increase of triglycerides and lactate and the decrease of glycogen in rat liver tissue, together with an elevation of the triglyceride level and a depletion of glycerophosphocholine and betaine in kidney tissues. The target lesions of Ce (NO3)(3) on liver and kidney were found by MAS NMR-based metabonomic method. This study demonstrates that the combination of MAS H-1 NMR and pattern recognition analysis can be an effective method for studies of biochemical effects of rare earths.

Preparation of poly(thionine) modified screen-printed carbon electrode and its application to determine NADH in flow injection analysis system

A poly(thionine) modified screen-printed carbon electrode has been prepared by an electrooxidative polymerization of thionine in neutral phosphate buffer. The modified electrodes are found to give stable and reproducible electrocatlytic responses to NADH and exhibit good stability. Several techniques, including cyclic voltammetry, X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM), have been employed to characterize the poly(thionine) film. Further, the modified screen-printed carbon electrode was found to be promising as an amperometric detector for the flow injection analysis (FIA) of NADH, typically with a dynamic range of 5-100 muM.

H-1 NMR analysis for metabolites in serum and urine from rats administrated chronically with La(NO3)(3)

H-1 NMR spectroscopy has been used to assess long-term toxicological effects of a rare earth. Male Wistar rats were administrated orally with La(NO3)(3) at doses of 0.1, 0.2, 2.0, 10, and 20 mg/kg body wt, resp., for 3-6 months. Urine was collected at 1, 2, and 3 months and serum samples were taken after 6 months. Numerous low-M-r metabolites in rats serum and rats urine, including creatinine, citrate, glucose, ketone bodies, trimethylamine N-oxide (TMAO), and various amino acids, were identified on 400- and 500-MHz H-1 NMR spectra. La3+-induced renal and liver damage is characterized by an increase in the amounts of the excreted ketone bodies, amino acids, lactate, ethanol, succinate, TMAO, dimethylamine, and taurine and a decrease in citrate, glucose, urea, and allantoin. Information on the molecular basis of the long-term toxicity of La(NO3)(3) was derived from the abnormal patterns of metabolite excretions. An assay of some biochemical indexes and analysis of some enzymes in plasma supported NMR results.