Effect of phenol red and steroid hormones on cystic fibrosis transmembrane conductance regulator in mouse endometrial epithelial cells

Previous studies have demonstrated that cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-mediated Cl(-)channel found in most epithelia including reproductive tract, could be regulated by various culture conditions. The present study further investigated the effect of phenol red, a pH indicator widely used in growth medium, and steroid hormones, present in the supplement fetal bovine serum (FBS), on primary cultured endometrial epithelial cells by monitoring ion channel activities using the short-circuit current technique. When compared to the results obtained with normal medium supplemented with regular FBS, the forskolin-stimulated I(SC), presumably mediated by CFTR, obtained in phenol red-free medium was significantly reduced, from 16.95+/-1.53 microA/cm(2)(control) to 9.72+/-0.89 microA/cm(2)(medium without phenol red, P< 0.05). The forskolin-activated I(SC)was further attenuated to 5.29+/-0.46 microA/cm(2)in the phenol red-free medium when supplemented with charcoal/ dextran-treated FBS where steroid hormones were removed. Our data suggest that phenol red and steroid hormones present in culture medium and FBS supplement, respectively, may somehow upregulate CFTR expression in vitro. Our study demonstrates the need for carefully choosing the culture media and supplements due to the effect of steroid hormones.     

Phenol removal in packed bed reactor under denitrifying conditions

Detailed studies on the efficiency of phenol degradation by a biofilm in an anaerobic packed bed reactor were carried out. The efficiency of phenol degradation depended on both the concentration of phenol in the medium and the phenol load in anaerobic packed bed reactor. Increasing phenol concentrations from 200 to 1,250 mg l(-1) and retention time (Tr)= 12 h were paralleled by increasing efficiency of the process, which reached a maximum value of 1,390 mg l(-1) day(-1) at 700 mg phenol l(-1). The highest concentration of phenol used inhibited growth by approximately 95%. When the phenol load in medium containing 200, 300, 400 and 500 mg l(-1) was increased through a shortening of the retention time (Tr from 24 to 2 h) a maximum efficiency of phenol degradation of 2,200 mg l(-1) day(-1) was obtained at Tr=4 h and phenol concentrations in the medium of 200 mg l(-1). Phenol in concentrations from 300 to 500 mg l(-1) was fully degraded at Tr>9 h and phenol load reaching 530-1330 mg l(-1) day(-1) for the individual concentrations. The post-denitrification effluent leaving packed bed reactor in spite of the absence or even trace amounts of phenol in it requires further purification.     

Isolation and Properties of Phenol Utilizing Microorganisms with Special Reference to Catechol 1,2-Oxygenase

Phenol utilizing yeasts were isolated from soil. The relationship were examined between distribution of phenol uptake rate using intact cells and distribution of the activities of catechol 1,2-oxygenase which is one of the key enzymes in phenol metabolism. Two of the isolates showed catechol 1,2-oxygenase activity even when grown in glucose medium, though the enzyme activity was about 1% of the full activity induced by phenol. Partially constitutive mutants for catechol 1,2-oxygenase were obtained by mutagenesis of an inducible strain. The level of mutant enzyme activity was close to that of the isolated constitutive strain. One isolate, Trichosporon cutaneum, preferentially utilized phenol to glucose in medium containing both phenol (200 ppm) and glucose (0.1%), until the concentration of phenol decreased to 10–20 ppm.     

Characterization of the phenol monooxygenase gene from Chromobacterium violaceum: Potential use for phenol biodegradation

In this work, the biodegradation mechanism of phenol and sub products (such as catechol and hydroquinone) in Chromobacterium violaceum was investigated by cloning and molecular characterization of a phenol monooxygenase gene in Escherichia coli. This gene (Cvmp) is very similar (74 and 59% of similarity and identity, respectively) to the ortholog from Ralstonia eutropha bacteria capable of utilizing phenol as the sole carbon source. The phenol biodegradation ability of E. coli recombinant strains was tested by cell-growth in a minimal medium containing phenol as the sole source of carbon and release of intermediary metabolites (catechol and hydroquinone). Interestingly, during the growth of these strains on phenol, catechol, and hydroquinone accumulated transiently in the medium. These metabolites were further analyzed by HPLC. These results indicated that phenol can be initially orto or para hydroxylated to produce cathecol or hydroquinone, respectively, followed by meta-cleavage of aromatic rings. To verify this information, the metabolites obtained from HPLC were submitted to LC/MS to confirm their chemical structure, thereby indicating that the recombinant strains utilize two different routes simultaneously, leading to different ring-fission substrates for the metabolism of phenol.     

Separation of a phenol carboxylating organism from a two-member, strict anaerobic co-culture

In a culture converting phenol to benzoic acid under anaerobic conditions and previously described as being constituted of only a Clostridium-like strain 6, another bacterium (strain 7) was observed. Each organism was enriched by centrifugation on a Percoll gradient. Strain 6 was purified by dilution and plating. Strain 7 did not grow on solid media, but a strain 7 culture, cleared of strain 6, was obtained by subculturing in the presence of ampicillin and by dilution. In fresh medium, phenol was transformed by the reconstituted co-culture but not by each strain alone. In a supernatant from a co-culture or from a strain 6 culture, strain 7 alone transformed phenol but not strain 6. Maintenance of an active strain 7 in fresh medium instead of co-culture supernatant became possible when phenol was replaced by 4-hydroxybenzoate (4-OHB), which is decarboxylated to phenol before being transformed to benzoate. Even with 4-OHB, the use of co-culture (or strain 6 culture) supernatant resulted in faster transformation activity and growth rate. A phylogenetic analysis placed strain 7 in a cluster of uncultivated or nonisolated bacteria (92-96% homology). Strain 7 is also related to Desulfotomaculum, Desulfitobacterium, Desulfosporosinus, Moorella, and Sporotomaculum genera (87-92% homology).     

Reusable System for Phenol Detection in an Aqueous Medium Based on Nanodiamonds and Extracellular Oxidase from Basidiomycete Neonothopanus nambi

Doklady Biochemistry and Biophysics - A reusable system for phenol determination in an aqueous medium was obtained by adsorption of extracellular oxidase from fungus Neonothopanus nambi onto...     

Engineering of solvent-tolerant Pseudomonas putida S12 for bioproduction of phenol from glucose

Efficient bioconversion of glucose to phenol via the central metabolite tyrosine was achieved in the solvent-tolerant strain Pseudomonas putida S12. The tpl gene from Pantoea agglomerans, encoding tyrosine phenol lyase, was introduced into P. putida S12 to enable phenol production. Tyrosine availability was a bottleneck for efficient production. The production host was optimized by overexpressing the aroF-1 gene, which codes for the first enzyme in the tyrosine biosynthetic pathway, and by random mutagenesis procedures involving selection with the toxic antimetabolites m-fluoro-dl-phenylalanine and m-fluoro-l-tyrosine. High-throughput screening of analogue-resistant mutants obtained in this way yielded a P. putida S12 derivative capable of producing 1.5 mM phenol in a shake flask culture with a yield of 6.7% (mol/mol). In a fed-batch process, the productivity was limited by accumulation of 5 mM phenol in the medium. This toxicity was overcome by use of octanol as an extractant for phenol in a biphasic medium-octanol system. This approach resulted in accumulation of 58 mM phenol in the octanol phase, and there was a twofold increase in the overall production compared to a single-phase fed batch.     

Feasibility study of degradation of phenol in a fluidized bed bioreactor with a cyclodextrin polymer as biofilm carrier

This work is focused on the evaluation of a beta-cyclodextrin polymer as a carrier medium in a fluidized bed bioreactor treating aqueous phenol as a model pollutant. The insoluble polymer support was obtained in the shape of spherical beads by crosslinking beta-cyclodextrin with epichlorohydrin. A batch of swollen polymer particles was loaded into the reactor and inoculated with a mixed bacterial culture. Bacterial growth on the polymer beads was initially stimulated by glucose addition to the medium, and then gradually replaced with phenol. The operational variables studied after the acclimation period included phenol load, hydraulic residence time and recirculation flow rate. Low hydraulic residence times and moderate phenol loads were applied. The elimination capacity was usually about 1.0 kg-phenol/m(3)d, although a maximum of 2.8 kg-phenol/m(3)d was achieved with a retention time of only 0.55 h. The depuration efficiency was not affected by the recirculation flow rate in the range studied. Neither operational nor support stability problems were detected during the operation. A high degree of expansion was achieved in the bioreactor due to the hydrogel nature of the cyclodextrin polymer and, consequently, a low energy requirement was necessary to fluidize the bed.     

Adsorption of phenol on formaldehyde-pretreated Pinus pinaster bark: equilibrium and kinetics

This work studies phenol adsorption on Pinus pinaster bark that has been previously treated with formaldehyde in acid medium. The influence of several variables such as solid/liquid ratio, pH and initial concentration of phenol in the solution on the adsorption capacity of the bark has been analysed. A kinetic model based on phenol diffusion within the pores of the adsorbent was in agreement with the results obtained for high initial concentrations of phenol, allowing the determination of diffusion coefficients. Adsorption equilibrium data were fitted by the Freundlich and BET isotherms. From their parameters phenol adsorption capacity and intensity, as well as the specific surface (BET) of the adsorbent, were determined.     

Protection of transfective poliovirion RNA by histidine

Transfectivity titers of RNA preparations obtained from purified poliovirions in phosphate-buffered saline using phenol were low. Addition of tissue culture nutrient medium to the virions prior to extractin with phenol increased the RNA titers 100-1000-fold. The 32 solute differences between the phosphate-buffered saline and the nutrient medium were divided into three blocs for testing. Only the bloc containing the 13 amino acids of the nutrient medium enabled the preparation of high-titer RNA. Tests of the individuals amino acids revealed that L-histidine has high activity, L-cystine and L-glutamine moderate activity, and the remaining ten zero or very slight activity. Five congeners of L-histidine, viz. D-histidine, histamine, L-histidine methyl ester, alpha-N-acetyl-L-histidine, and L-histidyl-glycine, also had high activity; but imidazole had no activity. The histidine effect was obtained whether transfection was enhanced by DEAE-dextran or by bentonite. Histidine was fully effective only when it was added to the virions before or very shortly after the phenol; later additions of histidine were progressively less effective. Without added histidine, RNA preparations made very rapidly and inoculated promptly showed high transfectivity titers, but the transfectivity was highly labile; with histidine present, the high RNA titers were stable. Histidine did not reactivate the inactivated RNA.     

Effect of Co‐Substrates on Aerobic Phenol Degradation by Acclimatized and Non‐acclimatized Enrichment Cultures

Aerobic degradation of 7 mmol/L phenol in the presence of alternative carbon sources (7 mmol/L glucose or acetate or 1–2 mmol/L 2‐chlorophenol) was investigated using non‐acclimatized and acclimatized sewage sludges and enrichment cultures. The substrates represented an intermediate of phenol degradation (acetate), an independent substrate (glucose) or a “precursor‐substrate” of phenol degradation (2‐chlorophenol). Bacteria from sewage sludge, not pre‐adapted to phenol (2 mmol/L), rapidly respired acetate and glucose in the presence of phenol, whereas phenol was only bioconverted to any unknown aromatic metabolite after 24 h. In the presence of phenol and 2‐chlorophenol, no removal of both substances was observed when using the unacclimatized sludge. Sludge that was acclimatized to the degradation of phenol showed an initial preference for easily degradable co‐substrates such as glucose or acetate with only a slow concomitant respiration of phenol. Respiration of phenol increased rapidly after the co‐substrates were depleted. The highest phenol degradation rates were 51.6 mmol/L d, when phenol was the sole carbon substrate. Vice versa, phenol was preferentially respired in the presence of a less easily degradable co‐substrate such as 2‐chlorophenol at a rate of around 7 mmol/L d. Further studies with an enrichment culture that was obtained after 7 successive transfers of phenol‐adapted sludge into mineral medium with phenol as the only carbon source indicated that the acetate and glucose‐degrading capabilities were diminished or almost completely lost. In these enrichment cultures, phenol degradation was not affected by the presence of glucose, but glucose was not degraded. In contrary, the presence of acetate slightly slowed down the phenol degradation rate of the enrichment culture. Growth of the microorganisms apparently occurred at the expense of phenol and acetate respiration. The result of this work may be of practical importance in determining the feeding strategy, which is the key factor for most biological wastewater treatment systems. When acetate was present together with phenol in a wastewater, the phenol degradation rates were influenced by acetate, since acetate was an intermediate of phenol degradation. Glucose as an “independent substrate” was apparently degraded by other bacteria via acetate, and in this way it also influenced the phenol degradation rates. Glucose‐degrading bacteria could be “washed out” from the acclimatized sludge during several transfers into mineral medium with phenol as the sole carbon source. If later on, glucose was added again, it remained undegraded and did not influence phenol degradation. 2‐Chlorophenol degradation also requires other bacteria than phenol degraders.     

Ovulation in the perfused ovary in vitro: further evidence that estrogen is not required

The effect of inhibition of estrogen synthesis on ovulation in rat ovaries perfused in vitro with medium without phenol red was examined. The addition of luteinizing hormone (LH, 0.1 microgram/mL) plus 3-isobutyl-1-methylxanthine (IBMX, 0.2 mM) to phenol red-free perfusion medium (M199 + 4% bovine serum albumin) induced ovulation. The number of ovulations was similar to that found in medium containing phenol red. There was a similar increase in estradiol (1, 3, 5 (10)-estratriene-3, 17 beta-diol) levels in the medium in both groups. The addition of 4-hydroxy-4-androstene-3, 17-dione (4-OH-A, 5 microM) to phenol red-free medium blocked the increase in estradiol levels induced by LH + IBMX, but did not prevent ovulation. There was no significant difference in the number of ovulations in the three groups. In conclusion, phenol red in the perfusion medium does not influence ovulation induced by LH + IBMX. Furthermore, an increase in estrogen is not required during the immediate preovulatory period for ovulation to occur.     

Degradation of phenol by Rhodococcus erythropolis UPV-1 immobilized on Biolite in a packed-bed reactor

A strain of Rhodococcus erythropolis has been isolated and identified by 16S rRNA sequencing. Cells acclimated to phenol can be adsorbed on the external surface of beads of the ceramic support Biolite where they grow forming a network of large filaments. Exponentially-growing cells were adsorbed faster than their stationary-phase counterparts. Immobilization resulted in a remarkable enhancement of the respiratory activity of cells and a shorter lag phase preceding the active phenol degradation. Under optimum operation conditions, the immobilized cells in a laboratory-scale column reactor packed with support beads were able to degrade completely phenol in defined mineral medium at a maximum rate of 18 kg phenol m(-3) per day. The performance of the bioreactor in long-term continuous operation was characterized by pumping defined mineral medium which contained different concentrations of phenol at different flow-rates. Once phenol biodegradation in defined mineral medium was well established, an industrial wastewater from a resin manufacturing company, which contained both phenol and formaldehyde, was tested. In this case, after wastewater conditioning (i.e. pH, nitrogen source and micronutrient amendments) the immobilized cells were able to remove completely formaldehyde and to partly biodegrade phenols at a rate of 1 kg phenol m(-3) per day.     

Mutation of Candida tropicalis by irradiation with a He-Ne laser to increase its ability to degrade phenol

Candida tropicalis isolated from acclimated activated sludge was used in this study. Cell suspensions with 5 x 10(7) cells ml(-1) were irradiated by using a He-Ne laser. After mutagenesis, the irradiated cell suspension was diluted and plated on yeast extract-peptone-dextrose (YEPD) medium. Plates with approximately 20 individual colonies were selected, and all individual colonies were harvested for phenol biodegradation. The phenol biodegradation stabilities for 70 phenol biodegradation-positive mutants, mutant strains CTM 1 to 70, ranked according to their original phenol biodegradation potentials, were tested continuously during transfers. Finally, mutant strain CTM 2, which degraded 2,600 mg liter(-1) phenol within 70.5 h, was obtained on the basis of its capacity and hereditary stability for phenol biodegradation. The phenol hydroxylase gene sequences were cloned in wild and mutant strains. The results showed that four amino acids were mutated by irradiation with a laser. In order to compare the activity of phenol hydroxylase in wild and mutant strains, their genes were expressed in Escherichia coli BL21(DE3) and enzyme activities were spectrophotometrically determined. It was clear that the activity of phenol hydroxylase was promoted after irradiation with a He-Ne laser. In addition, the cell growth and intrinsic phenol biodegradation kinetics of mutant strain CTM 2 in batch cultures were also described by Haldane's kinetic equation with a wide range of initial phenol concentrations from 0 to 2,600 mg liter(-1). The specific growth and degradation rates further demonstrated that the CTM 2 mutant strain possessed a higher capacity to resist phenol toxicity than wild C. tropicalis did.     

Estrogen mitogenic action. III. Is phenol red a “red herring”?

The reported estrogenic action of phenol red and/or its lipophilic contaminants has led to the widespread use of indicator-free culture medium to conduct endocrine studies in vitro. Because we have recently developed methods to measure large-magnitude estrogen effects in the tissue culture medium containing phenol red, we concluded that the indicator issue required further evaluation. To do this, we selected nine estrogen receptor positive (ER+) cell lines representing four target tissues and three species. We investigated phenol red using five different experimental protocols. First, 17beta-estradiol (E2) responsive growth of all nine ER+ cells lines was compared in the medium with and without the indicator. Second, using representative lines we asked if phenol red was mitogenic in the indicator-free medium. The dose-response effects of phenol red were compared directly to those of E2. Third, we asked if tamoxifen-inhibited growth equally in phenol red-containing and indicator-free medium. This study was based on a report indicating that antiestrogen effects should be seen only in phenol red-containing medium. Fourth, we asked if phenol red displaced the binding of 3H-E2 using ERK intact human breast cancer cells. Fifth, we compared E2 and phenol red as inducers of the progesterone receptor using a human breast cancer cell line. All the experiments presented in this report support the conclusion that the concentration of phenol red contaminants in a standard culture medium available today is not sufficient to cause estrogenic effects. In brief, our studies indicate that the real issue of how to demonstrate estrogenic effects in culture resides elsewhere than phenol red. We have found that the demonstration of sex steroid hormone-mitogenic effects in culture depends upon conditions that maximize the effects of a serum-borne inhibitor(s). When the effects of the inhibitor are optimized, the presence or absence of phenol red makes no everyday difference to the demonstration of estrogen mitogenic effects with several target cell types from diverse species.     

U-Shape Suppressive Effect of Phenol Red on the Epileptiform Burst Activity via Activation of Estrogen Receptors in Primary Hippocampal Culture

Phenol red is widely used in cell culture as a pH indicator. Recently, it also has been reported to have estrogen-like bioactivity and be capable of promoting cell proliferation in different cell lines. However, the effect of phenol red on primary neuronal culture has never been investigated. By using patch clamp technique, we demonstrated that hippocampal pyramidal neurons cultured in neurobasal medium containing no phenol red had large depolarization-associated epileptiform bursting activities, which were rarely seen in neurons cultured in phenol red-containing medium. Further experiment data indicate that the suppressive effect of the phenol red on the abnormal epileptiform burst neuronal activities was U-shape dose related, with the most effective concentration at 28 µM. In addition, this concentration related inhibitory effect of phenol red on the epileptiform neuronal discharges was mimicked by 17-β-estradiol, an estrogen receptor agonist, and inhibited by ICI-182,780, an estrogen receptor antagonist. Our results suggest that estrogen receptor activation by phenol red in the culture medium prevents formation of abnormal, epileptiform burst activity. These studies highlight the importance of phenol red as estrogen receptor stimulator and cautions of careful use of phenol red in cell culture media.     

Enhancement of biodegradation of phenol and a nongrowth substrate 4-chlorophenol by medium augmentation with conventional carbon sources

The enhancement of biodegradation of phenol and 4-chlorophenol (4-cp) as a cometabolised compound by Pseudomonas putida ATCC 49451 was accomplished by augmenting the medium with conventional carbon sources such as sodium glutamate and glucose. Compared with phenol as the sole carbon source, the addition of 1 gl(-1) sodium glutamate increased the toxicity tolerance of cells toward 4-cp and significantly improved the biodegradation rates of both phenol and 4-cp even when the initial concentration of 4-cp was as high as 200 mgl(-1). On the other hand, supplementation of glucose caused a significant drop in the medium pH from 7.2 to 4.3 resulting in a reduction of degradation rate, leaving a considerable amount of 4-cp undegraded when the initial concentration of 4-cp was higher than 100 mgl(-1). By regulating the pH of the medium, however, enhancement of degradation rates of phenol and 4-cp in the presence of glucose was achieved with a concomitant complete degradation of phenol and 4-cp.     

Rate of Biodegradation of Phenol by Klebsiella oxytoca in Minimal Medium and Nutrient Broth Conditions

The rate of biodegradation of phenol by Klebsiella oxytoca strain was studied in the nutrient broth and M9 minimal medium. It was found that K. oxytoca degrade phenol at elevated phenol concentration where 75% of initial phenol concentration of 100 ppm will degrade within 72 h. This rate was increased with increasing the initial cell densities, increasing the aeration rate and increasing the time required for complete degradation. At phenol concentration above 400 ppm, the cells were unable to degrade the substrate efficiently due to the increasing concentration of phenol in the medium. The culture conditions were also showed a significant impact on the ability of these cells to remove phenol. The optimum solution pH and temperature were 6.8 and 37°C, respectively. The growth of these cells in the presence and absence of phenol was modeled and it was found that the Recatti equation best fit the growth in the absence of phenol whereas the Voltera equation accounted for the history of the cell population in the presence of phenol.     

Preferential utilization of phenol rather than glucose by Trichosporon cutaneum possessing a partially constitutive catechol 1,2-oxygenase.

A phenol-utilizing yeast, Trichosporon cutaneum POB 14, which has a partially constitutive activity of catechol 1,2-oxygenase, utilized phenol in preference to glucose in a medium containing both phenol (200 mg/liter) and glucose (0.15%) as carbon sources. The glucose consumption was not observed until the concentration of phenol decreased to around 10 mg/liter. This phenomenon was confirmed by [U-14C]glucose uptake experiments. The intracellular activities of hexokinase (EC 2.7.1.1) and catechol 1,2-oxygenase (EC 1.13.1.1) changed inversely when phenol was added during growth in the glucose medium.     

Intensification of phenol biodegradation by humic substances

Phenol biodegradation was carried out in a batch system by the bacterial strain Cupriavidus metallidurans in the presence of potassium humate that was prepared by alkaline extraction from oxyhumolite. The experiments were focused on the assessment of the humate effect on biodegradation activity of the tested bacterial strain. The achieved results demonstrated that the humate has a positive influence on the biodegradation of phenol and reduces the incubation time necessary for phenol removal. Higher biodegradation rate and more intensive growth were observed during the cultivation in presence of humate in comparison to the cultivation without its addition. Adsorption of the humate on bacterial biomass was observed as well. Subsequently, a phenol biodegradation testing in a continuous-flow system using a biofilm reactor was also carried out. Although the reactor was inoculated by C. metallidurans only, the microbial composition under an aerobic non-aseptic condition during this long-term cultivation changed. The phenol removal efficiency obtained in the biofilm reactor was higher than 92% when phenol concentration in a treated medium was 1200 mg l⁻¹.