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PubMed2005

A highly efficient preparative-scale generic immunoaffinity chromatography method for the purification of antibodies to hydrophobic haptens: purification procedure and monitoring tests.

A generic affinity chromatography purification protocol for the isolation of preparative quantities of pure and stable polyclonal antibodies to hydrophobic haptenic analytes is described together with a panel of tests to monitor the purification process and assess the functional and structural purity of isolated antibodies. The purification method is based on the use of a mixture of acetonitrile and propionic acid to elute bound antibodies from Sepharose 4B-based immunoabsorbent gels. Highly specific and pure antibodies to steroid estrogens, pentachlorophenol and Irgarol 1051 were isolated in 50-150 mg quantities per preparation in a batch-wise method using appropriate ligands linked to the solid phase via a hydrophilic chemical arm, tetraethylene pentamine. The panel of ELISA tests together with SDS-PAGE enabled the monitoring of the absorption and elution steps and provided data relevant to the assessment of the degree of structural and functional purity of the isolated antibody preparations. The study demonstrates that the affinity purification procedure is practical, simple, generic for antibodies to hydrophobic haptens and suitable for scaling up. In addition, the study showed that the functional properties of the affinity-purified antibodies indicated improvements on the operational properties (specificity and assay detection limits) of the source antisera. The isolated IgG antibodies showed near 100% functional and structural purity and no deterioration of activity on storage for long periods. The method provides critical reagents for labelled-antibody immunoassays and immunosensors and antibody-dependent sample purification techniques.

Animals↗
PubMed2004

Growth substrate selection and biodegradation of PCP by New Zealand white-rot fungi.

Nine New Zealand native white-rot fungi were studied for their ability to grow and survive on different substrates formulated from bark, wheat straw, sawdust, apple pomace and maize products in order to identify their pentachlorophenol (PCP) biodegradation potential and to select a fungal carrier for bioaugmentation of polluted soils. Isolates were also evaluated to mineralize (14)C-PCP in liquid culture and in soil. The American fungus Phanerochaete chrysosporium outgrew the native fungi on the substrates tested, but the high colonisation did not result in superior PCP dechlorination as measured by chloride release. Whilst Trametes versicolor inocula produced on wheat straw and SCS (sawdust-corn meal-starch-mix) gave the highest chloride release, colonization of these two substrates as measured by biological potential was lower compared to the pomace and pomace-sawdust-mix. Neither lignin peroxidase nor manganese peroxidase production were measured for New Zealand white-rot fungi during the experiments. Laccase was the only enzyme detected. In liquid culture, the mineralisation rate was higher for T. versicolor isolates compared to P. chyrysoporium. Very little to no pentachloroanisole (PCA) was captured in the volatile fraction of T. versicolor isolates, whereas 75% of the volatile fraction of P. chrysosporium consisted of PCA. The soil microcosms studies, using contaminated soil from a timber treatment site, clearly showed that the New Zealand T. versicolor isolates mineralized PCP. Degradation of PCP in non-sterile soil was higher in the presence of white-rot fungi than in soil without white-rot fungus. This demonstrates that viable white-rot fungus is necessary for significant PCP degradation and that T. versicolor isolates showed PCP remediation potential. Wheat straw and SCS could be suitable carriers for New Zealand native T. versicolor isolates for bioremediation of PCP polluted soil sites.

Biodegradation, Environmental↗
PubMed2006

Use of EAF dust as heterogeneous catalyst in Fenton oxidation of PCP contaminated wastewaters.

In this study, chemical oxidation tests using H2O2 were performed on a solution contaminated with 100 mg l(-1) of pentachlorophenol (PCP). The effectiveness of electric arc furnace dust and hematite as heterogeneous catalysts was evaluated. Reactions were conducted at pH 2 for 24 h. Either H2O2 stabilized with KH2PO4 or un-stabilized H2O2 was used. Total organic carbon (T.O.C.) removal and chloride release from PCP molecule were monitored. Results showed that the maximum removal yields for electric arc furnace (EAF) dust (49.2% T.O.C., 56.7% Cl) were achieved when H2O2:PCP ratio was 10:1 and Fe:H2O2 = 1:5 for unstabilized H2O2 and when H2O2:PCP = 10:1 and Fe:H2O2 = 1:1 for stabilized H2O2 (48% T.O.C., 60.6% Cl). The maximum yield using hematite (45.2% T.O.C., 55.2% Cl) was obtained when H2O2:PCP ratio was 10:1 and Fe:H2O2 was 1:2. When EAF dust was used and Fe:H2O2 > 1:5, Cl release was higher than the one expected from T.O.C. removal.

Catalysis↗
PubMed2004

Anti-estrogenic activity of fifty chemicals evaluated by in vitro assays.

We examined the anti-estrogenic activity of 50 chemicals by the yeast two-hybrid assay and detected the activity of hexachlorophene, pentachlorophenol, and vitamin K3 (menadione), in that order. These chemicals were also observed to inhibit the transcriptional activity of 17beta-estradiol in a reporter gene assay system using MCF-7 cells, estrogen receptor-positive breast cancer cells, and to bind directly to estrogen receptor alpha in a competitive binding assay system, although the order of the activity was slightly different among the 3 assays. These findings suggested that three of fifty chemicals could inhibit estrogen activity by competitive binding with 17beta-estradiol to the estrogen receptor.

Binding, Competitive↗
PubMed2005

Insight into pollutant bioavailability and toxicity using Raman confocal microscopy.

Raman confocal microscopy was used to discriminate between cultures of Burkholderia xenovorans LB400 exposed to four different common environmental pollutants: phenanthrene, dodecane, 3-chlorobiphenyl and pentachlorophenol. Evidence is presented for the application of Raman spectroscopy as a bioassay for pollutant bioavailability and toxicity.

Alkanes↗
PubMed2006

DNA damage and repair in haemolymph cells of golden mussel (Limnoperna fortunei) exposed to environmental contaminants.

The development of methodologies for biomonitoring freshwater ecosystems is of particular relevance in view of the serious problem of aquatic environmental pollution. The mussel species Limnoperna fortunei (golden mussel) was chosen to be tested as a biomonitor organism based on its population data and distribution. L. fortunei individuals were exposed to UV radiation in vitro, and in vivo to pentachlorophenol (PCP) and copper sulphate (CuSO(4)), with the aim of standardizing comet assay and micronucleus test methodologies and evaluating the potential of this organism as a biomonitor. Haemolymph cells immobilized in agarose on slides exposed to UV radiation showed a dose-response relationship with maximum damage at 4.2 J/m(2). For the chemical tests, individuals were exposed for 2h for the comet assay and 24 and 48 h for the micronucleus test. A dose-response relationship was observed for both chemicals. 3x10(-5) M CuSO(4) induced high genotoxicity, also producing some toxicity after 48 h of exposure. PCP induced maximum damage in both assays at 150 μg/L. Individuals exposed to PCP showed 100% repair 2 h after the exposure period, as assessed by the comet assay. Exposure to an environmental sample over 7 days confirmed the mussel sensitivity to water contaminants, detected both by the comet assay and the micronucleus test. The results allow us to suggest the golden mussel as a potential biomonitor organism.

Animals↗
PubMed2005

Purification of a new manganese peroxidase of the white-rot fungus Irpex lacteus, and degradation of polycyclic aromatic hydrocarbons by the enzyme.

The white-rot fungus Irpex lacteus has been reported to be an efficient degrader of polycyclic aromatic hydrocarbons, polychlorinated biphenyls and pentachlorophenol. The fungus produces ligninolytic enzymes laccase, lignin peroxidase and manganese peroxidase (MnP), the latter being the major one produced. MnP was purified using anion exchange and size exclusion chromatography. SDS-PAGE showed the purified MnP to be a monomeric protein of 37 kDa (37.5 kDa using MALDI-TOF) with an isoelectric point at 3.55. The pH optimum was relatively broad, from 4.0 to 7.0 with a peak at pH 5.5. Kinetic constants K(m) were 8 microM for H(2)O(2) and 12 or 31 microM for Mn(2+) depending on the substrate. The enzyme did not perform oxidation in the absence of H(2)O(2) or Mn(2+). MnP was active at 5-70 degrees C with an optimum between 50-60 degrees C. At temperatures above 65 degrees C the enzyme rapidly lost activity. Degradation of four representatives of PAHs (phenanthrene, anthracene, fluoranthene, and pyrene) was tested and the enzyme showed the ability to degrade them in vitro. Major degradation products of anthracene were identified. The results confirm the role of MnP in PAH degradation by I. lacteus, including cleavage of the aromatic ring.

Basidiomycota↗
PubMed2005

Antimutagenic effect of neem leaves extract in freshwater fish, Channa punctatus evaluated by cytogenetic tests.

Neem (Azadirachta indica), an indigenous plant commonly grown in India and its sub-continent is a multipurpose plant well known for its insecticidal and biomedical properties, however, its antimutagenic effects in vertebrate organisms are lacking. The present work is therefore, focused on possible antimutagenic potential of ethanolic extract of neem leaves evaluated on the clastogenicity induced by Pentachlorophenol (PCP) and 2, 4-dichlorophenoxyacetic acid (2,4-D) in freshwater fish, Channa punctatus used as a vertebrate model, by cytogenetic endpoints: chromosome aberration (CA) and micronucleus (MN) test. In the first set of experiment, fish were exposed by medium treatment to a single treatment of each chemical (PCP, 0.6 ppm; 2,4-D, 75 ppm; neem extract, 3 ppm) along with the controls. The chromosome preparations were made after processing kidney cells and micronucleus slides were prepared from peripheral blood at multiple duration (48, 72 and 96 h). PCP and 2,4-D when used alone, induced significant CA and MN in a time dependent manner. Neem extract did not show genotoxic potential in both assays. The maximum frequency of CA were recorded as 18.58% and 15.17%, while frequency of MN reached to 8.08% and 4.62% by PCP and 2,4-D respectively, after 96 h exposure. In the second set of experiment, three concentrations of neem extract (1, 2 and 3 ppm) were run simultaneously with the same concentration of PCP (0.6 ppm) and 2,4-D (75 ppm) for antimutagenicity estimates. In mixed treatment, neem extract significantly reduced the frequency of CA and MN. The reduction in the frequency of CA ranged from 40-75% and 45.4-83.3% and similar values for MN were 40.2-75.3% and 44.1-65.8% for PCP and 2,4-D respectively. Although the reductions were significant but not dependent on concentration and time intervals employed. Results suggested that under present experimental conditions, neem extract exhibit strong antimutagenic activity in this fish model, which could further contribute to study its benefit in humans.

2,4-Dichlorophenoxyacetic Acid↗
PubMed2004

Evaluation of natural and enhanced PCP biodegradation at a former pesticide manufacturing plant.

Pentachlorophenol (PCP) has been used in the past as a pesticide, herbicide, antifungal agent, bactericide, and wood preservative. Thus, PCP is among the most ubiquitous chlorinated compounds found in groundwater contamination. A former pesticide manufacturing plant located in southern Taiwan has been identified as a PCP spill site. In this study, groundwater samples collected from the PCP site were analyzed to assess the occurrence of natural PCP biodegradation. Microcosm experiments were conducted to (1) evaluate the feasibility of biodegrading PCP by indigenous microbial consortia under aerobic and cometabolic conditions, and (2) determine the potential of enhancing PCP biodegradation using cane molasses and biological sludge cake as the substitute primary substrates under cometabolic conditions. The inocula used in this microcosm study were aquifer sediments collected from the PCP site and activated sludges collected from the municipal and industrial wastewater treatment plants. Results from this field investigation indicate that the natural biodegradation of PCP is occurring and causing the decrease in PCP concentration. Microcosm results show that the indigenous microorganisms can biodegrade PCP under both aerobic and aerobic cometabolism conditions. A PCP-degrading bacterium was isolated from the collected aquifer sediments and identified as Pseudomonas mendocina NSYSU via some biochemical tests and further conformation of DNA sequencing. In batch cultures, P. mendocina NSYSU used PCP as its sole source of carbon and energy. The isolated bacterium, P. mendocina NSYSU, was capable of completely degrading PCP as indicated by the increase in biomass formation with the decrease in PCP concentrations occurred in the carbon-free medium simultaneously. Results indicate that the in situ or on-site aerobic bioremediation using indigenous microorganisms or inoculated bacteria would be a feasible technology to clean up the studied PCP-contaminated site. Results from this study will be useful in designing a scale-up in situ or on-site PCP bioremediation system (e.g., on-site bioreactor) for field application.

Accidents↗
PubMed1986

pH measurements by 31p NMR in bacterial suspensions using phenyl phosphonate as a probe.

Phenylphosphonate was used as an internal reference for 31p NMR measurements of E. coli cytoplasmic pH. Phenylphosphonate diffused into the cells allowing determination of pH, independent of magnetic susceptibility differences between intercellular and extracellular pools. Phenylphosphonate was used to measure pH changes during succinate uptake and metabolism, as well as during uncoupling of the transmembrane pH gradient by pentachlorophenol.

Diphosphates↗
PubMed2000

Relationship of MTT reduction to stimulants of muscle metabolism.

MTT, a positively charged tetrazolium salt, is widely used as an indicator of cell viability and metabolism and has potential for histochemical identification of tissue regions of hypermetabolism. In the present study, MTT was infused in the constant-flow perfused rat hindlimb to assess the effect of various agents and particularly vasoconstrictors that increase muscle metabolism. Reduction of MTT to the insoluble formazan in muscles assessed at the end of experiments was linear over a 30 min period and production rates were greater in red fibre types than white fibre types. The vasoconstrictors, norepinephrine (100 nM) and angiotensin (10 nM) decreased MTT formazan production in all muscles but increased hindlimb oxygen uptake and lactate efflux. Veratridine, a Na(+) channel opener that increases hindlimb oxygen uptake and lactate efflux without increases in perfusion pressure, also decreased MTT formazan production. Membrane stabilizing doses (100 microM) of (+/-)-propranolol reversed the inhibitory effects of angiotensin and veratridine on MTT formazan production. Muscle contractions elicited by stimulation of the sciatic nerve, reversed the norepinephrine-mediated inhibitory effects on MTT formazan production, even though oxygen consumption and lactate efflux were further stimulated. Stimulation of hindlimb muscle oxygen uptake by pentachlorophenol, a mitochondrial uncoupler, was not associated with alterations in MTT formazan production. It is concluded that apart from muscle contractions MTT formazan production does not increase with increased muscle metabolism. Since the vasoconstrictors angiotensin and norepinephrine as well as veratridine activate Na(+) channels and the Na(+)/K(+) pump, energy required for Na(+) pumping may be required for MTT reduction. It is unlikely that vasoconstrictors that stimulate oxygen uptake do so by uncoupling respiration.

Angiotensin II↗
PubMed1998

Cooperative ligand binding by bovine phenol sulfotransferase.

Although several phenol sulfotransferases (PSTs) that metabolize hormones and xenobiotics have been purified and examined by steady state kinetic methods, little is known about ligand binding and subunit interactions in these enzymes. Inhibition of a purified recombinant homodimeric bovine PST by 2,6-dichloro-4-nitrophenol (DCNP) and pentachlorophenol (PCP) displayed very sharp titration curves that required modeling with Hill equations with slope factors of 2 and 3, respectively. This observation suggested positive cooperative ligand binding during catalytic turnover. The binding of PCP was also monitored by intrinsic protein fluorescence, which was quenched up to 36% upon saturation with the inhibitor. In the absence of 3'-phosphoadenosine-5'-phosphosulfate (PAPS), quenching curves were fit with the Hill equation and an interaction factor of 1. In contrast, inclusion of PAPS increased the association of PCP and resulted in positive cooperative binding with an interaction factor of 1.6-1.9. Whereas adenosine-3',5'-diphosphate (PAP) also facilitated cooperative binding of PCP, adenosine-5'-monophosphate (AMP) was not effective. This correlated to inhibition of PST by PAP, whereas AMP was not inhibitory up to 1 mM. Therefore, occupation of the PST nucleotide binding site(s) facilitates a subunit interaction that can promote subsequent binding of phenolic ligands.

Animals↗
PubMed2000

Conjugation of 1-naphthol in primary cell cultures of rat ovarian cells.

The present study concerns conjugation of 1-naphthol in primary cultures of rat ovarian cells. Two phase II enzymes catalyzing conjugation, i.e. phenol sulfotransferase (P-SULT) and phenol UDP-glucuronosyltransferase (P-UGT), were measured using 1-naphthol as substrate. The rates of conjugation by the different cell types of the rat ovary were the same at low concentrations and short incubation times. However, after 20 h of incubation the rate of conjugation in cells isolated from ovaries enriched in corpora lutea (CL) exceeded the rate in cells isolated from ovaries enriched in preovulatory follicles. In addition, when the granulosa cells were removed from the preovulatory follicles, the rate of conjugation was 1.7-fold higher, i.e. in the theca/stroma cells. When the cells were incubated with 1-[14C]naphthol and conjugates were subsequently separated by thin-layer chromatography, naphthyl glucuronide was the only conjugate observed. Pentachlorophenol (PCP), a commonly used inhibitor of P-SULT, inhibited 1-naphthol conjugation 50% in cell cultures, as well as in microsomal preparations. alpha-Naphthoflavone (ANF) and ellipticine (ELP), both cytochrome P450 (CYP) inhibitors, affected the conjugation of 1-naphthol in different ways; ANF did not affect P-UGT activity in microsomal preparations, but inhibited 1-naphthol conjugation in cell cultures by as much as 90%. On the other hand, ELP inhibited the conjugation of 1-naphthol up to 99% in the cell cultures, but only 75% in microsomal fractions. Testosterone (TST) and estradiol inhibited this activity approximately equal 50% in both of these experimental systems. Clomiphene citrate (CLF), a drug used to induce ovulation and demonstrating both estrogenic and antiestrogenic effects, did not influence the conjugation of 1-naphthol significantly in the cell cultures. The present findings demonstrate that P-UGT is by far the major enzyme conjugating 1-naphthol in the rat ovary and that commonly used inhibitors of P-SULT and CYPs also inhibit P-UGT activity, either directly or via other mechanisms.

Animals↗
PubMed2003

Exposure assessment of residents living near a wood treatment plant.

We report the results of environmental sampling and modeling in a neighborhood adjacent to a wood processing plant. This plant used creosote and pentachlorophenol (PCP) to treat wood for over 70 years. Between 1999 and 2001, environmental samples were obtained to quantify the level of environmental contamination from the wood processing plant. Blood from 10 residents was measured for chlorinated dioxins and dibenzofurans. Soil sediment samples from drainage ditches and attic/dust samples from nearby residents' homes were tested for polychlorinated dioxins, furans, and polycyclic aromatic hydrocarbons (PAH). The dioxin congeners analysis of the 10 residents revealed elevated valued for octachlorodibenzo-p-dioxin and heptachlorodibenzo-p-dioxin compatible with PCP as the source. The levels of carcinogenic PAHs were higher than background levels and were similar to soil contamination on wood preserving sites. Wipe sampling in the kitchens of 11 homes revealed that 20 of the 33 samples were positive for octachlorinated dioxins with a mean value of 10.27 ng/m2. The soil, ditch samples, and positive wipe samples from the homes indicate a possible ongoing route of exposure to the contaminants in the homes of these residents. Modeled air exposure estimated for the wood processing waste chemicals indicate some air exposure to combustion products. The estimated air levels for benzo(a)pyrene and tetrachlorodibenzodiozin in this neighborhood exceeded the recommended levels for these compounds in some states. The quantitative data presented suggest a significant contamination of a neighborhood by wood processing waste chemicals. These findings suggest the need for more stringent regulations on waste discharges from wood treatment plants.

Air Pollutants↗
PubMed1997

Isolation of mRNAs induced by a hazardous chemical in white-rot fungus, Coriolus versicolor, by differential display.

White-rot fungus Coriolus versicolor, a ligninolytic basidiomycete, has been studied because of its ability to degrade hazardous chemicals. In this study, we searched for genes that are induced by a hazardous chemical using the mRNA differential-display technique and C. versicolor IFO30340 that has been exposed to pentachlorophenol (PCP). Five cDNA fragments were cloned and the DNA sequences of two fragments were analyzed in further detail. The clones corresponded to novel genes that have not previously been identified in C. versicolor. One of the cDNAs exhibited strong sequence homology to the gene for an enolase and the other exhibited homology to a heat shock protein. The expression of the two genes was up-regulated in PCP-treated C. versicolor.

Amino Acid Sequence↗
PubMed2003

The effect of endocrine disrupting chemicals on thyroid hormone binding to Japanese quail transthyretin and thyroid hormone receptor.

We investigated the effect of endocrine disrupting chemicals (EDCs), including medical, industrial, and agricultural chemicals, on 3,3',5-L-[125I]triiodothyronine ([125I]T3) binding to purified Japanese quail transthyretin (qTTR), a major thyroid hormone-binding protein in plasma, and to the ligand-binding domain of thyroid hormone receptor beta (qTR LBD). Scatchard plots of T3 binding to qTTR and qTR LBD revealed two classes of binding sites, with Kd values of 6.9 and 185 nM, and a single class of binding sites, with Kd value of 0.31 nM, respectively. Among the test chemicals, diethylstilbestrol was the most powerful inhibitor of [125I]T3 binding to qTTR (IC50 < 0.4 nM). Diethylstilbestrol, ioxynil (IC50 =1.1+/-0.5 nM) and pentachlorophenol (IC50 = 6.3+/-3.8 nM) displaced [125I]T3 from qTTR more effectively than unlabeled T3 (IC50 = 9.7+/-0.9 nM) did. Although malathion, 4-nonylphenol, bisphenol A and n-butylbenzyl phthalate were effective inhibitors of [125I]T3 binding to qTTR, their potency was two orders of magnitude less than that of T3. All test chemicals except for diethylstilbestrol had either a weak or no effect on [125I]T3 binding to qTR LBD. These results show that several EDCs tested in this study target qTTR rather than qTR LBD.

Animals↗
PubMed2001

Protection from drug-induced hepatocellular changes by pretreatment with conjugating enzyme inhibitors in rats.

The present paper describes the role of conjugating enzymes in the development of hepatotoxicity after administration of repeated doses of a novel monoamine oxidase type-A (MAO-A) inhibitor, (5R)-3-[2-(( 1S)-3-cyano-1-hydroxypropyl)benzothiazol-6-yl]-5-methoxymethyl-2-oxazolidinone (E2011). The effects of pretreatment with three kinds of conjugating enzyme inhibitors on hepatic lesions induced by E2011 were evaluated in female Sprague-Dawley rats. The inhibitors used were 2,6-dichloro-4-nitrophenol (DCNP; inhibitor of sulfotransferase (ST)), pentachlorophenol (PCP; inhibitor of both ST and acetyltransferase (AT)) or ranitidine (inhibitor of UDP-glucuronosyltransferase (UDP-GT)). Two weeks treatment of E2011 alone at an oral dosage of 150 mg/kg induced hepatocellular changes characterized by nuclear enlargement. Daily pretreatment with DCNP (10 mg/kg, i.p.) enhanced the E2011-induced hepatocellular changes accompanied by single cell necrosis. On the other hand, the hepatotoxicity was clearly diminished by PCP (5 mg/kg, i.p.). Ranitidine pretreatment had no effect. Protection by PCP was attributed to the inhibitory effects of AT in addition to ST; it was considered that the hepatocellular changes caused by E2011 were largely dependent on the formation of acetyl conjugate(s).

Acetyltransferases↗
PubMed2001

Effectiveness of an anaerobic granular activated carbon fluidized-bed bioreactor to treat soil wash fluids: a proposed strategy for remediating PCP/PAH contaminated soils.

An integrated system has been developed to remediate soils contaminated with pentachlorophenol (PCP) and polycyclic aromatic hydrocarbons (PAHs). This system involves the coupling of two treatment technologies, soil-solvent washing and anaerobic biotreatment of the extract. Specifically, this study evaluated the effectiveness of a granular activated carbon (GAC) fluidized-bed reactor to treat a synthetic-waste stream of PCP and four PAHs (naphthalene, acenaphthene, pyrene, and benzo(b)fluoranthene) under anaerobic conditions. This waste stream was intended to simulate the wash fluids from a soil washing process treating soils from a wood-preserving site. The reactor achieved a removal efficiency of greater than 99.8% for PCP with conversion to its dechlorination intermediates averaging 46.5%. Effluent, carbon extraction, and isotherm data also indicate that naphthalene and acenaphthene were removed from the liquid phase with efficiencies of 86 and 93%, respectively. Effluent levels of pyrene and benzo(b)fluoranthene were extremely low due to the high-adsorptive capacity of GAC for these compounds. Experimental evidence does not suggest that the latter two compounds were biochemically transformed within the reactor.

Acenaphthenes↗