Ssu-Ching Chen

Ellagic acid protects human keratinocyte (HaCaT) cells against UVA-induced oxidative stress and apoptosis through the upregulation of the HO-1 and Nrf-2 antioxidant genes.

UV radiation from the sun is a potent environmental risk factor in the pathogenesis of skin damage. Much of the skin damage caused by ultraviolet A (UVA) irradiation from the sun is associated with oxidative stress. The aim of this study was to investigate the protective role of ellagic acid (25-75 μM), a natural antioxidant, against UVA (5-20 J/cm(2))-induced oxidative stress and apoptosis in human keratinocyte (HaCaT) cells and to reveal the possible mechanisms underlying this protective efficacy. Ellagic acid pre-treatment markedly increased HaCaT cell viability and suppressed UVA-induced ROS generation and MDA formation. Moreover, ellagic acid pre-treatment prevented UVA-induced DNA damage as evaluated by the comet assay. Ellagic acid treatment also significantly inhibited the UVA-induced apoptosis of HaCaT cells, as measured by a reduction of DNA fragmentation, mitochondria dysfunction, ER stress, caspase-3 activation, and Bcl-2/Bax deregulation. Notably, the antioxidant potential of ellagic acid was directly correlated with the increased expression of HO-1 and SOD, which was followed by the downregulation of Keap1 and the augmented nuclear translocation and transcriptional activation of Nrf2 with or without UVA irradiation. Nrf2 knockdown diminished the protective effects of ellagic acid. Therefore, ellagic acid may be useful for the treatment of UVA-induced skin damage.

Biotransformation of cyanide to methane and ammonia by Klebsiella oxytoca

Klebsiella oxytoca, isolated from cyanide-containing industrial wastewater, was shown to be able to biodegrade cyanide to non-toxic endproducts using cyanide as the sole nitrogen source. In this study, ammonia was one of the detected endproduct of cyanide biodegradation by the concentrated resting cells of K. oxytoca. Moreover, cyanide has been shown to be biotransformed to methane through the actions of concentrated resting cells. Biodegradation of cyanide by cell-free extracts was not observed, which might be due to the inactivation of nitrogenase (an oxygen-labial enzyme) caused by the oxygen exposure after cell disruption. Results show that the cyanide consumption by resting cells of K. oxytoca was induced when the pretreatment of these cells with cyanide was conducted. However, the cyanide-degrading capability of resting cells pretreated with ammonia was inhibited. The inhibition of cyanide degradation by resting cells of K. oxytoca was affected by the ammonia concentration. This might result from the suppression of nitrogenase activity of K. oxytoca by ammonia since nitrogenase was suggested to be the sole cyanide-degrading enzyme during the cyanide degradation process. Results from this study also show that the processes of cyanide biodegradation and ammonia production by resting cells occurred simultaneously. This suggests that the utilization of cyanide as nitrogen source by K. oxytoca might proceed using ammonia as an assimilatory substrate.

Remediation of PCE-contaminated aquifer by an in situ two-layer biobarrier: laboratory batch and column studies

The industrial solvent tetrachloroethylene (PCE) is among the most ubiquitous chlorinated compounds found in groundwater contamination. The objective of this study was to develop an in situ two-layer biobarrier system consisting of an organic-releasing material layer followed by an oxygen-releasing material layer. The organic-releasing material, which contained sludge cakes from a domestic wastewater treatment plant, is able to release biodegradable organics continuously. The oxygen-releasing material, which contained calcium peroxide, is able to release oxygen continuously upon contact with water. The first organic-releasing material layer was to supply organics (primary substrates) to reductively dechlorinate PCE in situ. The second oxygen-releasing material layer was to release oxygen to aerobic biodegrade or cometabolize PCE degradation byproducts from the first anaerobic layer. Batch experiments were conducted to design and identify the components of the organic and oxygen-releasing materials, and evaluate the organic substrate (presented as chemical oxygen demand (COD) equivalent) and oxygen release rates from the organic-releasing material and oxygen-releasing materials, respectively. The observed oxygen and COD release rates were approximately 0.0368 and 0.0416 mg/d/g of material, respectively. A laboratory-scale column experiment was then conducted to evaluate the feasibility of this proposed system for the bioremediation of PCE-contaminated groundwater. This system was performed using a series of continuous-flow glass columns including a soil column, an organic-releasing material column, two consecutive soil columns, and an oxygen-releasing material column, followed by two other consecutive soil columns. Anaerobic acclimated sludges were inoculated in the first four columns, and aerobic acclimated sludges were inoculated in the last three columns to provide microbial consortia for contaminant biodegradation. Simulated PCE-contaminated groundwater with a flow rate of 0.25 L/d was pumped into this system. Effluent samples from each column were analyzed for PCE and its degradation byproducts. Results show that up to 99% of PCE removal efficiency was obtained in this passive system. Thus, the biobarrier treatment scheme has the potential to be developed into an environmentally and economically acceptable remediation technology for the in situ treatment of PCE-contaminated aquifer.

Effects of soil pH, temperature and water content on the growth of Burkholderia pseudomallei.

Optimum conditions were determined for the growth ofBurkholderia pseudomallei in natural soils or waters. It grows better in paddy soil, crop-covered and fallow field than in fresh and salty water. Although the optimal temperature and pH for the growth were 37 or 42 °C, and 6.5 or 7.5 in an environmental-mimicking soil medium, this bacterium can still grow at 4 °C, which was suggested to be related with the occurrence of melioidosis in some cold areas. In soil media with water content <15.B. pseudomallei did not grow until 60 d of incubation, suggesting that water contents of soils in which it dwelled would be one important factor in determining the growth rate.

Antioxidant and Anti-Inflammatory Potential of Hesperetin Metabolites Obtained from Hesperetin-Administered Rat Serum: An Ex Vivo Approach

In recent years much attention has been focused on the pharmaceutical relevance of bioflavonoids, especially hesperidin and its aglycon hesperetin in terms of their antioxidant and anti-inflammatory actions. However, the bioactivity of their metabolites, the real molecules in vivo hesperetin glucuronides/sulfates produced after ingestion, has been poorly understood. Thus, the study using an ex vivo approach is aimed to compare the antioxidant and anti-inflammatory activities of hesperidin/hesperetin or hesperetin metabolites derived from hesperetin-administered rat serum. We found that hesperetin metabolites (2.5-20 μM) showed higher antioxidant activity against various oxidative systems, including superoxide anion scavenging, reducing power, and metal chelating effects, than that of hesperidin or hesperetin. The data also showed that pretreatment of hesperetin metabolites (1-10 μM) within the range of physiological concentrations, compared to hesperetin, significantly inhibited nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production, as evidenced by the inhibition of their precursors, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein levels without appreciable cytotoxicity on LPS-activated RAW264.7 macrophages or A7r5 smooth muscle cells. Concomitantly, hesperetin metabolites dose-dependently inhibited LPS-induced intracellular reactive oxygen species (ROS). Furthermore, hesperetin metabolites significantly downregulate LPS-induced nuclear factor-κB (NF-κB) activation followed by the suppression of inhibitor-κB (I-κB) degradation and phosphorylation of c-Jun N-terminal kinase1/2 (JNK1/2) and p38 MAPKs after challenge with LPS. Hesperetin metabolites ex vivo showed potent antioxidant and anti-inflammatory activity in comparison with hesperidin/hesperetin.

Application of real-time PCR, DGGE fingerprinting, and culture-based method to evaluate the effectiveness of intrinsic bioremediation on the control of petroleum-hydrocarbon plume.

Real-time polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), and the culture-based method were applied in the intrinsic bioremediation study at a petroleum-hydrocarbon contaminated site. The genes of phenol hydroxylase (PHE), ring-hydroxylating toluene monooxygenase (RMO), naphthalene dioxygenase (NAH), toluene monooxygenase (TOL), toluene dioxygenase (TOD), and biphenyl dioxygenase (BPH4) were quantified by real-time PCR. Results show that PHE gene was detected in groundwater contaminated with benzene, toluene, ethylbenzene, xylene isomers (BTEX) and methyl tert-butyl ether (MTBE), and this indicates that intrinsic bioremediation occurred at this contaminated site. Results from DGGE analyses reveal that the petroleum-hydrocarbon plume caused the variation in microbial communities. In this study, MTBE degraders including Pseudomonas sp. NKNU01, Bacillus sp. NKNU01, Klebsiella sp. NKNU01, Enterobacter sp. NKNU01, and Enterobacter sp. NKNU02 were isolated from the contaminated groundwater using the cultured-based method. Results from MTBE biodegradation experiment show that the isolated bacteria were affected by propane. This indicates that propane may influence the metabolic pathway of MTBE by these bacteria. Knowledge and comprehension obtained from this study will be helpful in evaluating the occurrence and effectiveness of intrinsic bioremediation on the remediation of petroleum-hydrocarbon contaminated groundwater.

Antrodia camphorata inhibits proliferation of human breast cancer cells in vitro and in vivo.

Antrodia camphorata (A. camphorata) has been shown to induce apoptosis in cultured human breast cancer cells (MDA-MB-231). In this study, we report the effectiveness of the fermented culture broth of A. camphorata in terms of tumor regression as determined using both in vitro cell culture and in vivo athymic nude mice models of breast cancer. We found that the A. camphorata treatment decreased the proliferation of MDA-MB-231 cells by arresting progression through the G1 phase of the cell cycle. This cell cycle blockade was associated with reductions in cyclin D1, cyclin E, CDK4, cyclin A, and proliferating cell nuclear antigen (PCNA), and increased CDK inhibitor p27/KIP and p21/WAF1 in a dose and time-dependent manner. Furthermore, the A. camphorata treatment was effective in delaying tumor incidence in the nude mice inoculated with MDA-MB-231 cells as well as reducing the tumor burden when compared to controls. A. camphorata treatment also inhibited proliferation (cyclin D1 and PCNA) and induced apoptosis (Bcl-2 and TUNEL) when the tumor tissue sections were examined histologically and immunohistochemically. These results suggest that the A. camphorata treatment induced cell cycle arrest and apoptosis of human breast cancer cells both in vitro and in vivo.

Evaluation of TCDD biodegradability under different redox conditions

Polychlorinated dibenzo-p-dioxins have been generated as unwanted by-products in many industrial processes. Although their widespread distribution in different environmental compartments has been recognized, little is known about their fate in the ultimate environment sinks. The highly stable dioxin isomer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been called the most toxic compound known to man. In this laboratory microcosm study, TCDD bioavailability was evaluated under five reduction/oxidation (redox) conditions including aerobic biodegradation, aerobic cometabolism, methanogenesis, iron reduction, and reductive dechlorination. Activated sludge and aquifer sediments from a TCDD and a pentachlorophenol (PCP) contaminated site were used as the inocula. Acetate, sludge cake, and cane molasses were used as the primary substrates (carbon sources) in cometabolism and reductive dechlorination microcosms. After a 90-day incubation period, microcosms constructed under reductive dechlorination conditions were the only treatment showing promising remediation results. The highest TCDD degradation rate [up to 86% of TCDD removal (with an initial concentration of 96 microg/kg of soil)] was observed in the microcosms with anaerobic activated sludge as the microbial inocula and sludge cakes as the primary substrates. Except for reductive dechlorination microcosms, no significant TCDD removal was observed in the microcosms prepared under other conditions. Thus, application of an effective primary substrate to enhance the reductive dechlorination process is a feasible method for TCDD bioremediation. Bioremediation expense can be significantly reduced by the supplement of some less expensive alternative substrates (e.g., sludge cakes, cane molasses). Results would be useful in designing a scale-up in situ or on-site bioremediation system such as bioslurry reactor for field application.

Biodegradation of propionitrile by Klebsiella oxytoca immobilized in alginate and cellulose triacetate gel

A microbial process for the degradation of propionitrile by Klebsiella oxytoca was studied. The microorganism, K. oxytoca, was isolated from the discharged wastewater of metal plating factory in southern Taiwan and adapted for propionitrile biodegradation. The free and immobilized cells of K. oxytoca were then examined for their capabilities on degrading propionitrile under various conditions. Alginate (AL) and cellulose triacetate (CT) techniques were applied for the preparation of immobilized cells. The efficiency and produced metabolic intermediates and end-products of propionitrile degradation were monitored in bath and continuous bioreactor experiments. Results reveal that up to 100 and 150 mM of propionitrile could be removed completely by the free and immobilized cell systems, respectively. Furthermore, both immobilized cell systems show higher removal efficiencies in wider ranges of temperature (20-40 degrees C) and pH (6-8) compared with the free cell system. Results also indicate that immobilized cell system could support a higher cell density to enhance the removal efficiency of propionitrile. Immobilized cells were reused in five consecutive degradation experiments, and up to 99% of propionitrile degradation was observed in each batch test. This suggests that the activity of immobilized cells can be maintained and reused throughout different propionitrile degradation processes. A two-step pathway was observed for the biodegradation of propionitrile. Propionamide was first produced followed by propionic acid and ammonia. Results suggest that nitrile hydratase and amidase were involved in the degradation pathways of K. oxytoca. In the continuous bioreactor, both immobilized cells were capable of removing 150 mM of propionitriles completely within 16h, and the maximum propionitriles removal rates using AL and CT immobilized beads were 5.04 and 4.98 mM h(-1), respectively. Comparing the removal rates obtained from batch experiments with immobilized cells (AL and CT were 1.57 and 2.18 mM h(-1) at 150 mM of propionitrile, respectively), the continuous-flow bioreactor show higher potential for practical application.

Anti-metastatic activities of Antrodia camphorata against human breast cancer cells mediated through suppression of the MAPK signaling pathway

The fermented culture broth of Antrodia camphorata (A. camphorata) has been shown to promote cell cycle arrest and apoptosis of human estrogen-nonresponsive MDA-MB-231 cells. Herein, we demonstrate that non-cytotoxic concentrations (20-80 μg/mL) of A. camphorata markedly inhibited the invasion/migration of highly metastatic MDA-MB-231 cells as shown by an in vitro transwell and a wound-healing repair assay. The results of a gelatin zymography assay showed that A. camphorata suppressed the activity of matrix metalloproteinase (MMP)-9 and urokinase plasminogen activator (uPA). Western blot results demonstrated that treatment with A. camphorata decreased the expression of MMP-9, MMP-2, uPA, uPA receptor (uPAR) and vascular endothelial growth factor (VEGF); while the expression of the endogenous inhibitors of these proteins, i.e., tissue inhibitors of MMP (TIMP-1 and TIMP-2), and plasminogen activator inhibitor (PAI)-1, increased. Further investigation revealed that A. camphorata suppressed the phosphorylation of ERK1/2, p38, and JNK1/2. A. camphorata treatment also led to a dose-dependent inhibition on NF-κB binding and activation. This is the first report confirming the anti-metastatic activity of this potentially beneficial mushroom against human breast cancer.