Intracellular oxidative stress and cytotoxicity in rat primary cortical neurons exposed to cholesterol secoaldehyde

Cholesterol secoaldehyde (ChSeco or 3β-hydroxy-5-oxo-5,6-secocholestan-6-al) has been shown to induce Aβ aggregation and apoptosis in GT1-7 hypothalamic neurons. The present study was undertaken to evaluate the effects of ChSeco on rat primary cortical neuronal cells. ChSeco was cytotoxic at concentrations ranging from 5 to 20 μM, while cholesterol of comparable concentrations showed little or no toxicity. In ChSeco-exposed neuronal cells, there was an increased formation of intracellular peroxide or peroxide-like substance(s), the levels of which were comparable to those found in typical menadione exposures. There was a loss in the mitochondrial transmembrane potential, the extent of which was dependent on concentration of ChSeco employed. Pre-treatment with N-acetyl-l-cysteine (5 mM; 1 h) offered protection against the cytotoxicity and the generation of intracellular oxidants. Cytotoxicity of ChSeco was evidenced by the loss of axonal branches and also condensed apoptotic nuclei in these cells. Immunohistochemical analysis revealed a decreased intracellular Aβ42 staining proportional to the loss in the axonal out growth and dendritic branches. The observed decrease in Aβ42 has been suggested to be due to loss of integrity of dendrites and the plasma membrane, possibly resulting from increased production of reactive oxygen species.     

Differential methotrexate hepatotoxicity on rat hepatocytes in 2-D monolayer culture and 3-D gel entrapment culture

It has been reported that 3-D cultures of hepatocytes or HepG2 cells were less susceptible to methotrexate (MTX) than their 2-D counterparts. Such a mechanism was addressed in this study by investigation of MTX hepatotoxicity in gel entrapped (3-D) rat hepatocytes vs. traditional monolayer culture (2-D). Similarly, gel entrapped hepatocytes showed higher drug resistance to MTX than hepatocyte monolayers in whatever culture medium with or without modification by hormone supplements (dexamethasone, glucagon and insulin). It was also found that medium modification by hormones greatly increased drug resistance of hepatocyte monolayers but has only a slight effect on 3-D cultured hepatocytes. These differential MTX toxicities regarding culture medium and culture models were assumed to correlate with multidrug resistance associated protein 2 (Mrp2). The involvement of Mrp2 was confirmed directly by the fact that MTX intracellularly accumulated less in gel entrapped hepatocytes than in hepatocyte monolayer but could be enhanced by Mrp2 inhibitors accompanied by reduced drug resistance. Furthermore, the expression of Mrp2 on gene level and transportation activity together with bile-duct-like structure were more significantly evidenced in 3-D gel entrapment culture than in 2-D monolayer culture. In conclusion, the highly preserved Mrp2 in 3-D gel entrapped hepatocytes determines its high drug resistance to MTX. Gel entrapped hepatocytes could be useful for investigation of hepatic transportation and hepatotoxicity.     

Serum-free collagen sandwich cultures of adult rat hepatocytes maintain liver-like properties long term: A valuable model for in vitro toxicity and drug-drug interaction studies

Cultures of primary hepatocytes from various species, including human, are used in several applications during pre-clinical drug development. Their use is however limited by cell survival and conservation of liver-specific functions in vitro. The differentiation status of hepatocytes in culture strongly depends on medium formulation and the extracellular matrix environment. We incubated primary rat hepatocytes for 10 days on collagen monolayer and in collagen sandwich cultures with or without serum. Restoration of polygonal cell shape and formation of functional bile canaliculi-like structures was stable only in serum-free sandwich cultures. Variations in general cell viability, as judged by the cellular ATP content, LDH release or apoptosis, were less pronounced between alternative cultures. The intracellular glutathione content was preserved close to in vivo levels especially in serum-free sandwich cultures. Basal activities of cytochrome P450 enzymes (P450) varied strongly between cultures. There was a minor effect on CYP1A but CYP2B activity was only detectable in the serum-free sandwich culture after 3 days and beyond. CYP2C activity was slightly elevated in both sandwich cultures, whereas CYP3A showed increased levels in both serum-free cultures. Inducibility of these P450s was fully maintained over time in serum-free collagen sandwich only. Gene expression was largely constant over time in serum-free sandwich cultures that was closest to liver. This liver-like property was supported by protein profiling results. Taken together, the serum-free collagen sandwich culture of primary rat hepatocytes maintained liver-like features over 10 days and is therefore a suitable model for long-term toxicity and drug-drug interaction studies.     

Phosphorus dynamics in shallow eutrophic lakes: an example from Zeekoevlei,South Africa

Zeekoevlei is the largest freshwater lake in South Africa and has been suffering from hyper-eutrophic conditions since last few decades. We have used total P (TP), dissolved phosphate (PO₄ ³⁻), organic P (OP), calcium (Ca) and iron (Fe) bound P fractions to investigate the relevant physical, chemical and biological processes responsible for sedimentation and retention of P and to study phosphorus (P) dynamics in this shallow lake. In addition, redox proxies (V/Cr and Th/U ratios) are used to study the prevailing redox conditions in sediments. Adsorption by CaCO₃ and planktonic assimilation of P are found to control P sedimentation in Zeekoevlei. Low concentration of the labile OP fraction in surface sediments restricts the release of P by bacterial remineralisation. Low molar Ca/P and Fe/P ratios indicate low P retention capacity of sediments, and P is most likely released by desorption from wind-induced resuspended sediments and mixing of pore water with the overlying water column. Handling editor: J. Saros     

Summer sedimentation in six shallow arctic lakes

The movement of sediment between the lake bottom and water column of shallow lakes can be sizeable due to the large potential for resuspension in these systems. Resuspended sediments have been shown to alter phytoplankton community composition and elevate water column production and nutrient concentrations. We measured the summer sedimentation rates of two lakes in 2003 and six lakes in 2004. All lakes were shallow and located in the Alaskan Arctic. In 2004, turbidity, light attenuation, total sediment:chlorophyll a mass in the sediment traps, and thermal stratification were also measured in each of the lakes. The sediment:chlorophyll a mass was much greater than if the sediment was derived from phytoplankton production in all of the lakes, indicating that the source of the sedimenting material was resuspension and allochthonous inputs. Consistent with these findings, the temporal variation in sedimentation rate was synchronous between most lakes, and sedimentation rate was positively related to wind speed and rainfall suggesting that sedimentation rate was strongly influenced by landscape-scale factors (e.g., wind and rain events). Two of the lakes are located on deposits of loess that accumulated during past glacial periods. These two lakes had sedimentation rates that were significantly greater and more variable than any of the other lakes in the study, as well as high turbidity and light attenuation. Our results indicate that sedimentation in these shallow arctic lakes is supported primarily by allochthonous inputs and resuspension and that landscape-scale factors (e.g., weather and geology) impact on the transport of materials between the lake bottom and water column. Handling editor: J. Saros     

Generation of primary amide glucosides from cyanogenic glucosides

The cyanogenic glucoside-related compound prunasinamide, (2R)-β-d-glucopyranosyloxyacetamide, has been detected in dried, but not in fresh leaves of the prunasin-containing species Olinia ventosa, Prunus laurocerasus, Pteridium aquilinium and Holocalyx balansae. Experiments with leaves of O. ventosa indicated a connection between amide generation and an excessive production of reactive oxygen species. In vitro, the Radziszewski reaction with H₂O₂ has been performed to yield high amounts of prunasinamide from prunasin. This reaction is suggested to produce primary amides from cyanogenic glycosides in drying and decaying leaves. Two different benzoic acid esters which may be connected to prunasin metabolism were isolated and identified as the main constituents of chlorotic leaves from O. ventosa and P. laurocerasus.     

Insights into plant metabolic networks from steady-state metabolic flux analysis

Steady-state metabolic flux analysis (MFA) is an experimental approach that allows the measurement of multiple fluxes in the core network of primary carbon metabolism. It is based on isotopic labelling experiments, and although well established in the analysis of micro-organisms, and some mammalian systems, the extension of the method to plant cells has been challenging because of the extensive subcellular compartmentation of the metabolic network. Despite this difficulty there has been substantial progress in developing robust protocols for the analysis of heterotrophic plant metabolism by steady-state MFA, and flux maps have now been published that reflect the metabolic phenotypes of excised root tips, developing embryos and cotyledons, hairy root cultures, and cell suspensions under a variety of physiological conditions. There has been a steady improvement in the quality, extent and statistical reliability of these analyses, and new information is emerging on the performance of the plant metabolic network and the contributions of specific pathways.     

Summer-drought constrains the phenology and growth of two coexisting Mediterranean oaks with contrasting leaf habit: implications for their persistence and reproduction

This study analyses how coexisting evergreen and deciduous oaks adjust their phenology to cope with the stressful Mediterranean summer conditions. We test the hypothesis that the vegetative and reproductive growth of the winter deciduous (Quercus faginea Lam.) is more affected by summer drought than that of the evergreen [Quercus ilex L. subsp. ballota (Desf.) Samp.]. First, we assessed the complete aboveground phenology of both species during two consecutive years. Shoot and litter production and bud, acorn and secondary growth were monitored monthly. Second, we identified several parameters affected by summer conditions: apical bud size, individual leaf area (LA), leaf mass per area (LMA) and acorn yield in both species, and leaf-fall in Q. faginea; and analysed their variation over 10 years. Q. ilex performed up to 25% of shoot growth and most leaf development during summer, whereas Q. faginea completed most of both phenophases during spring. Secondary growth was arrested in summer under drought conditions. Approximately, 30–40% of bud and 40–50% of acorn growth was undertaken during summer in both species. Summer drought related to differences in LA, LMA and leaf senescence, but not to acorn yield. Both species had similar year-to-year patterns of acorn production, though yields were always lower in Q. faginea. Bud size decreased severely in both species during extremely dry years. In Q. ilex, bud size tended to alternate between years of large and small buds, and these patterns were followed by opposite trends in stem length. In Q. faginea, bud size was more stable through time. Q. ilex was more phenologically active during summer than Q. faginea, indicating a higher tolerance to drought. Furthermore, bud and fruit growth (the only two phenophases that both species performed during summer) were more severely affected by summer drought in Q. faginea than in the evergreen. The differential effects of summer drought on key phenophases for the persistence (bud growth) and colonization ability (fruit production) of both species may have consequences for their coexistence.     

Life cycle assessment of primary magnesium production using the Pidgeon process in China

Background, aims, and scope  China has been the largest primary magnesium producer in the world since year 2000 and is an important part of the global magnesium supply chain. Almost all of the primary magnesium in China is produced using the Pidgeon process invented in the 1940s in Canada. The environmental problems of the primary magnesium production with the Pidgeon process have already attracted much attention of the local government and enterprises. The main purposes of this research are to investigate the environmental impacts of magnesium production and to determine the accumulative environmental performances of three different scenarios. System boundary included the cradle-to-gate life cycle of magnesium production, including dolomite ore extraction, ferrosilicon production, the Pidgeon process, transportation of materials, and emissions from thermal power plant. The life cycle assessment (LCA) case study was performed on three different fuel use scenarios from coal as the overall fuel to two kinds of gaseous fuels, the producer gas and coke oven gas. The burden use of gaseous fuels was also considered. Methods  The procedures, details, and results obtained are based on the application of the existing international standards of LCA, i.e., the ISO 14040. Depletion of abiotic resources, global warming, acidification, and human toxicity were adopted as the midpoint impact categories developed by the problem-oriented approach of CML to estimate the characterized results of the case study. The local characterization and normalization factors of abiotic resources were used to calculate abiotic depletion potential (ADP). The analytic hierarchy process was used to determine the weight factors. Using the Umberto version 4.0, the emissions of dolomite ore extraction were estimated and the transportation models of the three scenarios were designed. Results and conclusions  The emissions inventory showed that both the Pidgeon process of magnesium production and the Fe–Si production were mainly to blame for the total pollutant emissions in the life cycle of magnesium production. The characterized results indicated that ADP, acidification potential, and human toxicity potential decreased cumulatively from scenarios 1 to 3, with the exception of global warming potential. The final single scores indicated that the accumulative environmental performance of scenario 3 was the best compared with scenarios 1 and 2. The impact of abiotic resources depletion deserves more attention although the types and the amount of mineral resources for Mg production are abundant in China. This study suggested that producer gas was an alternative fuel for magnesium production rather than the coal burned directly in areas where the cost of oven gas-produced coke is high. The utilization of “clean” energy and the reduction of greenhouse gases and acidic gases emission were the main goals of the technological improvements and cleaner production of the magnesium industry in China. Recommendation and perspective  This paper has demonstrated that the theory and method of LCA are actually helpful for the research on the accumulative environmental performance of primary magnesium production. Further studies with “cradle-to-cradle” scheme are recommended. Furthermore, other energy sources used in magnesium production and the cost of energy production could be treated in further research.