Kinetics of chlorinated hydrocarbon degradation by Methylosinus trichosporium OB3b and toxicity of trichloroethylene

The kinetics of the degradation of trichloroethylene (TCE) and seven other chlorinated aliphatic hydrocarbons by Methylosinus trichosporium OB3b were studied. All experiments were performed with cells grown under copper stress and thus expressing soluble methane monooxygenase. Compounds that were readily degraded included chloroform, trans-1,2-dichloroethylene, and TCE, with Vmax values of 550, 330, and 290 nmol min-1 mg of cells-1, respectively. 1,1-Dichloroethylene was a very poor substrate. TCE was found to be toxic for the cells, and this phenomenon was studied in detail. Addition of activated carbon decreased the acute toxicity of high levels of TCE by adsorption, and slow desorption enabled the cells to partially degrade TCE. TCE was also toxic by inactivating the cells during its conversion. The degree of inactivation was proportional to the amount of TCE degraded; maximum degradation occurred at a concentration of 2 mumol of TCE mg of cells-1. During conversion of [14C]TCE, various proteins became radiolabeled, including the alpha-subunit of the hydroxylase component of soluble methane monooxygenase. This indicated that TCE-mediated inactivation of cells was caused by nonspecific covalent binding of degradation products to cellular proteins.     

Kinetics of chlorinated hydrocarbon degradation by Methylosinus trichosporium OB3b and toxicity of trichloroethylene.

The kinetics of the degradation of trichloroethylene (TCE) and seven other chlorinated aliphatic hydrocarbons by Methylosinus trichosporium OB3b were studied. All experiments were performed with cells grown under copper stress and thus expressing soluble methane monooxygenase. Compounds that were readily degraded included chloroform, trans-1,2-dichloroethylene, and TCE, with Vmax values of 550, 330, and 290 nmol min-1 mg of cells-1, respectively. 1,1-Dichloroethylene was a very poor substrate. TCE was found to be toxic for the cells, and this phenomenon was studied in detail. Addition of activated carbon decreased the acute toxicity of high levels of TCE by adsorption, and slow desorption enabled the cells to partially degrade TCE. TCE was also toxic by inactivating the cells during its conversion. The degree of inactivation was proportional to the amount of TCE degraded; maximum degradation occurred at a concentration of 2 mumol of TCE mg of cells-1. During conversion of [14C]TCE, various proteins became radiolabeled, including the alpha-subunit of the hydroxylase component of soluble methane monooxygenase. This indicated that TCE-mediated inactivation of cells was caused by nonspecific covalent binding of degradation products to cellular proteins.     

Correlation of biochemical and morphological changes induced by chemical injury to the lung

Comparison has been made of injury to the rat pulmonary alveolar parenchyma evoked by intravenous injection of N-nitrosomethylurethane, intratracheal instillation of 3-methylcholanthrene or repeated inhalation for up to 15 days of carbon tetrachloride, trichloroethylene or gasoline vapour. Biochemical analyses, including assessment of rates of RNA and DNA synthesis and secretion of pulmonary surfactant, were correlated with morphological changes determined by electron microscopy. Single doses of N-nitrosomethylurethane or 3-methylcholanthrene inhibited incorporation of [14C] orotate into lung RNA 1--3 days after treatment. Daily exposure for 30 min to carbon tetrachloride or trichloroethylene vapour caused less marked reduction in orotate incorporation. Ultrastructural examination revealed that 3-methylcholanthrene toxicity was characterised by cytoplasmic change including disruption of surfactant lamellaie of Type 2 pneumocytes and variable degenerative changes Type 1 pneumocytes. Eight to ten days after treatment, the morphological evidence of hypertrophy/hyperplasia and transformation of Type 2 pneumocytes correlated well with biochemical evidence of stimulated incorporation of [3H]thymidine. Inhalation of carbon tetrachloride or trichloroethylene vapour produced milder responses including occasional degenerative changes in Type 1 pneumocytes, reduced numbers of surfactant lamellae in Type 2 pneumocytes and no change in [3H]thymidine incorporation. In contrast to the gradation of injury produced by the various chemicals, all procedures caused a marked and reproducible reduction in secretion of pulmonary surfactant as determined by endobronchial lavage. Following solvent inhalation, reduced recovery of surfactant was detected within 5 days of repeated exposure and thereafter no further change in this depressed level resulted from continued exposure for a further 10 days. The data are discussed in terms of a generalised pattern of response by pulmonary alveolar tissue to chemical injury and the apparent sensitivity of surfactant secretion as an indicator of damage to the lung.     

Trichloroethylene degradation by Escherichia coli containing the cloned Pseudomonas putida F1 toluene dioxygenase genes

Toluene dioxygenase from Pseudomonas putida F1 has been implicated as an enzyme capable of degrading trichloroethylene. This has now been confirmed with Escherichia coli JM109(pDTG601) that contains the structural genes (todC1C2BA) of toluene dioxygenase under the control of the tac promoter. The extent of trichloroethylene degradation by the recombinant organism depended on the cell concentration and the concentration of trichloroethylene. A linear rate of trichloroethylene degradation was observed with the E. coli recombinant strain. In contrast, P. putida F39/D, a mutant strain of P. putida F1 that does not contain cis-toluene dihydrodiol dehydrogenase, showed a much faster initial rate of trichloroethylene degradation which decreased over time.     

Trichloroethylene degradation by Escherichia coli containing the cloned Pseudomonas putida F1 toluene dioxygenase genes.

Toluene dioxygenase from Pseudomonas putida F1 has been implicated as an enzyme capable of degrading trichloroethylene. This has now been confirmed with Escherichia coli JM109(pDTG601) that contains the structural genes (todC1C2BA) of toluene dioxygenase under the control of the tac promoter. The extent of trichloroethylene degradation by the recombinant organism depended on the cell concentration and the concentration of trichloroethylene. A linear rate of trichloroethylene degradation was observed with the E. coli recombinant strain. In contrast, P. putida F39/D, a mutant strain of P. putida F1 that does not contain cis-toluene dihydrodiol dehydrogenase, showed a much faster initial rate of trichloroethylene degradation which decreased over time.     

Trichloroethylene and Human Cancer

Evidence to suggest that trichloroethylene may be a human carcinogen comes mainly from two small epidemiological studies with supporting evidence from human toxicity and genotoxicity studies and from rodent cancer bioassays. Careful analysis of these data reveal marked inconsistencies between the data, differences in the conclusions drawn by various authorities reviewing the same data and, for certain key human studies, a complete absence of exposure data. Much of the rodent cancer data may be dismissed as not indicative of a human hazard because the tumors result from either peroxisome proliferation, or as a consequence of exceptionally high metabolic rates that are found uniquely in mouse tissues, or because the tumors only occur in the presence of overt toxicity. A common mechanism invoked to account for the development of renal tumors in both rats and humans is unproven, there is contrary evidence to suggest that this mechanism does not result in renal cancer, and alternative mechanisms have been proposed. Overall, the uncertainty and lack of consistency throughout the trichloroethylene studies, whether they are in humans, in animals, or in tests in vitro, lead to the conclusion that it would be wholly inappropriate to classify trichloroethylene as a human carcinogen.     

An anaesthetic-induced phosphatidylcholine hexagonal phase

The interaction of trichloroethylene, an inhalational general anaesthetic, with phosphatidylcholine model membranes has been studied by ³¹P- and ²H-nuclear magnetic resonance spectroscopy. The incorporation of increasing amounts of trichloroethylene induces a phase transition from a multilamellar bilayer to a hexagonal phase. The effect is enhanced by increasing temperature. No indication of an intermediate cubic (C_II) phase was discovered.     

Characterization of the adaptive response to trichloroethylene-mediated stresses in Ralstonia pickettii PKO1

In Ralstonia pickettii PKO1, a denitrifying toluene oxidizer that carries a toluene-3-monooxygenase (T3MO) pathway, the biodegradation of toluene and trichloroethylene (TCE) by the organism is induced by TCE at high concentrations. In this study, the effect of TCE preexposure was studied in the context of bacterial protective response to TCE-mediated toxicity in this organism. The results of TCE degradation experiments showed that cells induced by TCE at 110 mg/liter were more tolerant to TCE-mediated stress than were those induced by TCE at lower concentrations, indicating an ability of PKO1 to adapt to TCE-mediated stress. To characterize the bacterial protective response to TCE-mediated stress, the effect of TCE itself (solvent stress) was isolated from TCE degradation-dependent stress (toxic intermediate stress) in the subsequent chlorinated ethylene toxicity assays with both nondegradable tetrachloroethylene and degradable TCE. The results of the toxicity assays showed that TCE preexposure led to an increase in tolerance to TCE degradation-dependent stress rather than to solvent stress. The possibility that such tolerance was selected by TCE degradation-dependent stress during TCE preexposure was ruled out because a similar extent of tolerance was observed in cells that were induced by toluene, whose metabolism does not produce any toxic products. These findings suggest that the adaptation of TCE-induced cells to TCE degradation-dependent stress was caused by the combined effects of solvent stress response and T3MO pathway expression.     

Dichloromethane and trichloroethylene inhibition of methane oxidation by the membrane-associated methane monooxygenase of Methylosinus trichosporium OB3b

Whole-cell assays were used to measure the effect of dichloromethane and trichloroethylene on methane oxidation by Methylosinus trichosporium OB3b synthesizing the membrane-associated or particulate methane monooxygenase (pMMO). For M. trichosporium OB3b grown with 20 μM copper, no inhibition of methane oxidation was observed in the presence of either dichloromethane or trichloroethylene. If 20 mM formate was added to the reaction vials, however, methane oxidation rates increased and inhibition of methane oxidation was observed in the presence of dichloromethane and trichloroethylene. In the presence of formate, dichloromethane acted as a competitive inhibitor, while trichloroethylene acted as a noncompetitive inhibitor. The finding of noncompetitive inhibition by trichloroethylene was further examined by measuring the inhibition constants K _iE and K _iES. These constants suggest that trichloroethylene competes with methane at some sites, although it can bind to others if methane is already bound. Whole-cell oxygen uptake experiments for active and acetylene-treated cells also showed that provision of formate could stimulate both methane and trichloroethylene oxidation and that trichloroethylene did not affect formate dehydrogenase activity. The finding that different chlorinated hydrocarbons caused different inhibition patterns can be explained by either multiple substrate binding sites existing in pMMO or multiple forms of pMMO with different activities. The whole-cell analysis performed here cannot distinguish between these models, and further work should be done on obtaining active preparations of the purified pMMO. Received: 3 November 1998 / Accepted: 1 March 1999     

Characterization of the Adaptive Response to Trichloroethylene-Mediated Stresses in Ralstonia pickettii PKO1

In Ralstonia pickettii PKO1, a denitrifying toluene oxidizer that carries a toluene-3-monooxygenase (T3MO) pathway, the biodegradation of toluene and trichloroethylene (TCE) by the organism is induced by TCE at high concentrations. In this study, the effect of TCE preexposure was studied in the context of bacterial protective response to TCE-mediated toxicity in this organism. The results of TCE degradation experiments showed that cells induced by TCE at 110 mg/liter were more tolerant to TCE-mediated stress than were those induced by TCE at lower concentrations, indicating an ability of PKO1 to adapt to TCE-mediated stress. To characterize the bacterial protective response to TCE-mediated stress, the effect of TCE itself (solvent stress) was isolated from TCE degradation-dependent stress (toxic intermediate stress) in the subsequent chlorinated ethylene toxicity assays with both nondegradable tetrachloroethylene and degradable TCE. The results of the toxicity assays showed that TCE preexposure led to an increase in tolerance to TCE degradation-dependent stress rather than to solvent stress. The possibility that such tolerance was selected by TCE degradation-dependent stress during TCE preexposure was ruled out because a similar extent of tolerance was observed in cells that were induced by toluene, whose metabolism does not produce any toxic products. These findings suggest that the adaptation of TCE-induced cells to TCE degradation-dependent stress was caused by the combined effects of solvent stress response and T3MO pathway expression.     

Antioxidant and pro-oxidant effects of a manganese porphyrin complex against CYP2E1-dependent toxicity

Superoxide dismutases (SOD) mimetics have been shown to be protective against cell injury caused by reactive oxygen species. The objective of this study was to investigate the effects of the manganese (III) tetrakis(N-methyl-2-pyridyl)porphyrin (MnTMPyP) on CYP2E1-dependent toxicity. The synergistic toxicity of iron and arachidonic acid has been associated with oxidative stress and lipid peroxidation in HepG2 cells that overexpress CYP2E1. Iron plus arachidonic acid caused loss of viability, increased lipid peroxidation and reactive oxygen species generation, and mitochondrial membrane injury in these cells. MnTMPyP partially protected against the decrease in cell viability, the enhanced lipid peroxidation and oxygen radical production, and the loss of mitochondrial membrane potential. The effect of MnTMPyP on arachidonic acid (absence of iron) toxicity was also evaluated. Arachidonic acid also caused toxicity, lipid peroxidation and reduction of the mitochondrial membrane potential. However, in this model, all of these alterations were actually enhanced by MnTMPyP. MnTMPyP also enhanced toxicity in CYP2E1-expressing HepG2 cells depleted of reduced glutathione (GSH). MnCl(2) had little or no effect on the toxicity by arachidonic acid, and MnTMPyP itself did not peroxidize arachidonic acid. MnTMPyP, an SOD mimetic that also scavenges hydrogen peroxide and peroxynitrite, thus showed an antioxidant and protective effect against iron plus arachidonic acid toxicity, but a pro-oxidant and cytotoxic effect against arachidonic acid toxicity in CYP2E1-expressing cells. These different actions may relate to the ability of MnTMPyP to either scavenge or produce free radicals in cells depending upon the prevailing MnTMPyP oxidation-reduction pathways. MnTMPyP and related manganese porphyrin compounds may have potential clinical utility against diseases associated with the overproduction of reactive oxygen species such as ethanol-induced liver injury but it is clear that further investigation of all the pathways of manganese porphyrin oxidation-reduction are necessary.     

Hsp105alpha suppresses the aggregation of truncated androgen receptor with expanded CAG repeats and cell toxicity

Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disorder caused by the expansion of a polyglutamine tract in the androgen receptor (AR). The N-terminal fragment of AR containing the expanded polyglutamine tract aggregates in cytoplasm and/or in nucleus and induces cell death. Some chaperones such as Hsp40 and Hsp70 have been identified as important regulators of polyglutamine aggregation and/or cell death in neuronal cells. Recently, Hsp105alpha, expressed at especially high levels in mammalian brain, has been shown to suppress apoptosis in neuronal cells and prevent the aggregation of protein caused by heat shock in vitro. However, its role in polyglutamine-mediated cell death and toxicity has not been studied. In the present study, we examined the effects of Hsp105alpha on the aggregation and cell toxicity caused by expansion of the polyglutamine tract using a cellular model of SBMA. The transient expression of truncated ARs (tARs) containing an expanded polyglutamine tract caused aggregates to form in COS-7 and SK-N-SH cells and concomitantly apoptosis in the cells with the nuclear aggregates. When Hsp105alpha was overexpressed with tAR97 in the cells, Hsp105alpha was colocalized to aggregates of tAR97, and the aggregation and cell toxicity caused by expansion of the polyglutamine tract were markedly reduced. Both beta-sheet and alpha-helix domains, but not the ATPase domain, of Hsp105alpha were necessary to suppress the formation of aggregates in vivo and in vitro. Furthermore, Hsp105alpha was found to localize in nuclear inclusions formed by ARs containing an expanded polyglutamine tract in tissues of patients and transgenic mice with SBMA. These findings suggest that overexpression of Hsp105alpha suppresses cell death caused by expansion of the polyglutamine tract without chaperone activity, and the enhanced expression of the essential domains of Hsp105alpha in brain may provide an effective therapeutic approach for CAG repeat diseases.     

炎症标记物与缺血性中风预后评价

缺血性中风触发的炎症反应是一个级联放大过程,不仅可直接对缺血脑组织造成继发性损伤,还可通过与其他病理生理通路的相互影响、相互促进,共同对缺血后脑组织造成不可逆损伤。因此,采用炎症标记物对脑缺血损伤及其预后进行评价,具有重要临床意义。临床研究发现,多炎症标记物法用于缺血性中风的诊治和预后评价比单炎症标记物法更全面、更准确,故更具明显优势。综述脑缺血引发的炎症机制、脑缺血所致炎症通路与其他病理生理通路( 如氧化应激、细胞凋亡和兴奋性毒性) 的关联以及炎症标记物在缺血性中风预后评价中的应用。     

鸡脚参醇提物药理活性研究

对鸡脚参醇提物进行了抗炎、止咳作用的研究。结果表明其活性部位对大鼠蛋清性足肿胀、小鼠耳廓肿胀有明显的抑制作用,有抑制毛细血管通透性作用的趋势。可延长二氧化硫引起的小白鼠咳嗽的潜伏期。鸡脚参醇提物的急性毒性较小,抗炎、止咳作用明显。     

Biodegradation of trichloroethylene by Methylosinus trichosporium OB3b

The methanotroph Methylosinus trichosporium OB3b, a type II methanotroph, degraded trichloroethylene at rates exceeding 1.2 mmol/h per g (dry weight) following the appearance of soluble methane monooxygenase in continuous and batch cultures. Cells capable oxidizing trichloroethylene contained components of soluble methane monooxygenase as demonstrated by Western blot (immunoblot) analysis with antibodies prepared against the purified enzyme. Growth of cultures in a medium containing 0.25 microM or less copper sulfate caused derepression of the synthesis of soluble methane monooxygenase. In these cultures, the specific rates of methane and methanol oxidation did not change during growth, while trichloroethylene oxidation increased with the appearance of soluble methane monooxygenase. M. trichosporium OB3b cells that contained soluble methane monooxygenase also degraded vinyl chloride, 1,1-dichloroethylene, cis-1,2-dichloroethylene, and trans-1,2-dichloroethylene.     

Biodegradation of trichloroethylene by Methylosinus trichosporium OB3b.

The methanotroph Methylosinus trichosporium OB3b, a type II methanotroph, degraded trichloroethylene at rates exceeding 1.2 mmol/h per g (dry weight) following the appearance of soluble methane monooxygenase in continuous and batch cultures. Cells capable oxidizing trichloroethylene contained components of soluble methane monooxygenase as demonstrated by Western blot (immunoblot) analysis with antibodies prepared against the purified enzyme. Growth of cultures in a medium containing 0.25 microM or less copper sulfate caused derepression of the synthesis of soluble methane monooxygenase. In these cultures, the specific rates of methane and methanol oxidation did not change during growth, while trichloroethylene oxidation increased with the appearance of soluble methane monooxygenase. M. trichosporium OB3b cells that contained soluble methane monooxygenase also degraded vinyl chloride, 1,1-dichloroethylene, cis-1,2-dichloroethylene, and trans-1,2-dichloroethylene.     

Evaluation of bacterial aerotaxis for its potential use in detecting the toxicity of chemicals to microorganisms

Bacterial aerotaxis (the movement of a cell toward oxygen) was evaluated for its potential use in detecting the toxicity of chemicals to microorganisms. The level of toxicity was determined by the concentration of test chemicals resulting in a 50% inhibition of aerotaxis of Pseudomonas aeruginosa PAO1 after 40 min of exposure. The aerotactic responses of P. aeruginosa were measured by using chemotaxis well chambers. Each clear acrylic chamber had a lower and upper well separated by a polycarbonate filter with a uniform pore size of 8.0 microm. To automatically detect bacterial cells that crossed the filter in response to a gradient of oxygen, P. aeruginosa PAO1 was marked with green fluorescent protein (GFP), and the GFP fluorescence intensity in the upper well was continuously monitored by using a fluorescence spectrometer. By using this technique, volatile chlorinated aliphatic compounds, including trichloroethylene (TCE), trichloroethane, and tetrachloroethylene, were found to be inhibitory to bacterial aerotaxis, suggesting their possible toxicity to microorganisms. We also examined more than 20 potential toxicants for their ability to inhibit the aerotaxis of P. aeruginosa. Based on these experimental results, we concluded that bacterial aerotaxis has potential for use as a fast and reliable indicator in assessing the toxicity of chemicals to microorganisms.     

Mitochondria‐mediated mitigatory role of curcumin in cisplatin‐induced nephrotoxicity

Cisplatin (CP) is one of the most potent chemotherapeutic anti‐tumour drugs, and it has been implicated in renal toxicity. Oxidative stress has been proven to be involved in CP‐induced toxicity including nephrotoxicity. However, there is paucity of literature involving role of mitochondria in mediating CP‐induced renal toxicity, and its underlying mechanism remains unclear. Therefore, the present study was undertaken to examine the antioxidant potential of curcumin (CMN; a natural polyphenolic compound) against the mitochondrial toxicity of CP in kidneys of male rats. Acute toxicity was induced by a single intra‐peritoneal injection of CP (6 mg kg^?1). We studied the ameliorative effect of CMN pre‐treatment (200 mg kg^?1) on the toxicity of CP in rat kidney mitochondria. CP caused a significant elevation in the mitochondrial lipid peroxidation (LPO) levels and protein carbonyl (PC) content. Pre‐treatment of rat with CMN significantly replenished the mitochondrial LPO levels and PC content. It also restored the CP‐induced modulatory effects on altered enzymatic and non‐enzymatic antioxidants in kidney mitochondria. We hypothesize that the reno‐protective effects of CMN may be related to its predisposition to scavenge free radicals, and upregulate antioxidant machinery in kidney mitochondria. Copyright © 2013 John Wiley & Sons, Ltd.     

Arsenate toxicity and stress responses in the freshwater ciliate Tetrahymena pyriformis

The arsenic metabolism in different biological organisms has been studied extensively. However, little is known about protozoa. Herein, we investigated the cell stress responses of the freshwater ciliate Tetrahymena pyriformis to arsenate toxicity. An acute toxicity assay revealed an 18-h EC(50) arsenate concentration of ca. 40 μM, which caused significant changes in the cell shape, growth and organism mobility. Whereas, under exposure to 30 μM arsenate, T. pyriformis could grow reasonably well, indicating a certain resistance of this organism. Arsenic speciation analysis revealed that 94-98% of the total arsenate in cells of T. pyriformis could be transformed to monomethylarsonic acid, dimethylarsinic acid and a small proportion of arsenite after 18 h of arsenate exposure, thus indicating the major detoxification pathway by arsenic oxidation/reduction and biomethylation. Finally, comparative proteomic analysis unveiled significant changes in the expression of multiple proteins involved in anti-oxidation, sugar and energy metabolism, proteolysis, and signal transduction. Our results revealed multiple pathways of arsenate detoxification in T. pyriformis, and indicated that protozoa may play important roles in the biogeochemical cycles of arsenic.     

Computer simulation of the distribution of aluminum speciation in soil solutions in equilibrium with the mineral phase imogolite.

The speciation of aluminum (Al) is a critical issue when evaluating the environmental and biological significance of elevated Al concentrations in soil solutions caused by acidic precipitation. Numerous studies have revealed that, with increased concentrations of silica acid in soil, the activity of Al species in soil solutions is greatly modified by SiO(4)(2-). However, thus far there has been little thorough theoretical modeling of this subject. This paper reports a computer simulation of the distribution of Al speciation in soil solutions in equilibrium with the mineral phase imogolite based on a chemical equilibrium calculation. The unique characteristic associated with imogolite reported by previous researchers can be explained theoretically by the proposed model. The dissolved silica has a remarkable influence on Al speciation: increasing concentrations of silica acid may effectively inhibit the formation of polymeric alumino-hydroxo species, and, furthermore, detoxify Al toxicity to plants.