Genotoxicity of 2,4- and 2,6-dinitrotoluene as measured by the Tradescantia micronucleus (Trad-MCN) bioassay

The phytogenotoxicity of 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT) was assessed using the Tradescantia micronucleus (Trad-MCN) bioassay. Tradescantia cuttings bearing young inflorescences were exposed for 6h to 2,4- or 2,6-DNT amended water solutions up to their respective solubilities. The nominal concentrations were 0, 1.9, 3.8, 7.5, 15, 30, 60, 100, 150, 200mg/l of 2,4-DNT, and 0, 7.5, 15, 30, 60, 90, 120, 180mg/l of 2,6-DNT. Each treatment was repeated three or four times. Chemical concentrations in test solutions were analyzed prior to and after the exposure. Cadmium chloride (0-20mM) was used as the positive control. Micronuclei (MCN) were scored in the tetrad-stage pollen mother cells. The MCN frequency (%), i.e. the number of micronuclei scored in 100 tetrads, was the measurement endpoint. Results indicated that both 2,4-DNT and 2,6-DNT were genotoxic with the minimum effective dose (MED) of 30 and 135mg/l, respectively. Longer exposure (30h) without recovery time at 150mg/l of 2,4-DNT and 180mg/l of 2,6-DNT did not induce significantly higher MCN frequencies.     

Assessment of the in vivo genotoxicity of isomers of dinitrotoluene using the alkaline Comet and peripheral blood micronucleus assays

Dinitrotoluene (DNT) is a nitroaromatic explosive that exists as six isomers; two major isomers (2,4- and 2,6-DNT) and four minor isomers (2,3-, 2,5-, 3,4-, and 3,5-DNT). DNT has been found in soil, surface water, and groundwater near ammunition production plants. The major isomers of DNT are classified as "likely to cause cancer in humans."In vitro studies have provided conflicting data regarding the genotoxicity of the minor isomers. Studies indicate that metabolism in the gut and liver are necessary to convert DNT to genotoxic compounds. As such, in the present study the genotoxicity of isomers of DNT was assessed using two in vivo genotoxicity assays. The Comet assay was used to detect DNA damage in liver cells from male Sprague-Dawley rats following oral exposure (14-day) to individual isomers of DNT. The micronucleus assay was conducted using flow cytometric analysis to detect chromosomal damage in peripheral blood. Treatment with 2,3-, 3,4-, 2,4-, 2,5- and 3,5-DNT did not induce DNA damage in liver cells or increase the frequency of micronucleated reticulocytes (MN-RET) in peripheral blood at the doses tested. Treatment with 2,6-DNT induced DNA damage in liver tissue at all doses tested, but did not increase the frequency of micronucleated reticulocytes (MN-RET) in peripheral blood. Thus, 2,4-DNT and the minor isomers were not genotoxic under these test conditions, while 2,6-DNT was genotoxic in the target tissue, the liver. These results support previous research which indicated that the hepatocarcinogenicity of technical grade DNT (TG-DNT) could be attributed to the 2,6-DNT isomer.     

Kinetic analysis of simultaneous 2,4-dinitrotoluene (DNT) and 2, 6-DNT biodegradation in an aerobic fluidized-bed biofilm reactor.

We previously reported on the mineralization of 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT) in an aerobic fluidized-bed bioreactor (FBBR) (Lendenmann et al. 1998 Environ Sci Technol 32:82-87). The current study examines the kinetics of 2, 4-DNT and 2,6-DNT mineralization at increasing loading rates in the FBBR with the goal of obtaining system-independent kinetic parameters. At each steady state, the FBBR was subjected to a set of transient load experiments in which substrate flux in the biofilm and bulk substrate concentrations were measured. The pseudo-steady-state data were used to estimate the biokinetic parameters for 2,4-DNT and 2,6-DNT removal using a mechanistic mathematical biofilm model and a routine that minimized the sum of the squared residuals (RSS). Estimated kinetic parameters varied slightly for each steady-state; retrieved parameters for qm were 0. 83 to 0.98 g DNT/g XCOD d for 2,4-DNT removal and 0.14 to 0.33 g DNT/g XCOD d for 2,6-DNT removal. Ks values for 2,4-DNT removal (0. 029 to 0.36 g DNT/m3) were consistently lower than Ks values for 2, 6-DNT removal (0.21 to 0.84 g DNT/m3). A new approach was introduced to estimate the fundamental biofilm kinetic parameter S*b,min from steady-state performance information. Values of S*b,min indicated that the FBBR performance was limited by growth potential. Adequate performance of the examined FBBR technology at higher loading rates will depend on an improvement in the growth potential. The obtained kinetic parameters, qm, Ks, and S*b,min, can be used to aid in the design of aerobic FBBRs treating waters containing DNT mixtures.     

Voltammetric behaviour of DNA bases at a screen-printed carbon electrode and its application to a simple and rapid voltammetric method for the determination of oxidative damage in double stranded DNA

Screen-printed carbon electrodes (SPCEs) have been investigated as possible sensors to identify gamma-irradiation induced oxidative damage in double stranded (ds) DNA. Studies were undertaken to explore the possibility of using both cyclic voltammetry and differential pulse voltammetry to identify changes due to oxidative damage. Initially, guanine, adenine and 8-oxoguanosine were examined and it was found possible to differentiate them from their voltammetric responses. The voltammetric response of 8-oxoguanosine was found to be linear over the concentration range 1-400 microM, with a slope of 0.0296 microA microM(-1) (R2 value of 0.9984), in the presence of 2mM concentrations of guanine and adenine. Investigations were made into harnessing these findings to identify oxidative damage in gamma-irradiated dsDNA. The presence of oxidative damage in these samples was readily identifiable, and the magnitude of the voltammetric response was found to be dose dependant (R2=0.9919). A simple sample preparation step involving only the dissolution of double stranded DNA sample in the optimised electrolyte (0.1M acetate buffer pH 4.5) was required. This report appears to be first describing the use of a SPCE to detect DNA damage which can be related to the dose of gamma-radiation used.     

Determination of defensin HNP-1, HNP-2, and HNP-3 in human saliva by using LC/MS

The mass spectral profiling of saliva by liquid chromatography mass spectrometry in relation to particular types of pain is being examined. The aim is to develop a profile that could be useful for the assessment of patients and their treatment programs, as well as identifying unknown compounds observed in saliva. Defensin human neutrophil peptide-1 (HNP-1) and defensin HNP-2 were identified and confirmed, whereas defensin HNP-3 was tentatively identified. Linear calibration range of defensin HNP-1 and HNP-2 was 0.25 to 3 microg/ml with R(2) values of > 0.99 for both. The detection limit for defensin HNP-1 and HNP-2 was estimated at 0.1 microg/ml. The healthy subjects surveyed in this study had readily measurable salivary concentrations of defensin HNP-1 (8.6 +/- SD 8.0 microg/ml) and defensin HNP-2 (5.6 +/- SD 5.2 microg/ml).     

A comparison of leptin and ghrelin levels in plasma and saliva of young healthy subjects

In the last 10 years, saliva has been increasingly used as a diagnostic fluid and in predictions of disease progression. Leptin and ghrelin are synthesized in several tissues including the salivary glands. The action of ghrelin is antagonistic to that of leptin. This study was undertaken to measure and compare the saliva ghrelin-leptin and plasma ghrelin-leptin levels in healthy young subjects. In 30 healthy subjects, after an overnight fast, saliva and plasma leptin levels were measured using the ELISA method while saliva and plasma immunoreactive ghrelin levels were measured using a commercial radioimmunoassay (RIA). The latter uses 125I-labeled bioactive ghrelin as a tracer and a rabbit polyclonal antibody raised against full-length octanoylated human ghrelin (Phoenix, Europe, Karlsruhe, Germany). The results of this investigation revealed that saliva leptin levels (6.19+/-2.10 microg/l) were lower than plasma levels (7.39+/-3.23 microg/l) while saliva ghrelin levels (188.5+/-84.7 pg/ml) were higher than plasma levels (126.4+/-38.5 pg/ml), when male and female subjects were considered together. Saliva leptin levels (5.93+/-1.94 microg/l) were lower than plasma levels (6.22+/-2.92 pg/ml) while saliva ghrelin levels (190.3+/-80.2 pg/ml) were higher than plasma levels (120.4+/-35.7 pg/ml) in young males. Saliva leptin levels (6.47+/-2.29 microg/l) were lower than plasma levels (8.73+/-3.14 microg/l) while saliva ghrelin levels (183.2+/-90.2 pg/ml) were higher than plasma levels (129.3+/-42.8 pg/ml) in young females, and both saliva and plasma leptin levels were slightly lower in male subjects in comparison with female subjects. Also, Immunohistochemistry study indicated that ghrelin positivity was found in ductus epithelium of salivary gland. We have demonstrated for the first time that saliva ghrelin levels were higher than in plasma while saliva leptin levels were almost the same as in plasma. Measurements of ghrelin and leptin in saliva is non-invasive, simple, and generally much preferred by patients and thus may be an acceptable alternative to plasma sampling.     

Saturation mutagenesis of 2,4-DNT dioxygenase of Burkholderia sp. strain DNT for enhanced dinitrotoluene degradation

2,4-Dinitrotoluene (2,4-DNT) and 2,6-DNT are priority pollutants, and 2,4-DNT dioxygenase of Burkholderia sp. strain DNT (DDO) catalyzes the initial oxidation of 2,4-DNT to form 4-methyl-5-nitrocatechol and nitrite but has significantly less activity on other dinitrotoluenes and nitrotoluenes (NT). Hence, oxidation of 2,3-DNT, 2,4-DNT, 2,5-DNT, 2,6-DNT, 2NT, and 4NT were enhanced here by performing saturation mutagenesis on codon I204 of the alpha subunit (DntAc) of DDO and by using a membrane agar plate assay to detect catechol formation. Rates of degradation were quantified both by the formation of nitrite and by the formation of the intermediates with high performance liquid chromatography. The degradation of both 2,3-DNT and 2,5-DNT were achieved for the first time (no detectable activity with the wild-type enzyme) using whole Escherichia coli TG1 cells expressing DDO variants DntAc I204L and I204Y (0.70 +/- 0.03 and 0.22 +/- 0.02 nmol/min/mg protein for 2,5-DNT transformation, respectively). DDO DntAc variant I204L also transformed both 2,6-DNT and 2,4-DNT 2-fold faster than wild-type DDO (0.8 +/- 0.6 nmol/min/mg protein and 4.7 +/- 0.5 nmol/min/mg protein, respectively). Moreover, the activities of DDO for 2NT and 4NT were also enhanced 3.5-fold and 8-fold, respectively. Further, DntAc variant I204Y was also discovered with comparable rate enhancements for the substrates 2,4-DNT, 2,6-DNT, and 2NT but not 4NT. Sequencing information obtained during this study indicated that the 2,4-DNT dioxygenases of Burkholderia sp. strain DNT and B. cepacia R34 are more closely related than originally reported. This is the first report of engineering an enzyme for enhanced degradation of nitroaromatic compounds and the first report of degrading 2,5-DNT.     

Mutagenicity of 2,6-dinitrotoluene and its metabolites, and their related compounds in Salmonella typhimurium

The mutagenic activities of 2,6-dinitrotoluene (2,6-DNT) and its 6 metabolites, and their 8 related compounds were examined using Salmonella typhimurium strains TA98 and TA100 in the absence or presence of S9 mix. 2,6-DNT itself showed no mutagenicity toward either strain, but 2,6-dinitrobenzaldehyde (2,6-DNBAl), one of the metabolites of 2,6-DNT, showed the highest mutagenic activity in strain TA100. 2,6-DNBAl was a direct-acting mutagen, not requiring metabolic activation. The other compounds containing nitro groups showed weak or no mutagenic activity. This result suggests that the direct-acting mutagenicity of 2,6-DNBAl is mainly due to the aldehyde group of the 2,6-DNBAl molecule.     

Aerobic Degradation of Dinitrotoluenes and Pathway for Bacterial Degradation of 2,6-Dinitrotoluene

An oxidative pathway for the mineralization of 2,4-dinitrotoluene (2,4-DNT) by Burkholderia sp. strain DNT has been reported previously. We report here the isolation of additional strains with the ability to mineralize 2,4-DNT by the same pathway and the isolation and characterization of bacterial strains that mineralize 2,6-dinitrotoluene (2,6-DNT) by a different pathway. Burkholderia cepacia strain JS850 and Hydrogenophaga palleronii strain JS863 grew on 2,6-DNT as the sole source of carbon and nitrogen. The initial steps in the pathway for degradation of 2,6-DNT were determined by simultaneous induction, enzyme assays, and identification of metabolites through mass spectroscopy and nuclear magnetic resonance. 2,6-DNT was converted to 3-methyl-4-nitrocatechol by a dioxygenation reaction accompanied by the release of nitrite. 3-Methyl-4-nitrocatechol was the substrate for extradiol ring cleavage yielding 2-hydroxy-5-nitro-6-oxohepta-2,4-dienoic acid, which was converted to 2-hydroxy-5-nitropenta-2,4-dienoic acid. 2,4-DNT-degrading strains also converted 2,6-DNT to 3-methyl-4-nitrocatechol but did not metabolize the 3-methyl-4-nitrocatechol. Although 2,6-DNT prevented the degradation of 2,4-DNT by 2,4-DNT-degrading strains, the effect was not the result of inhibition of 2,4-DNT dioxygenase by 2,6-DNT or of 4-methyl-5-nitrocatechol monooxygenase by 3-methyl-4-nitrocatechol.     

Aerobic degradation of dinitrotoluenes and pathway for bacterial degradation of 2,6-dinitrotoluene

An oxidative pathway for the mineralization of 2,4-dinitrotoluene (2, 4-DNT) by Burkholderia sp. strain DNT has been reported previously. We report here the isolation of additional strains with the ability to mineralize 2,4-DNT by the same pathway and the isolation and characterization of bacterial strains that mineralize 2, 6-dinitrotoluene (2,6-DNT) by a different pathway. Burkholderia cepacia strain JS850 and Hydrogenophaga palleronii strain JS863 grew on 2,6-DNT as the sole source of carbon and nitrogen. The initial steps in the pathway for degradation of 2,6-DNT were determined by simultaneous induction, enzyme assays, and identification of metabolites through mass spectroscopy and nuclear magnetic resonance. 2,6-DNT was converted to 3-methyl-4-nitrocatechol by a dioxygenation reaction accompanied by the release of nitrite. 3-Methyl-4-nitrocatechol was the substrate for extradiol ring cleavage yielding 2-hydroxy-5-nitro-6-oxohepta-2,4-dienoic acid, which was converted to 2-hydroxy-5-nitropenta-2,4-dienoic acid. 2, 4-DNT-degrading strains also converted 2,6-DNT to 3-methyl-4-nitrocatechol but did not metabolize the 3-methyl-4-nitrocatechol. Although 2,6-DNT prevented the degradation of 2,4-DNT by 2,4-DNT-degrading strains, the effect was not the result of inhibition of 2,4-DNT dioxygenase by 2,6-DNT or of 4-methyl-5-nitrocatechol monooxygenase by 3-methyl-4-nitrocatechol.     

Surface plasmon resonance immunosensor for highly sensitive detection of 2,4,6-trinitrotoluene

We have examined the sensing characteristics of a surface plasmon resonance (SPR) immunoassay for the detection of 2,4,6-trinitrotoluene (TNT) using an immunoreaction between 2,4,6-trinitrophenol-ovalbumin (TNP-OVA) conjugate and anti-2,4,6-trinitrophenol antibody (anti-TNP antibody). TNP-OVA conjugate was attached to a SPR-gold sensing surface by means of physical immobilization, which undergoes binding interaction with anti-TNP antibody. Both the immobilization and binding processes were studied from a change in the SPR-resonance angle. The quantification of TNT is based on the principle of indirect competitive immunoassay, in which the immunoreaction between the TNP-OVA conjugate and anti-TNP antibody was inhibited in the presence of free TNT in solution. The decrease in the resonance angle shift is proportional to an increase in concentration of TNT used for incubation. The immunoassay exhibited excellent sensitivity for the detection of TNT in the concentration range from 0.09 to 1000 ng/ml with good stability and reproducibility. The immunosensor developed could detect TNT as low as 0.09 ng/ml, within a response time of approximately 22 min. The sensor surface was regenerated by a brief flow of pepsin solution, which disrupts the antigen-antibody complex without destroying the conjugate biofilm. Cross-reactivity of the SPR sensor to some structurally related nitroaromatic derivative and the detection of TNT in the presence of these nitroaromatic compounds were investigated. The cross-reactivity of the SPR sensor to 2,4-dinitrotoluene (2,4-DNT), 1,3-dinitrobenzene (1,3-DNB), 2-amino-4,6-dinitrotoluene (2A-4,6-DNT) and 4-amino-2,6-dinitrotoluene (4A-2,6-DNT) were very low (< or =1.1%). The analytical characteristics of the proposed immunosensor are highly promising for the development of new field-portable sensors for on-site detection of landmines.     

Immunosensor for Mycobacterium tuberculosis on screen-printed carbon electrodes

In this work, two methods have been compared to produce enzymatic voltammetric immunosensors for the determination of Mycobacterium tuberculosis antigens (Ag360 and Ag231), using a pre-oxidised screen-printed carbon electrode (SPCE) as a signal transduction element. The enzyme alkaline phosphatase (AP) was used in combination with the substrate 3-indoxyl phosphate (3-IP). In one design, the immune complexes between M. tuberculosis antigens and monoclonal antibodies against M. tuberculosis were formed out of the electrode surface. Then, the immune complexes were captured by biotinylated rabbit anti-M. tuberculosis antibodies, immobilised on the streptavidin modified SPCEs through the streptavidin:biotin reaction. Finally, an alkaline phosphatase (AP) labelled rabbit IgG anti-mouse immunoglobulin G was used as a detector antibody. In the other design, the M. tuberculosis antigens were captured by monoclonal antibodies against M. tuberculosis, which were immobilised on the electrode surface through the reaction with rabbit IgG passively adsorbed on the SPCEs. The biotinylated rabbit anti-M. tuberculosis antibodies were used with an alkaline phosphatase labelled streptavidin as detector antibodies. The best results for M. tuberculosis antigen determination were obtained using the immunosensor on the streptavidin modified SPCEs and the immune complexes between antigen Ag231 and monoclonal antibodies MabF184-3, with a detection limit of 1.0 ng/ml. The immunosensor was also applied to Ag231 spiked proteic matrices.     

Analysis of antibiotics in urine and wipe samples from environmental and biological monitoring--comparison of HPLC with UV-, single MS- and tandem MS-detection

Results of the simultaneous determination of the structurally different antibiotics cefazoline, cefotiame, cefuroxime, chloramphenicol, ciprofloxacin, ofloxacin, sulfamethoxazole and trimethoprim from environmental and biological monitoring using high-performance liquid chromatography with UV, single mass and tandem mass spectrometry were compared. For sample enrichment and clean-up a SPE method using bakerbond C18 cartridges was developed. Mean recovery rates were above 70%. Because of the complex urine matrix, only the wipe samples could be analyzed by UV-detection. However, UV-detection and single MS-detection are useful for control measurements after spillage, e.g. (LOD=1-2 ng/cm(2)). Samples from biological monitoring of occupational uptake should be analyzed by LC-MS/MS. The limits of detection (LOD) in urine ranged from 0.4 to 70 microg/L for LC-MS and 0.01 to 0.9 microg/L for LC-MS/MS detection. The limits of detection in wipe samples ranged from 0.003 to 0.13 ng/cm(2).     

Mutagenicities of 2,4- and 2,6-dinitrotoluenes and their reduced products in Salmonella typhimurium nitroreductase- and O-acetyltransferase-overproducing Ames test strains

Mutagenicities of 2,4- and 2,6-dinitrotoluene (2,4-and 2,6-DNT), and reduced metabolites formed by the incubation of 2,4- and 2,6-DNT with Salmonella typhimurium TA98, were tested using S. typhimurium YG strains possessing high level of nitroreductase (NR) and/or O-acetyltransferase (OAT) activities. All compounds tested showed greatest mutagenic activities toward strains YG1041 and YG1042, which possess high levels of NR and OAT activities. The relative mutagenic activities of 2,4-DNT and its related compounds toward YG1041 and YG1042 were aminonitrotoluenes (2A4NT, 4A2NT)<2,4-DNT<2,2′-dimethyl-5,5′-dinitroazoxybenzene (2,2′-DM-5,5′-DNAOB)4-hydroxylamino-2-nitrotoluene (4HA2NT)4,4′-dimethyl-3,3′-dinitroazoxybenzene (4,4′-DM-3,3′-DNAOB), and aminonitrotoluenes (2A4NT, 4A2NT)<2,4-DNT<4HA2NT4,4′-dimethyl-3,3′-dinitroazoxybenzene (4,4′-DM-3,3′-DNAOB)<2HA4NT, respectively. In addition, the relative mutagenic activities of 2,6-DNT and its related compounds toward YG1041 and YG1042 were 2,6-DNT<2-hydroxylamino-6-nitrotoluene (2HA6NT)<2,2′-dimethyl-3,3′-dinitroazoxybenzene (2,2′-DM-3,3′-DNAOB), and 2-amino-6-nitrotoluene (2A6NT)<2,6-DNT<2HA6NT, respectively. These results, together with previous findings, suggested that aminohydroxylamino dimethylazoxybenzenes or aminohydroxylamino dimethylazobenzenes produced either by the reduction of hydroxylaminonitrotoluenes or by the reduction of dimethyl dinitroazoxybenzenes are active metabolites responsible for the mutagenic activities of 2,4- and 2,6-DNT.     

Microbial Transformation of Nitroaromatics in Surface Soils and Aquifer Materials

Microorganisms indigenous to surface soils and aquifer materials collected at a munitions-contaminated site transformed 2,4,6-trinitrotoluene (TNT), 2,4-dinitrotoluene (2,4-DNT), and 2,6-dinitrotoluene (2,6-DNT) to amino-nitro intermediates within 20 to 70 days. Carbon mineralization studies with both unlabeled (TNT, 2,4-DNT, and 2,6-DNT) and radiolabeled ([¹⁴C]TNT) substrates indicated that a significant fraction of these source compounds was degraded to CO₂.     

Liquid chromatographic determination of isoniazid, pyrazinamide and rifampicin from pharmaceutical preparations and blood

Isoniazid (IN), pyrazinamide (Pz) and rifampicin (Rf) are separated on YMC-ODS column. IN was derivatized with 2-fluorene-carboxaldehyde (FA). The separation was achieved using ethanol-chloroform-acetonitrile water by isocratic elution and detected at 337 nm. The detection limits were 0.11 ng, 0.2 ng and 13 ng/injection (5 microl) for IN, Pz and Rf, respectively. The method of analysis was applied to the pharmaceutical preparations and in the blood samples of the patients suffering from tuberculosis after undergoing chemotherapy with IN, Pz and Rf. The amounts quantitated in blood showed 0.97 to 1.58 microg/ml IN, 3.44 to 4.09 microg/ml Pz and 1.98 to 3.5 microg/ml Rf with coefficient of variations 0.8-1.8%, 0.9-1.3% and 0.8-2.1%, respectively.     

Biodegradation of 2,4,6-trinitrotoluene (TNT) by the white-rot basidiomycete Phlebia radiata

Phlebia radiatatransformed 2,4,6-trinitrotoluene (TNT), as well as its first reduction products, the aminodinitrotoluenes, into 4-hydroxylamino-2,6-dinitrotoluene (4-OHA-2,6-DNT) and 4-amino-2,6-dinitrotoluene (4-A-2,6-DNT). No extracellular peroxidases were involved in this step. The ligninolytic extracellular fluid, assumed to contain peroxidases, did not reduce TNT. However, ligninolytic peroxidases are implicated in the transformation of the first reduction products of TNT.     

Voltammetric sensor for vanillylmandelic acid based on molecularly imprinted polymer-modified electrodes

Despite the increasing number of applications of molecularly imprinted polymers (MIPs) in analytical chemistry, the construction of a biomimetic voltammetric sensor remains still challenging. This work investigates the development of a voltammetric sensor for vanillylmandelic acid (VMA) based on acrylic MIP-modified electrodes. Thin layers of MIPs for VMA have been prepared by spin coating the surface of a glassy carbon electrode with the monomers mixture (template, methacrylic acid, a cross-linking agent and solvent), followed by in situ photopolymerisation. After extraction of the template molecule, the peak current recorded with the imprinted sensor after rebinding was linear with VMA concentration in the range 19-350 microg ml(-1), whereas the response of the control electrode is independent of incubation concentration, and was about one-tenth of the value recorded with the imprinted sensor at the maximum concentration tested. Under the conditions used, the sensor is able to differentiate between VMA and other closely structural-related compounds, such as 3-methoxy-4-hydroxyphenylethylene glycol (not detected), or 3,4- and 2,5-dihydroxyphenilacetic acids, which are adsorbed on the bare electrode surface but not at the polymer layer. Homovanillic acid was detected with the imprinted sensors after incubation, indicating that the presence of both methoxy and carboxylic groups in the same position as in VMA is necessary for effective binding in the imprinted sites. Nevertheless, both species can be differentiated by the oxidation potential. It can be concluded that MIP-based voltammetric electrodes are very promising analytical tool for the development of highly selective analytical sensors.     

Bioremediation Potential of Rhodococcus pyridinivorans NT2 in Nitrotoluene-Contaminated Soils: The Effectiveness of Natural Attenuation,Biostimulation and Bioaugmentation Approaches

This work evaluated the effect of bioremediation treatments including natural attenuation, bioaugmentation, biostimulation as well as combined biostimulation and bioaugmentation on degradation of 4-nitrotoluene (4-NT), 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT) in soil microcosms. Bioaugmentation with a previously isolated NTs-degrading bacterium, Rhodococcus pyridinivorans NT2, showed an 86–88% decrease in 4-NT, 2,4-DNT or 2,6-DNT after 60 days. Irrespective of the substrate types, least degradation (6–6.5%) was observed in abiotic control. The addition of β-cyclodextrin or rhamnolipid significantly improved NTs degradation efficiency in soil (18.5–74%) than natural attenuation (22–25%). Exogenous addition of preselected bacterial isolate NT2 along with β-cyclodextrin/rhamnolipid resulted in the greatest number (1.8× and 2.5× high) of total heterotrophic aerobic bacteria and NT degraders, respectively, compared to natural attenuation. Irrespective of the treatment types, the population of NT degraders increased steadily in the first 5 weeks of incubation followed by a plateau within the next few weeks. The treatment BABS2 (Soil + rhamnolipid + NT2) yielded highest microbial-C and -N and dehydrogenase activity, consistent with results of NTs degradation and microbial counts in combined bioaugmentation and biostimulation. Thus the results of this study suggest that bioaugmentation by R. pyridinivorans NT2 may be a promising bioremediation strategy for nitroaromatics-contaminated soils.     

Effect of bolus fluid intake on energy expenditure values as determined by the doubly labeled water method.

The doubly labeled water (DLW, 2H(2)18O) method is a highly accurate method for measuring energy expenditure (EE). A possible source of error is bolus fluid intake before body water sampling. If there is bolus fluid intake immediately before body water sampling, the saliva may reflect the ingested water disproportionately, because the ingested water may not have had time to mix fully with the body water pool. To ascertain the magnitude of this problem, EE was measured over a 5-day period by the DLW method. Six subjects were dosed with 2H2(18)O. After the reference salivas for the two-point determination were obtained, subjects drank water (700-1,000 ml), and serial saliva samples were collected for the next 3 h. Expressing the postbolus saliva enrichments as a percentage of the prebolus value, we found 1) a minimum in the saliva isotopic enrichments were reached at approximately 30 min with the minimum for 2H (95.48 +/- 0.43%) being significantly lower than the minimum for 18O (97.55 +/- 0.44, P less than 0.05) and 2) EE values calculated using the postbolus isotopic enrichments are appreciably higher (19.9 +/- 7.5%) than the prebolus reference values. In conclusion, it is not advisable to collect saliva samples for DLW measurements within approximately 1 h of bolus fluid intake.