An integrative investigation of the therapeutic mechanism of Ainsliaea fragrans Champ. in cervicitis using liquid chromatography tandem mass spectrometry based on a rat plasma metabolomics strategy.

Cervicitis is an extremely common gynecological disease and can be induced by diverse factors such as Neisseria gonorrhoeae, Chlamydia trachomatis, and Mycoplasma genitalium infections. Long-term unhealed cervicitis may lead to a series of diseases including endometritis, salpingitis, pelvic inflammatory disease, and chorioamnionitis. However, the pathogenesis of cervicitis remains unknown. Ainsliaea fragrans Champ. (AFC) has been widely used in clinical treatment of cervicitis. In the present study, we performed an integrative investigation involving histopathology analysis and non-target plasma metabolomics analysis in a cervicitis rat model induced by phenol mucilage, using ultra-performance liquid chromatography coupled with a tandem quadrupole time-of-flight mass spectrometry approach. Based on the integrative investigation, marked metabolomic differences were identified between the cervicitis and control groups using multivariate analysis. As a result, 32 potential biomarkers were identified in the response to cervicitis, and were involved in arachidonic acid metabolism, linoleic acid metabolism, primary bile acid biosynthesis, taurine and hypotaurine metabolism, pantothenate and CoA biosynthesis, and glycerophospholipid metabolism. After treatment, a total of 27 potential biomarkers exhibited altered levels in the AFC group compared to the model group, and 12 metabolites including 1-stearoylglycerophosphoinositol, bolasterone, lysoPC(16:0), lysoPC(20:4), lysoPC(P-16:0), lysoPC(P-18:0), lysoPC(P-18:1), stearoylcarnitine, taurine, lysoPC(17:0), 20-hydroxyeicosatetraenoic acid, and 1-arachidonoylglycerophosphoinositol returned to their normal levels. This study suggested that the therapeutic mechanism of AFC is related to those altered endogenous metabolites.

An approach to study ultrastructural changes and adaptive strategies displayed by Acinetobacter guillouiae SFC 500-1A under simultaneous Cr(VI) and phenol treatment.

Acinetobacter guillouiae SFC 500-1A, a native bacterial strain isolated from tannery sediments, is able to simultaneously remove high concentrations of Cr(VI) and phenol. In this complementary study, high-resolution microscopy techniques, such as atomic force microscopy (AFM) and transmission electron microscopy (TEM), were used to improve our understanding of some bacterial adaptive mechanisms that enhance their ability to survive. AFM contributed in gaining insight into changes in bacterial size and morphology. It allowed the unambiguous identification of pollutant-induced cellular disturbances and the visualization of bacterial cells with depth sensitivity. TEM analysis revealed that Cr(VI) produced changes mainly at the intracellular level, whereas phenol produced alterations at the membrane level. This strain tended to form more extensive biofilms after phenol treatment, which was consistent with microscopy images and the production of exopolysaccharides (EPSs). In addition, other exopolymeric substances (DNA, proteins) significantly increased under Cr(VI) and phenol treatment. These exopolymers are important for biofilm formation playing a key role in bacterial aggregate stability, being especially useful for bioremediation of environmental pollutants. This study yields the first direct evidences of a range of different changes in A. guillouiae SFC 500-1A which seems to be adaptive strategies to survive in stressful conditions.

Marrubium vulgare ethanolic extract induces proliferation block, apoptosis, and cytoprotective autophagy in cancer cells in vitro.

Marrubium vulgare is a European medicinal plant with numerous beneficial effects on human health. The aim of the study was to isolate the plant ethanolic extract (MVE) and to investigate its anti-melanoma and anti-glioma effects. MVE was prepared by the modified pharmacopoeial percolation method and characterized by UHPLC-LTQ OrbiTrap MS. MVE dose-dependently reduced viability of melanoma (B16) and glioma (U251) cells, but not peripheral blood mononuclear cells. It arrested cell cycle in S+G2/M phase, which was associated with the activation of MAP kinase p38 and up-regulation of antiproliferative genes p53, p21 and p27. MVE induced oxidative stress, while antioxidants abrogated its antitumor effect. Furthermore, MVE induced mitochondrial depolarization, activation of caspase-9 and -3, Parp cleavage, phosphatidylserine exposure and DNA fragmentation. The mitochondrial apoptotic pathway was associated with the up-regulation of proapoptotic genes Pten, Bak1, Apaf1, and Puma and down-regulation of antiapoptotic genes survivin and Xiap. MVE also stimulated the expression of autophagy-related genes Atg5, Atg7, Atg12, Beclin-1, Gabarab and Sqstm1, as well as LC3-I conversion to the autophagosome associated LC3-II, while autophagy inhibitors exacerbated its cytotoxicity. Finally, the most abundant phenolic components of MVE, ferulic, p-hydroxybenzoic, caffeic and chlorogenic acids, did not exert a profound effect on viability of tumor cells, suggesting that other components individually or in concert are the mediators of the extracts' cytotoxicity. By demonstrating the ability of MVE to inhibit proliferation, induce apoptosis and cytoprotective autophagy, our results suggest that MVE, alone or combined with autophagy inhibitors, could be a good candidate for anti-melanoma and anti-glioma therapy.

Effect of biosurfactants on laccase production and phenol biodegradation in solid-state fermentation.

The effects of two biosurfactants, tea saponin (TS) and rhamnolipid (RL), on the production of laccase and the degradation of phenol by P. simplicissimum were investigated in solid-state fermentation consisting of rice straw, rice bran, and sawdust. Firstly, the effects of phenol on the fermentation process were studied in the absence of surfactants. Then, a phenol concentration of 3 mg/g in the fermentation was selected for detailed research with the addition of biosurfactants. The results showed that TS and RL at different concentrations had stimulative effects on the enzyme activity of laccase. The highest laccase activities during the fermentation were enhanced by 163.7%, 68.2%, and 23.3% by TS at concentrations of 0.02%, 0.06%, and 0.10%, respectively. As a result of the enhanced laccase activity, the efficiency of phenol degradation was also improved by both biosurfactants. RL caused a significant increase of fungal biomass in the early stage of the fermentation, while TS had an inhibitory effect in the whole process. These results indicated that RL could mitigate the negative effects of phenol on fungal growth and consequently improve laccase production and phenol degradation. TS was potentially applicable to phenol-polluted solid-state fermentation.

Candidate biomarker discovery in plasma of juvenile cod (Gadus morhua) exposed to crude North Sea oil, alkyl phenols and polycyclic aromatic hydrocarbons (PAHs).

In this study we have investigated protein changes in plasma of juvenile Atlantic cod (Gadus morhua) induced by crude North Sea oil and North Sea oil spiked with alkyl phenols and polycyclic aromatic hydrocarbons, a surrogate produced water composition. Using a proteomic approach, we identified 137 differentially expressed proteins at different levels of crude oil exposure. Many of the induced protein changes occurred at low levels of exposure. The results obtained with protein expression profiles after exposure to oil and surrogate produced water indicate effects on fibrinolysis and the complement cascade, the immune system, fertility-linked proteins, bone resorption, fatty acid metabolism as well as increased oxidative stress, impaired cell mobility and increased levels of proteins associated with apoptosis. Although the number of individuals and samples in this study is limited within each treatment group, the protein changes observed in this study represent a first screening for potential biomarker candidates in cod plasma reflecting potential effects of crude oil and produced water exposure on fish.

Acute toxicity evaluation of proliferol: a dose-escalating, placebo-controlled study in swine.

Prolotherapy is one of the many treatments available for chronic musculoskeletal disorders. A commonly used drug contains dextrose 12.5%, glycerin 12.5%, phenol 1.0%, and lidocaine hydrochloride 0.25% in aqueous solution (recently termed Proliferol). For chronic low back pain, this is injected into lumbosacral ligaments to stimulate connective tissue repair. Despite generally positive clinical results, the toxicity of this drug is not well characterized and was assessed in 48 (24 male, 24 female) Yucatan miniature swine randomly assigned to low (1x), medium (5x), or high (10x) dose or saline placebo. Outcomes included clinical observations, clinical chemistry, hematology, coagulation, urinalysis, toxicokinetics, and full gross and microscopic histopathology after 24 hours or 14 days. Findings attributable to Proliferol after 24 hours included dose-response elevations in alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and creatine kinase, which returned to normal after 14 days. There were no remarkable findings in hematology, coagulation, or urinalysis. Urine concentrations of lidocaine and phenol both peaked after 8 hours. Histopathology findings after 24 hours included hemorrhage, inflammation, necrosis, and vascular changes in the ligaments and adjacent soft tissues at the sites of injection. After 14 days, there was evidence of repair under way, with fibrosis and skeletal muscle regeneration at the injection sites.

Green tea attenuates benzene-induced oxidative stress in pump workers.

Workers exposed to benzene frequently suffer from toxicities of the bone marrow as well as the central nervous, immune, and reproductive systems. This toxicity most likely is a result of the oxidative metabolism of benzene to reactive products. As green tea possesses antioxidant effects, the objective of this study was to examine any amelioration of benzene-induced oxidative stress in pump workers drinking 6 cups (150 ml/cup) of freshly prepared tea daily. Sixty male non-smoking subjects, divided into four groups: no benzene exposure/no green tea; no exposure/tea; exposure/no tea; and, exposure/tea, were monitored after a 6 mo period. On the final day of the study, urine samples were collected for analyses of benzene, trans-trans muconic acid, and phenol. Blood was also collected at this time; plasma was assayed for total antioxidant activity, malondialdehyde (MDA), and glutathione (GSH) while erythrocytes were analyzed for activity of antioxidant enzymes glutathione peroxidase (GSHPX), superoxide dismutase (SOD), and catalase. The results demonstrated that urinary levels of benzene, trans-trans muconic acid, and phenol were elevated in all pump workers, and that this elevation was mitigated by consumption of green tea. The benzene exposures also led to significant reductions in plasma GSH levels and erythrocyte antioxidant enzyme activities; these effects were abrogated (to near-control levels) by the tea. Interestingly, among control subjects, tea ingestion itself caused significant increases in both GSHPX and catalase activities. Unlike with the other plasma parameters, while the benzene exposures also significantly increased plasma MDA levels and decreased total antioxidant activity, tea ingestion did not cause a near-total reversion to control values; the effects on these two endpoints were more like those noted with the urine parameters (mitigation, not abrogation). These studies demonstrate that drinking green tea during benzene exposure can reduce several parameters indicative of oxidative stress. As such, as a dietary supplement, green tea could represent a potential therapeutic agent in reducing certain aspects of benzene-induced toxicity.

Potential of vetiver (Vetiveria zizanoides L. Nash) for phytoremediation of phenol.

Aseptically grown Vetiveria zizanoides were evaluated for their potential for phytoremediation of phenol from Murashige and Skoog's liquid medium. Phenol was found to be completely removed from incubation medium at the end of 4 days by V. zizanoides plantlets, when medium was supplemented with 50 and 100 mg L(-1) phenol, while with 200, 500, and 1000 mg L(-1) of phenol, 89%, 76% and 70%, respectively, were removed. Phenol removal was found to be associated with inherent production of peroxidase and hydrogen peroxide. Coupled with H(2)O(2) formation, the levels of antioxidant enzymes like superoxide dismutase and peroxidase showed an enhancement when plants were exposed to phenol, whereas catalase levels initially showed a decline due to the utilization of H(2)O(2) by peroxidase for phenol oxidation. However, when peroxidase levels declined, there was an enhancement in catalase levels to minimize the presence of H(2)O(2) in the medium. Having confirmed that the removal of phenol was by V. zizanoides plantlets, in the next phase, micropropagated plantlets and well-developed plants grown in hydroponics were used under in vivo conditions to study the effect of phenol (200 mg L(-1)) on plant growth and reuse. Although plant growth was reduced in presence of phenol, the results of the reuse study indicated the possibility of plants getting adapted to phenol without any decline in potential for phenol remediation.

Evolution of ecotoxicity upon Fenton's oxidation of phenol in water.

This work deals with the evolution of intermediates and ecotoxicity upon Fenton's oxidation of phenol in aqueous solution. The EC50 values of the intermediates identified in the oxidation pathway of phenol have been measured. Some of these compounds, mainly hydroquinone and p-benzoquinone, showed toxicity levels much higher than phenol itself. Depending on the operating conditions, these intermediates could be completely transformed into organic acids, mainly oxalic and formic. Ecotoxicity values substantially lower than those expected from the chemical composition were measured in the reaction samples. This is explained by a reduction of the concentration of aromatic intermediates when the pH was adjusted at 6-8 (according to what is required by the standard bioassay ISO 11348-3). Formation of complexes between hydroquinone and p-benzoquinone at increasing pH can remove from solution those highly toxic intermediates whose very low EC50 values give rise to a high ecotoxicity even at fairly low concentrations. This together with the enhanced decomposition of residual H202 at increasing pH represent important beneficial effects of the neutralization step following Fenton treatment which allow a complementary cleaning of the effluent.

Bioenergetic effects of sodium sulfate on the freshwater crustacean, Ceriodaphnia dubia.

I tested the hypothesis that if sodium sulfate alters the bioenergetics of Ceriodaphnia dubia, concentrations that cause reduced fecundity in the short (7-day) and long (5 generations) term should also cause changes in feeding rate and/or metabolism, measured as oxygen consumption. In addition, to test the hypothesis that an altered bioenergetic level caused by sodium sulfate exposure will affect the response of that organism to another toxicant, I measured the acute toxicity of phenol to C. dubia in the presence and absence of both food and sodium sulfate. Sodium sulfate reduced the filter-feeding rate of C. dubia, which was associated with significantly reduced oxygen consumption. This decreased energy level appeared to result in a consistent but decreased level of fecundity over a number of generations and the reproductive impairment was dose-dependent. These effects occurred at concentrations much lower than those at which acute (mortality) effects have been observed, a finding that may have regulatory implications. In addition, whereas phenol toxicity to C. dubia was exacerbated by the addition of food, increased phenol toxicity, likely induced by an increase in filtering or metabolic rate due to food addition, was negated when sodium sulfate was added to the test medium.

Solar-based detoxification of phenol and p-nitrophenol by sequential TiO2 photocatalysis and photosynthetically aerated biological treatment.

Simulated solar UV/TiO(2) photocatalysis was efficient to detoxify a mixture of 100 mgphenoll(-1) and 50 mgp-nitrophenol (PNP) l(-1) and allow the subsequent biodegradation of the remaining pollutants and their photocatalytic products under photosynthetic aeration with Chlorella vulgaris. Photocatalytic degradation of phenol and PNP was well described by pseudo-first order kinetics (r(2)>0.98) with removal rate constants of 1.9x10(-4) and 2.8x10(-4)min(-1), respectively, when the pollutants were provided together and 5.7x10(-4) and 9.7x10(-4)min(-1), respectively, when they were provided individually. Photocatalytic pre-treatment of the mixture during 60 h removed 50+/-1% and 62+/-2% of the phenol and PNP initially present but only 11+/-3% of the initial COD. Hydroquinone, nitrate and catechol were identified as PNP photocatalytic products and catechol and hydroquinone as phenol photocatalytic products. Subsequent biological treatment of the pre-treated samples removed the remaining contaminants and their photocatalytic products as well as 81-83% of the initial COD, allowing complete detoxification of the mixture to C. vulgaris. Similar detoxification efficiencies were recorded after biological treatment of the irradiated mixture with activated sludge microflora or with an acclimated consortia composed of a phenol-degrading Alcaligenes sp. and a PNP-degrading Arthrobacter sp., although the acclimated strains biodegraded the remaining pollutants faster. Biological treatment of the non-irradiated mixture was inefficient due to C. vulgaris inhibition.

Report of the IWGT working group on strategies and interpretation of regulatory in vivo tests I. Increases in micronucleated bone marrow cells in rodents that do not indicate genotoxic hazards.

In vivo genotoxicity tests play a pivotal role in genotoxicity testing batteries. They are used both to determine if potential genotoxicity observed in vitro is realised in vivo and to detect any genotoxic carcinogens that are poorly detected in vitro. It is recognised that individual in vivo genotoxicity tests have limited sensitivity but good specificity. Thus, a positive result from the established in vivo assays is taken as strong evidence for genotoxic carcinogenicity of the compound tested. However, there is a growing body of evidence that compound-related disturbances in the physiology of the rodents used in these assays can result in increases in micronucleated cells in the bone marrow that are not related to the intrinsic genotoxicity of the compound under test. For rodent bone marrow or peripheral blood micronucleus tests, these disturbances include changes in core body temperature (hypothermia and hyperthermia) and increases in erythropoiesis following prior toxicity to erythroblasts or by direct stimulation of cell division in these cells. This paper reviews relevant data from the literature and also previously unpublished data obtained from a questionnaire devised by the IWGT working group. Regulatory implications of these findings are discussed and flow diagrams have been provided to aid in interpretation and decision-making when such changes in physiology are suspected.

Anaerobic biological treatment of phenol at 9.5-15 degrees C in an expanded granular sludge bed (EGSB)-based bioreactor.

The aims of this study were to demonstrate the (1) feasibility of psychrophilic, or low-temperature, anaerobic digestion (PAD) of phenolic wastewaters at 10-15 degrees C; (2) economic attractiveness of PAD for the treatment of phenol as measured by daily biogas yields and (3) impact on bioreactor performance of phenol loading rates (PLRs) in excess of those previously documented (1.2 kg phenol m(-3)d(-1)). Two expanded granular sludge bed (EGSB)-based bioreactors, R1 and R2, were employed to mineralise a volatile fatty acid-based wastewater. R2 influent wastewater was supplemented with phenol at an initial concentration of 500 mgl(-1) (PLR, 1 kgm(-3)d(-1)). Reactor performance was measured by chemical oxygen demand (COD) removal efficiency, CH(4) composition of biogas and phenol removal (R2 only). Specific methanogenic activity, biodegradability and toxicity assays were employed to monitor the physiological capacity of reactor biomass samples. The applied PLR was increased to 2 kgm(-3)d(-1) on day 147 and phenol removal by day 415 was 99% efficient, with 4 mgl(-1) present in R2 effluent. The operational temperature of R1 (control) and R2 was reduced by stepwise decrements from 15 degrees C through to a final operating temperature of 9.5 degrees C. COD removal efficiencies of c. 90% were recorded in both bioreactors at the conclusion of the trial (day 673), when the phenol concentration in R2 effluent was below 30 mgl(-1). Daily biogas yields were determined during the final (9.5 degrees C) operating period, when typical daily R2 CH4 yields of c. 3.3lCH4g(-1) COD(removed) d(-1) were recorded. The rate of phenol depletion and methanation by R2 biomass by day 673 were 68 mg phenol gVSS(-1)d(-1) and 12-20 ml CH(4) gVSS(-1)d(-1), respectively.

Uptake and transformation of phenol and chlorophenols by hairy root cultures of Daucus carota, Ipomoea batatas and Solanum aviculare.

Hairy root cultures of Daucus carota L., Ipomoea batatas L. and Solanum aviculare Forst were investigated for their susceptibility to the highly toxic pollutants phenol and chlorophenols and for the involvement of inherent peroxidases in the removal of phenols from liquid media. Roots of D. carota grew normally in medium containing 1000 micromol l(-1) of phenol, whilst normal growth of roots of I. batatas and S. aviculare was only possible at levels up to 500 micromol l(-1). In the presence of chlorophenols, normal root growth was possible only in concentrations not exceeding 50 micromol l(-1), except for I. batatas which was severely affected at all concentrations. Despite the reduction in biomass, the growth of S. aviculare cultures was sustained in medium containing up to 2000 micromol l(-1) of phenol or 2-chlorophenol, and up to 500 micromol l(-1) of 2,6-dichlorophenol. The amounts of phenol removed by the roots within 72 h of treatment were 72.7%, 90.7% and 98.6% of the initial concentration for D. carota, I. batatas and S. aviculare, respectively. For the removal of 2,6-dichlorophenol the values were, respectively, 83.0%, 57.7% and 73.1%. Phenols labelled with 14C were absorbed by the root tissues and condensed with highly polar cellular substances as well as being incorporated into the cell walls or membranes. The results suggest that S. aviculare, an ornamental plant, would be best suited for remediation trials under field conditions.

Phenol degradation and toxicity assessment upon biostimulation to an indigenous Rhizobium Ralstonia taiwanensis.

This study provides a first attempt from a toxicological perspective to put forward, in general terms and explanations, combined toxic interactions and biostimulation strategy upon nutrient medium to Ralstonia taiwanensis for bioremediation. Dose-response analysis clearly revealed that most of the supplemented nutrients tested (except for gluconic acid) synergistically interact with chronic toxicity to phenol, especially at low doses. Acute toxicity based upon adaptation lag is a more appropriate indicator for comparative analysis of toxicity due to similar toxic ranking at almost all effective concentrations. In addition, comparison upon acute and chronic toxicity for various nutrient media also suggests in parallel that acute toxicity is more significant than chronic toxicity possibly as the result of a more sensitive response of adaptation lag to growth in different media. Feasibility of adding extra nutrient substrates (e.g., phenol, gluconic acid, yeast extract, pyruvic acid, acetic acid, and glycerol) to stimulate proliferation of phenol degraders for better phenol degradation performance was also assessed. The results show that using acetic acid as the augmented nutrient source might be the most feasible biostimulation strategy for phenol degradation.

Describing phytotoxic effects on cumulative germination.

Phytotoxic studies strongly depend on evaluation of germination responses, which implies the need for adequate procedures to account for distinct aspects of the germinative process. For this, indices, comparisons among treatments at various times, and model fitting have been proposed. The objective of this work is to compare the three approaches and select the one providing the greatest insight and precision. Speed of germination, speed of accumulated germination, the coefficient of the rate of germination, comparisons at each determination time, including final germination, and the parameters of the Weibull function were examined. The Weibull function proved the best approach to describe the germination process, providing not only the same type of information about the speed of germination, with greater precision, but also additional information about the initiation and shape of the germination response curve.

Detoxication of phenol in annual plant seedlings.

Transformation of phenol (14C6H5OH) penetrating through the roots of mung bean (Phaseolus aureus) and wheat (Triticum vulgare) sterile seedlings has been studied. Phenol was coupled to low-molecular-weight peptides, producing phenol-peptide conjugates. Hydrolytic cleavage of the conjugates liberated initial labeled phenol and some unlabeled amino acids. Phenol- glutathione and phenol-homoglutathione were not found among the peptide conjugates. It is suggested that the conjugation is carried out via the hydroxyl group of phenol and functional groups of peptides. Conjugation with low-molecular-weight peptides is considered to be the main pathway for phenol detoxication, since about 60% of phenol absorbed by plants conjugates with peptides. In the plants treated with phenol, the amount of low-molecular-weight peptides is increased. The increase in peptide synthesis in plants seems to be induced by the penetration of toxic phenol molecules into the cell. The small amount of phenol molecules assimilated through roots is transformed via aromatic ring cleavage and bibasic carbonic acid formation.

A limited renal injury may cause a permanent form of neurogenic hypertension.

Previously, we have shown that an acute injury to the kidney produced by an intrarenal injection of phenol causes an immediate increase in blood pressure and in norepinephrine (NE) secretion from the posterior hypothalamus. The studies suggest that in this model afferent impulses from the kidney to central integrative structures in the brain may be responsible for the increase in blood pressure. To further evaluate whether a renal injury caused by the intrarenal injection of phenol leads to a permanent elevation of blood pressure and whether this is mediated by increased sympathetic nervous system activity, we examined the chronic effects (4 weeks) of an intrarenal injection of 50 microL of 10% phenol on blood pressure and NE secretion from the posterior hypothalamus. Systolic blood pressure increased from 128 +/- 2.1 to 176 +/- 1.5 mm Hg (P < .01) 4 weeks after receiving the intrarenal injection of phenol, but it did not change in rats that received the vehicle (128 +/- 2.4 and 135 +/- 1.7 mm Hg) and in rats that were subjected to renal denervation (127 +/- 3.4 and 124 +/- 1.0 mm Hg). The secretion of NE from the posterior hypothalamic nuclei was greater (P < .01) in rats that received phenol (253 +/- 9.6 pg/mL) than in controls (158 +/- 8.6 pg/mL) and denervated rats (170 +/- 2.1 pg/mL). These studies have shown that a limited injury to one kidney may cause a permanent elevation of blood pressure and this is associated with increased sympathetic nervous system activity.