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PubMed2000

Assessment of decalcifying protocols for detection of specific RNA by non-radioactive in situ hybridization in calcified tissues.

For the best performance of in situ analysis of specific RNA expression in calcified tissues, it is necessary to choose an appropriate protocol to decalcify the tissues. We evaluated the usefulness of various acid-based decalcifying reagents with reference to 28 S rRNA staining by in situ hybridization using a thymine-thymine dimerized oligonucleotide probe. The reagents evaluated were 10% nitric acid, 10% HCl, 5% formic acid, 5% trichloroacetic acid, Morse's solution, Plank-Rychlo's solution, and K-CX solution, all of which are commonly used to decalcify tissues, and their effects on retention of morphology and RNA were compared with EDTA-based solutions. When normal mouse mandible was used as a model tissue, well-preserved morphology of ameloblasts was obtained from sections decalcified with Morse's solution, 10% HCl, Plank-Rychlo's solution, and K-CX solution, and best retention of 28 S rRNA was obtained with 5% formic acid and Morse's solution. We recommend Morse's solution to decalcify tissues to be processed for the rapid analysis of specific RNA expression. Indeed, we detected specific mRNAs strongly in sections treated with Morse's solution, and quantitative analysis showed that the ratio of signal intensities of 28 S rRNA and the specific mRNAs correlated with each other depending on decalcifying solutions.

Animals↗

PubMed2002

The aerobic dechlorination activities of two bacterial species isolated from a refuse dumpsite in Nigeria.

Two bacterial species isolated using enrichment culture techniques from the topsoil of a main refuse dumpsite in Nigeria were assessed for their dehalogenation potentials. The bacterial isolates were identified as belonging to the Bacillus and Pseudomonas genera. Axenic cultures of the isolates utilized monochloroacetic acid (MCA), trichloroacetic acid (TCA), trichloromethane (CHCl3) and tetrachloromethane (CCl4) as the sole source of carbon for growth up to a final substrate concentration of 0.1% (w/v). The mean generation times of the isolates in all the growth media ranged significantly (P<0.05) from 2.41 to 10.04 h and were generally higher than that observed in glucose medium (1.46-1.51 h). The numbers of the chloride atoms in the different organochlorides were negatively correlated with the ability of the organisms to degrade the compounds. Dehalogenase specific activities of the cell-mediated cultures ranged from 0.1 to 0.96 microg ml(-1) chloride release (mg protein)(-1) h(-1) and were significantly (P<0.05) higher than that of the cell-free extract [0.09-0.8 microg ml(-1) chloride release (mg protein)(-1) h(-1)]. The optimal pH of the dehalogenase activity was found to be 8.0, and the optimal temperature was between 30 and 35 degrees C.

Aerobiosis↗

PubMed1985

Selective stage-specific changes in the permeability to small hydrophilic solutes of human erythrocytes infected with Plasmodium falciparum.

The permeability characteristics of Plasmodium falciparum-infected human erythrocytes to various 3H-labelled solutes were measured during the maturation of the parasites in sorbitol-synchronised cultures. Using [14C]inulin as the extracellular marker, estimates were made of the influx kinetics of [3H]amino acids into trichloroacetic acid (TCA)-soluble pools within the erythrocyte and concomitant incorporation into TCA-precipitable material. These measurements provided values of the rates of protein synthesis by the parasite and the initial influx rates for the transport of precursor amino acids into the erythrocyte. For about 12-15 h after parasitisation, the influx of L-[3H]glutamine remained at a low level comparable to that in the uninfected cell (2-9 nmol g-1 cells min-1). As pigment appeared in the trophozoite, the initial rate of influx of L-glutamine increased to a value up to 100-fold higher than in the uninfected erythrocyte. The increase in permeability affected only the parasitised cells in a culture of partially infected erythrocytes, and was selective with respect to substrate since the influx kinetics for both [3H]isoleucine and [3H]arginine were not affected by parasitisation. The permeability changes occurred mainly over a 4-8 h period in the development of the young trophozoite, during which time [3H]glycine influx was enhanced by a factor of 3-10, with a comparable increase in the uptake of myo-[3H]inositol. L-[3H]glutamate, which did not penetrate significantly into uninfected erythrocytes, entered red cells infected with mature trophozoites at a rate which was much less than 1% of the parasite-induced-L-glutamine influx. At the stages when the permeability to L-glutamine was markedly enhanced, parasitised cells remained impermeable to [3H]sucrose. An analysis of the relative 3H activities in glutathione and free amino acid pools indicated that, if L-glutamine permeation did not increase during parasite maturation beyond the ring stage, or was blocked by a potential antimalarial compound, an insufficient supply of L-glutamine would be available for the increased rates of parasite protein synthesis and glutathione turnover within the red cell.

Animals↗

PubMed1992

Toxicology of maternally ingested trichloroethylene (TCE) on embryonal and fetal development in mice and of TCE metabolites on in vitro fertilization.

Trichloroethylene (TCE), an industrial solvent, is a soil and ground water contaminant found across the United States. The metabolism and carcinogenic potential of TCE have been studied extensively in the past 15 years yet there is little information on the chemical's possible effects on reproduction. No reference to the reproductive effects in mice of TCE by oral administration exists in the literature. In this study, female B6D2F1 mice were gavaged from Days 1 to 5, 6 to 10, or 11 to 15 (Day 1 = vaginal plug) with TCE in corn oil at 0, 1/10, and 1/100 of the oral LD50. Weights of mice were recorded and the livers and kidneys were weighed and preserved in 10% buffered formalin. Litters were counted, sexed, weighed, and measured for crown-rump length until weaning on Day 21 and some animals were allowed to develop to 6 weeks of age. At this time, a minimum of two litters from each dose were killed and gonads removed, weighed, and preserved in Bouin's fixative. Litters were also assessed for developmental abnormalities. No maternal or reproductive effect of TCE was seen at either dose level. TCE, administered consecutively on Days 1 to 5, 6 to 10, and 11 to 15 of pregnancy, does not appear to be a potential reproductive toxicant up to 1/10 the oral LD50. In a second series of studies, TCE and its metabolites dichloroacetic acid (DCA), trichloroacetic acid (TCAA), and trichloroethanol (TCOH) were added to culture media to assess the toxic effects on in vitro fertilization (IVF) in mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

High-performance liquid chromatographic measurement of selected blood citric acid cycle intermediates.

We describe a high-performance liquid chromatographic (HPLC) method for analysis of the intermediates of the citric acid cycle. Using two Aminex HPX-87H columns in series at 36 degrees C, the early eluting compounds cis-aconitate, oxaloacetate, alpha-ketoglutarate and citrate-isocitrate can be resolved. Acetonitrile is used for extraction of citric acid cycle intermediates from blood as interfering ultraviolet absorbing peaks are present with perchloric acid or trichloroacetic acid extraction. Acetonitrile extraction is compared with perchloric acid extraction of citric acid cycle intermediates from plasma. Low recovery of some organic acids from blood seems not to be due to enzymatic degradation. Storage of acetonitrile extracts in liquid nitrogen led to a small but significant decrease in pyruvate levels in human blood. However, significant changes in other organic acids were not seen. HPLC methodology allows study of the citric acid cycle in tissue samples as well as blood and promises to facilitate the investigation of human disorders of energy metabolism.

Acetonitriles↗

PubMed1995

Quantitative chromatographic analysis of inositol phospholipids and related compounds.

The metabolism of phospholipids and the mobilization of second messengers such as inositol-1,4,5-trisphosphate, 1,2-diacylglycerol (DAG) and arachidonic acid (AA) from phospholipids is commonly studied by radiolabelling phospholipids with [3H]myo-inositol or [32P]ATP and measuring the incorporation of radioactivity in different phospholipids or their hydrolysis products. However, for the radiolabelling method to accurately reflect changes in the compound's mass, it is essential that the tissue is labelled to isotopic equilibrium which is difficult to achieve. To circumvent the disadvantages of the radiolabelling method, several analytical procedures have been developed for the mass analysis of phospholipids and inositolphosphates (IPs). Quantitation of the mass or the radiolabelling of phospholipids is a complex multi-step procedure that involves quantitative isolation of phospholipids, fractionation of individual phospholipids and either determination of radioactivity in each component or the measurement of their mass. Phospholipids, DAG and AA are extracted from tissue sample with organic solvents such as chloroform-methanol (2:1) containing HCl or formic acid. The extract is separated by TLC, cartridge-column chromatography or HPLC on a reversed-phase column. Phospholipids are quantitated by measuring inorganic phosphate, absorption at 200 nm or mass spectrometry. Inositol phosphates are extracted with perchloric acid or trichloroacetic acid and separated by ion-exchange cartridge-column or HPLC with an ion-exchange column. IPs are quantitated by measuring inorganic phosphate or by using enzymatic reaction, metal-dye coupling, NMR or mass spectrometry.

Arachidonic Acid↗

PubMed2004

Haloacetic acids in the aquatic environment. Part I: macrophyte toxicity.

Haloacetic acids (HAAs) are contaminants of aquatic ecosystems with numerous sources, both anthropogenic and natural. The toxicity of HAAs to aquatic plants is generally uncharacterized. Laboratory tests were conducted with three macrophytes (Lemna gibba, Myriophyllum sibiricum and Myriophyllum spicatum) to assess the toxicity of five HAAs. Myriophyllum spp. has been proposed as required test species for pesticide registration in North America, but few studies have been conducted under standard test conditions. The HAAs in the present experiments were monochloroacetic acid (MCA), dichloroacetic acid (DCA), trichloroacetic acid (TCA), trifluoroacetic acid (TFA) and chlorodifluoroacetic acid (CDFA). MCA was the most toxic to Myriophyllum spp. with EC50 values ranging from 8 to 12.4 mg/l depending on the endpoint, followed by DCA (EC50 range 62-722.5 mg/l), TCA (EC50 range 49.5-1702.6 mg/l), CDFA (EC50 range 105.3 to >10,000 mg/l) and with TFA (EC50 range 222.1 to 10,000 mg/l) the least toxic. Generally, L. gibba was less sensitive to HAA toxicity than Myriophyllum spp., with the difference in toxicity between them approximately threefold. The range of toxicity within Myriophyllum spp. was normally less than twofold. Statistically, plant length and node number were the most sensitive endpoints as they had the lowest observed coefficients of variation, but they were not the most sensitive to HAA toxicity. Toxicological sensitivity of endpoints varied depending on the measure of effect chosen and the HAA, with morphological endpoints usually an order of magnitude more sensitive than pigments for all plant species. Overall, mass and root measures tended to be the most sensitive indicators of HAA toxicity. The data from this paper were subsequently used in an ecological risk assessment for HAAs and aquatic plants. The assessment found HAAs to be of low risk to aquatic macrophytes and the results are described in the second manuscript of this series.

Acetates↗

PubMed2004

Haloacetic acids in the aquatic environment. Part II: ecological risk assessment.

Haloacetic acids (HAAs) are environmental contaminants found in aquatic ecosystems throughout the world as a result of both anthropogenic and natural production. The ecological risk posed by these compounds to organisms in freshwater environments, with a specific focus on aquatic macrophytes, was characterized. The plants evaluated were Lemna gibba, Myriophyllum spicatum and M. sibiricum and the HAAs screened were monochloroacetic acid (MCA), dichloroacetic acid (DCA), trichloroacetic acid (TCA), trifluoroacetic acid (TFA) and chlorodifluoroacetic acid (CDFA). Laboratory toxicity data formed the basis of the risk assessment, but field studies were also utilized. The estimated risk was calculated using hazard quotients (HQ), as well as effect measure distributions (EMD) in a modified probabilistic ecological risk assessment. EMDs were used to estimate HAA thresholds of toxicity for use in HQ assessments. This threshold was found to be a more sensitive measure of low toxicity than the no observed effect concentrations (NOEC) or the effective concentration (EC10). Using both deterministic and probabilistic methods, it was found that HAAs do not pose a significant risk to freshwater macrophytes at current environmental concentrations in Canada, Europe or Africa for both single compound and mixture exposures. Still, HAAs are generally found as mixtures and their potential interactions are not fully understood, rendering this phase of the assessment uncertain and justifying further effects characterization. TCA in some environments poses a slight risk to phytoplankton and future concentrations of TFA and CDFA are likely to increase due to their recalcitrant nature, warranting continued environmental surveillance of HAAs.

Acetates↗

PubMed2006

THM, HAA and CNCl formation from UV irradiation and chlor(am)ination of selected organic waters.

The formation of disinfection by-products (DBPs), including chloroform, dichloroacetic acid (DCAA), trichloroacetic acid (TCAA), and cyanogen chloride (CNCl) after sequential exposure of four organic waters to UV irradiation via either low- or medium-pressure lamps and free chlorine (or preformed monochloramine) under practical conditions was simulated. Statistically significant changes in the DBP formation from chlorination due to the additional UV irradiation are commonly observed under testing conditions, although some of these changes are not practically significant. The impacts from UV exposure were found to be most significant in chloroform formation (up to 40 microg/L) among the four tested DBPs. Organics from rivers were more sensitive to UV alteration than was the organic drawn from soil. This difference could not be explained by the specific UV absorbance (SUVA) values. In most cases, irradiation with the medium-pressure UV lamp gave similar or slightly larger changes in DBP yields, compared with the corresponding trials using the low-pressure lamp. Different application sequences could significantly change the relative quantities of DBPs but no general trend was identified. Case-specific evaluation of the formation of chloroform and CNCl is necessary.

Acetates↗

PubMed2001

Aerobic dehalogenation potentials of four bacterial species isolated from soil and sewage sludge.

Four bacterial species each were isolated from soil and a sewage oxidation pond using enrichment culture technique, and the bacterial isolates were identified to belong to the two genera Bacillus and Corynebacterium. The axenic cultures of the isolates utilized monochloroacetic acid (MCA), trichloroacetic acid (TCA), CHCl3 and CCl4 for growth up to 1 g substrate l(-1) (w/v) and growths were enhanced in the mixed cultures of the isolates. The specific growth rate constants in the media ranged from 0.144 to 0.475. This is lower than the comparatively high values observed for the glucose medium, which varied significantly (P < 0.05) between 0.699 and 0.792 h(-1). Serial adaptation of the individual isolates in the organochloride media significantly (P < 0.05) affected the bacteria growth yields. The dehalogenase specific activity observed in the cell-free extracts of the mixed cultures of the isolates was significantly higher (P < 0.05) than those of their respective monocultures. Optimal pH of the dehalogenase activity was between 7.6 and 8.0, while temperature optima were between 30 degrees C and 35 degrees C.

Bacillus↗

PubMed2003

Measurement of 13C/12C of chloroacetic acids by gas chromatography/combustion/isotope ratio mass spectrometry.

This paper describes a novel analytical methodology using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) to measure the 13C/12C ratios of chloroacetic acids (CAAs). CAAs are a major class of environmental pollutants that are widely distributed throughout the world, often at relatively high concentrations, and are of concern due to their toxic effects, particularly on plants. The 13C/12C of CAA reagents was measured by IRMS subsequent to offline combustion. Aqueous solutions of these CAAs were derivatized to the corresponding methyl chloroacetates (MCAAs) with acidic methanol with a known isotopic composition, extracted into pentane, and analyzed by GC/C/IRMS. Measured 13C/12C ratios of derivatized MCAAs were in agreement with calculated values within 1 per thousand for monochloroacetic acid and trichloroacetic acid and within 2 per thousand for dichloroacetic acid, suggesting that methylation and other analytical methodology steps do not isotopically fractionate derivatized MCAAs. 13C/12C ratios of reagent CAAs from different sources had varying isotopic signatures suggesting differences in source carbon and/or production methods. Our results underscore the potential of stable isotopes to serve as tracers of environmental sources of CAAs.

Acetates↗

PubMed1977

Histochemical methods for proteins in tissues submitted to decalcification. A histophotometric study.

4 histochemical methods for demonstration of protein acid groups: Nihydrin-Schiff, Millon, DDD and DMAB-nitrite were applied in sections of rat pancreas fixed in the following fixatives: 10% formalin, 10% formalin in saline, ZENKER and GENDRE, and treated with the following decalcifying solutions: 5% nitric acid, 5% trichloroacetic acid, 33% formic acid, 5% EDTA pH=7.0 JENKINS, GREEP and citrate buffer pH=4.5. The colour intensity of each reaction was measured photometrically over the zymogen granules region of the acinar cells, using a Zeiss histophotometer, and compared with controls, i.e., fixed tissue which have not been treated with decalcifying solutions. The results have shown that for each method there is more than one combination fixative-decalcifying solution which gives results similar to those of the control.

Animals↗

PubMed1975

[Incorporation of tritiated thymidine into DNA of rat liver: a critique of various evaluation methods].

We have measured the incorporation of 3H-(methyl)-thymidine by cell cultures of rat foetal liver and in vivo by the livers of young rats stimulated by casein, in order to compare three methods for the extraction of DNA. The DNA was extracted by three different techniques: perchloric acid precipitation, trichloroacetic acid precipitation and phenol extraction, and its specific activity was determined. The radioactive labelling was also determined for the lipid, ribonucleic acid and protein fractions for the two first methods, in both of which 70 p. cent of the incorporated tritium is found in the DNA fraction and about 10 p. cent in each of the other fractions. The determination of the specific radioactivity of DNA gives similar results for the three extraction methods. However, since larger yields were obtained by both acid precipitation techniques than by phenol extraction, we believe them to be more suitable for studies on cell cultures.

Animals↗

PubMed2002

Finnish quality assurance programme for biological monitoring of organic solvents.

The FIOH quality assurance programme for organic solvents and their metabolites consists of analyses for 2,5-hexadione, phenol, mandelic acid, methylenedianiline, methylhippuric acid, trans,trans-muconic acid and trichloroacetic acid in urine, and creatinine and relative density for standardisation. Four times a year two levels of spiked urine or urine specimens collected from occupationally exposed workers are distributed to the participants in 22 countries. RSD and recovery were studied during 1997-2000. Average RSDs of all participants varied between 23 and 56% and were clearly dependent on the analytical method used and the concentration level of the samples. Since 1997 the target values have been determined in reference laboratories for five of the analytes. Lower RSDs (9-21%) and good recoveries were obtained for all analytes in these laboratories, indicating that good performance can be achieved even in the complex analyses performed in biological monitoring of exposure to industrial chemicals.

Environmental Monitoring↗

PubMed1998

High-performance liquid chromatographic determination of beta-alanine, beta-aminoisobutyric acid and gamma-aminobutyric acid in tissue extracts and urine of normal and (aminooxy)acetate-treated rats.

A method is described for the simultaneous determination of beta-alanine, beta-aminoisobutyric acid and gamma-aminobutyric acid in biological materials. Amino acids including these beta- and gamma-amino acids were derivatized with 4-dimethylaminoazobenzene-4'-sulfonyl (dabsyl) chloride and dabsyl amino acids formed were separated by reversed-phase high-performance liquid chromatography. Dabsyl derivatives of these beta- and gamma-amino acids were well separated from other dabsyl-amino acids. The method was applied to the determination of these beta- and gamma-amino acids in trichloroacetic acid extracts of various tissues and to the urine of normal rats and those injected with (aminooxy)acetate (AOA). AOA injection (15 mg per kg of body mass) produced remarkable increase in beta-alanine contents in liver, kidney and urine (10.2, 4.6 and 25.7 times, respectively).

Aminoisobutyric Acids↗

PubMed1988

Enhanced inactivation of bacterial lipases and proteinases in whole milk by a modified ultra high temperature treatment.

Cultures of Pseudomonas spp. strains P10, P12 and P15 grown in whole milk which contained approximately 1 x 10(8) viable bacteria ml-1 demonstrated near linear increases in the concentration of short-chain free fatty acids and trichloroacetic acid soluble free amino groups at 20 degrees C, following either ultra high temperature (UHT) treatment (140 degrees C for 5 s) or dual heat treatments (140 degrees C followed by either 57, 60 or 65 degrees C). The dual heat treatments reduced the rates of lipolysis and proteolysis compared to the UHT treatment by up to 25-fold. The dual heat treatment utilizing 60 degrees C for 5 min also effectively limited both lipase and proteinase activities in raw milk culture samples which had contained either 6 x 10(6), 5 x 10(7) or 1 x 10(8) viable bacteria ml-1. In this system enzyme activities were reduced by up to 10-fold following dual heat treatment compared to UHT treatment alone.

Animals↗

PubMed2006

Trichloroethylene uptake by apple and peach trees and transfer to fruit.

A greenhouse study was conducted to quantify 14C-trichloroethylene (TCE) uptake and transfer into the edible fruit of apple and peach trees. Trees were subsurface irrigated with solutions of 14C [TCE] that bracketed groundwater concentrations (5 and 500 microg/L) found in residential areas surrounding Hill Air Force Base, UT, where trace amounts of TCE had been found in several fruits during a preliminary field survey. Nondosed control trees were grown within the canopy of the dosed trees and in a separate greenhouse. Tissue samples were analyzed for 14C and TCE using combustion/liquid scintillation counting (LSC) and headspace/gas chromatography/mass spectrometry (HS/GC/MS). Tissue was also extracted and analyzed by GC/MS for dichloroacetic acid (DCAA), trichloroacetic acid (TCAA), and trichloroethanol (TCEt), three specific TCE metabolites that have been previously identified in laboratory and field studies. No 14C was detected in the nonexposed control trees. Exposed trees contained levels of 14C that were proportional to the exposure concentration. 14C concentrations were greatest in leaves followed by branches and fruits. At the end of the study, TCE was detected only in roots implying that the 14C in the leaves, branches, and fruit was associated with unidentified nonvolatile TCE transformation products and/or is nonextractable. However, TCAA and DCAA were positively identified only in leaves collected during the first year from an apple tree exposed to the high dose treatment. Additional data for other chemicals and fruittrees are needed to better understand the potential transfer of organic compounds to edible fruit.

Carbon Isotopes↗

PubMed1978

Choice of precursors for the measurement of protein turnover by the double-isotope method. Application to the study of mitochondrial proteins.

1. The double-isotope concept [Arias, Doyle & Schimke (1969) J. Biol. Chem. 224, 3303--3315] for the measurement of protein turnover was used to estimate the turnover of proteins in subcellular and submitochondrial fractions prepared from rat liver. 2. Double-isotope experiments with [3H]leucine as first precursor and [14C]leucine as second precursor were used to measure the turnover rates of proteins in subcellular and submitochondrial fractions. Solvent extraction procedures designed to remove lipids and nucleic acids from trichloroacetic acid precipitates only changed the isotope ratio of the microsomal fraction. It was not possible to measure turnover of proteins in mitochondrial and submitochondrial fractions with these precursors. 3. Double-isotope experiments were designed to minimize first-precursor reutilization by employing NaH14CO3. [3H]Arginine was used as second precursor. The turnover rates of protein in subcellular and submitochondrial fractions was measured. Solvent extraction procedures designed to remove lipids and nucleic acids showed changes in the isotope ratio for all subcellular fractions, especially in microsomal and detergent-soluble mitochondrial fractions. Isotope ratios of precipitates after solvent extraction indicate that, whereas considerable heterogeneity exists for the average rates of protein turnover in subcellular fractions, little heterogeneity is observed in the average rates of protein turnover in submitochondrial fractions.

Animals↗