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PubMed1987

Metabolism of high-density lipoproteins in cultured rat luteal cells.

The uptake of cholesterol from high-density lipoproteins (HDL) labeled with 125I and [3H]cholesterol was examined in cultured rat luteal cells. Luteal cells were incubated with labeled HDL, following which the metabolic fate of the apolipoproteins and cholesterol moieties of the receptor-bound HDL were examined. About 50% of the originally bound HDL apolipoproteins were released into the medium in 24 h by a temperature-dependent process while only 5% of the HDL cholesterol was released unmetabolized. Inclusion of unlabeled HDL in the chase incubation resulted in increased release of apolipoprotein-derived radioactive products without significant change in the release of unmetabolized cholesterol. 60% of the apolipoprotein-derived radioactivity could be precipitated with trichloroacetic acid; the remaining trichloroacetic acid-soluble radioactive fraction was identified as [125I]iodotyrosine. Gel filtration chromatography of the chase-released material showed that the trichloroacetic acid-precipitable products, which contained no detectable amounts of cholesterol, eluted over a range of molecular sizes (9-80 kDa). No intact HDL was retroendocytosed. About 80% of trichloroacetic acid-precipitable products could be immunoadsorbed on anti-apolipoprotein A-I antibody immobilized on CNBr-activated Sepharose, suggesting the presence of fragments containing apolipoprotein A-I. This material was also capable of reassociating with native HDL. Lysosomal inhibitors were partially effective in inhibiting the amount of trichloroacetic acid-soluble products formed. The lysosomal degradation appeared to have no role in the uptake of HDL-derived cholesterol. These studies demonstrate preferential and total uptake of HDL cholesterol by luteal cells, with concomitant degradation of the lipoprotein.

Animals↗

PubMed1991

[Application of trichloroacetic acid-treated antigen for serodiagnosis of Q fever by indirect immunofluorescence technique].

It is well known that the etiologic agent, Coxiella burnetii, exhibits an antigenic phase variation (phase I to phase II), and the diagnostic significance of the relative antibody titers against phase I and phase II antigens is pointed out. Therefore both phase I and phase II antigens are necessary for the serological examination of Q fever. But it is not so easy to prepare and maintain the phase II antigen by the conventional method. In the present study we tried to prepare the phase II antigen for immunofluorescence test by chemical treatment of the phase I antigen. As a result, treatment of the TK-1 strain of C. burnetii (phase I) with 10% trichloroacetic acid for 4 hr at 4 degrees C modified the antigenicity. The modified antigen reacted strongly with anti-phase II antibody.

Animals↗

PubMedDate not supplied

Assessment of urban population exposure to trichloroethylene and tetrachloroethylene by means of biological monitoring.

Exposure of the general population to trichloroethylene and tetrachloroethylene under normal environmental conditions, achieved with biological monitoring, was assessed, and the possible influence of these compounds via drinking water on the body burden was revealed. A total of 79 subjects with no known solvent exposure was selected, by stratified sampling, from the residents of the city of Zagreb. Trichloroethylene and tetrachloroethylene were determined in blood, and trichloroethanol and trichloroacetic acid were determined in plasma and urine. Drinking water samples were also analyzed for trichloroethylene and tetrachloroethylene. Concentrations of trichloroethylene and tetrachloroethylene in blood, trichloroacetic acid in plasma, trichloroacetic acid in urine, trichloroethylene in drinking water, and tetrachloroethylene in drinking water were as follows: < 0.015 to 0.090 micrograms/l, < 0.010 to 0.239 micrograms/l, 8.6 to 148.1 micrograms/l, 1.67 to 102.3 micrograms/24 h, < 0.05 to 22.93 micrograms/l, and 0.21 to 7.80 micrograms/l, respectively. The variation in all results presented is probably a reflection of different environmental contamination with trichloroethylene and tetrachloroethylene in the different city areas. Correlation analyses revealed significant relationships between trichloroethylene and tetrachloroethylene in blood (r = .402, p = .0004); trichloroacetic acid in urine and in plasma (r = .522, p = .0000); and trichloroethylene and tetrachloroethylene in drinking water (r = .800, p = .0000). A division of all parameters into a subgroup (n = 58), taking drinking water concentrations of trichloroethylene above 3 micrograms/l as a basis, demonstrated the same significant relationships as mentioned above.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1999

NTP technical report on the toxicity and metabolism studies of chloral hydrate (CAS No. 302-17-0). Administered by gavage to F344/N rats and B6C3F1 mice.

Chloral hydrate is widely used as a sedative and a hypnotic in pediatric medicine. It is also a byproduct of water chlorination. Chloral hydrate has been shown to be genotoxic in numerous prokaryotic and eukaryotic assay systems including human lymphocytes in vitro. One of its metabolites, trichloroacetic acid, has demonstrated hepatocarcinogenic activity in mice. Trichloroethylene and perchloroethylene, both of which are metabolized to chloral hydrate, have been shown to be carcinogenic in rats and/or mice. Because of this evidence of carcinogenicity and because of the wide-spread use of chloral hydrate, 16- or 17-day range-finding toxicity studies and separate 16- or 17-day metabolism studies were performed in F344/N rats and B6C3F1 mice in preparation for further long-term rodent studies. In addition, in vitro studies of the metabolism and DNA-binding capacity of chloral hydrate and its metabolites were performed. Genetic toxicity studies were conducted in Salmonella typhimurium, cultured Chinese hamster ovary cells, Drosophila melanogaster, and mouse bone marrow cells. For the range-finding studies, groups of eight male and eight female F344/N Nctr BR rats and B6C3F1/Nctr BR (C57BL/6N x C3H/HeN MTV-) mice were administered 0, 50, 100, 200, 400, or 800 mg chloral hydrate per kg body weight in water by gavage 5 days per week for 17 days (rats) or 16 days (mice) for a total of 12 doses. One male rat receiving 800 mg/kg died after five doses. Two 800 mg/kg female rats died after dosing ended but before study termination. One male mouse in each group except the 400 mg/kg group died before the end of the study. Two 800 mg/kg female mice also died before the end of the study. The final mean body weight of 800 mg/kg male rats and the mean body weight gains of 400 and 800 mg/kg males were significantly less than those of the vehicle controls. The mean body weight gains of all groups of dosed male mice were significantly greater than that of the vehicle control group. The only clinical finding in rats and mice attributed to chloral hydrate treatment was light sedation in the 400 mg/kg groups and heavy sedation in the 800 mg/kg groups; sedation subsided within 30 minutes or 3 hours, respectively. The liver weights of 400 mg/kg male mice and 800 mg/kg male and female mice were significantly greater than those of the vehicle control groups. No chemical-related lesions were observed in rats or mice. Male and female rats and mice were administered a single dose of 50 or 200 mg chloral hydrate per kg body weight in water by gavage, or 12 doses of 50 or 200 mg/kg over 17 days (rats) or 16 days (mice). Plasma concentrations of chloral hydrate and its metabolites were determined 15 minutes, 1, 3, 6, and 24 hours, and 2, 4, 8, and 16 days after receiving 1 or 12 doses. Maximum concentrations of chloral hydrate were observed at the initial sampling point of 15 minutes. By 1 hour, the concentrations had dropped substantially, and by 3 hours, chloral hydrate could not be detected in rats or mice. Trichloroacetic acid was the major metabolite detected in the plasma. In rats, the concentrations rose slowly, with the peaks occurring between 1 and 6 hours after treatment. In mice, the peak concentrations were found 1 hour after dosing. The concentrations then slowly decreased such that by 2 days the metabolite could no longer be detected in rats or mice. Trichloroethanol was assayed both as the free alcohol and its glucuronide. In rats, the maximum concentrations of free trichloroethanol occurred at 15 minutes, while the peak concentrations of trichloroethanol glucuronide were found at 1 hour; by 3 hours, concentrations of both metabolites approached background levels. In mice, the maximum concentrations of both metabolites occurred at 15 minutes, and by 1 to 3 hours concentrations approached background levels. The plasma concentrations of chloral hydrate and its metabolites were dose dependent in rats and mice. In mice, plasma concentrations of trichloroacetic acid were significantly higher after a single dose than after 12 doses. None of the metabolic parameters appears to account for species differences that may exist in hepatocarcinogenicity. The data from the study of metabolism and DNA adduct formation indicated that in vitro metabolism of 200 microM to 5 mM chloral hydrate by male B6C3F1 mouse liver microsomes (control microsomes) generated free radical intermediates that resulted in endogenous lipid peroxidation, forming malondialdehyde, formaldehyde, acetaldehyde, acetone, and propionaldehyde. Similar concentrations of trichloroacetic acid and trichloroethanol, the primary metabolites of chloral hydrate, also generated free radicals and induced lipid peroxidation. Lipid peroxidation induced by trichloroacetic acid nearly equaled that induced by chloral hydrate, while that from trichloroethanol was three- to fourfold less. Metabolism of 200 microM to 5 mM chloral hydrate, trichloroacetic acid, and trichloroethanol by liver microsomes of B6C3F1 mice pretreated with pyrazole (pyrazole-induced microsomes) yielded lipid peroxidation products at concentrations two- to threefold greater than those from liver microsomes of untreated mice. Additionally, chloral hydrate-induced lipid peroxidation catalyzed by control and pyrazole-induced microsomes was reduced significantly by 2,4-dichloro-6-phenylphenoxyethylamine, a general cytochrome P450 inhibitor. Human lymphoblastoid transgenic cells expressing cytochrome P(450)2E1 metabolized 200 to 5,000 micrograms/mL chloral hydrate to reactants inducing mutations, whereas the parental cell line was inactive. The malondialdehyde-modified DNA adduct, 3-(2-deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2 alpha]purin-10(3H)-one (MDA-MG-1), formed from the metabolism of 1 mM chloral hydrate, trichloroacetic acid, and trichloroethanol by control B6C3F1 mouse liver microsomes, mouse pyrazole-induced microsomes, male F344/N rat liver microsomes, and human liver microsomes in the presence and absence of calf thymus DNA was also determined. When incubated in the absence of calf thymus DNA, the amount of malondialdehyde formed from metabolism by pyrazole-induced mouse microsomes was twice that from rat or human liver microsomes. Amounts of chloral hydrate-induced and trichloroacetic acid-induced lipid peroxidation products formed from metabolism by rat and human liver microsomes were similar, and these quantities were about twice those formed from the metabolism of trichloroethanol. The quantity of MDA-MG-1 formed from the metabolism of chloral hydrate, trichloroacetic acid, and trichloroethanol by mouse, rat, and human liver microsomes exhibited a linear correlation with the quantity of malondialdehyde formed under incubation conditions in the absence of calf thymus DNA. Chloral hydrate was shown to be mutagenic in vitro and in vivo. At doses from 1,000 to 10,000 micrograms/plate, it induced mutations in S. typhimurium strain TA100, with and without S9 activation; an equivocal response was obtained in S. typhimurium strain TA98 in the absence of S9, and no mutagenicity was detected with strain TA1535 or TA1537. Chloral hydrate at doses from 1,700 to 5,000 micrograms/mL induced sister chromatid exchanges; at doses from 1,000 to 3,000 micrograms/mL, chromosomal aberrations were induced in cultured Chinese hamster ovary cells, with and without S9. Results of a sex-linked recessive lethal test in D. melanogaster were unclear; administration of chloral hydrate by feeding produced an inconclusive increase in recessive lethal mutations, results of the injection experiment were negative. An in vivo mouse bone marrow micronucleus test with chloral hydrate at doses from 125 to 500 mg/kg gave a positive dose trend. In summary, due to the absence of chloral hydrate-induced histopathologic lesions in rats and mice, no-observed-adverse-effect levels (NOAELs) were based on body weights of rats and liver weights of mice. The NOAELs for rats and mice were 200 mg/kg. Chloral hydrate was rapidly metabolized by rats and mice, with trichloroacetic acid occurring as the major metabolite. Peak concentrations of trichloroacetic acid occurred more quickly in mice. Plasma concentrations of chloral hydrate were dose dependent, but metabolic rates were unaffected by dose or sex. Chloral hydrate was mutagenic in vitro and in vivo. Metabolism of chloral hydrate and its metabolites produced free radicals that resulted in lipid peroxidation in liver microsomes of mice, rats, and humans. Induction of cytochrome P(450)2E1 by pyrazole increased the concentrations of lipid peroxidation products; inhibition of cytochrome P(450)2E1 by 2,4-dinitrophenylhydrazine reduced these concentrations. Metabolism of chloral hydrate and its metabolites by mouse, rat, and human liver microsomes formed malondialdehyde, and in the presence of calf thymus DNA formed the DNA adduct MDA-MG-1.

Animals↗

PubMed2003

Rapid development of keratoacanthomas after a body peel.

Resurfacing techniques have been traditionally limited to the face because of a lack of predictability and standardization for peeling nonfacial skin. There is a need for medical and surgical intervention for treating nonfacial skin that is actinically damaged. Medium-depth chemical peels (Jessner +35% trichloroacetic acid) remove the photodamaged epidermis to stimulate the production of new collagen in the dermis and remove lesions associated with facial actinic damage, including lentigines and actinic keratoses. Widespread actinic damage is common on the arms and chest. A 70% glycolic acid gel plus 40% trichloroacetic acid peel (Cook Body Peel) is a controlled peel that predictably enables peeling of nonfacial skin in a uniform and safe fashion with specific clinical endpoints. An unusual complication of this body peel is reported.

Aged↗

PubMed1984

The degradation of monoiodotyrosyl insulin isomers by insulin protease.

The four single-site monoiodotyrosyl insulin isomers were synthesized by lactoperoxidase-catalyzed iodination of porcine insulin and were separated from one another by high performance liquid chromatography. The susceptibility of the four isomers (A14-, A19-, B16-, and B26-monoiodotyrosyl insulin) to degradation by purified insulin protease was examined using several different assay methods, including trichloroacetic acid precipitation, immunoprecipitation, and Sephadex G-50 chromatograpy. Using trichloroacetic acid precipitation, isomer susceptibility, determined from the initial rate of hydrolysis, was highest with the A14 isomer, lowest with the A19 isomer, and intermediate and roughly equal with the two B-chain-labeled isomers. Based upon the initial rate of isomer hydrolysis, the Michaelis Menten constant (Km) of insulin protease was higher for the B16 isomer (55 nM) than for the other three isomers, whose Km values were not different from one another (A14 = 24 nM; A19 = 35 nM; B26 = 29 nM). In addition, the values for maximum velocity (Vmax) were higher for the A14 and B26 isomers than for the A19 and B16 isomers. However, during incubation, the order of isomer susceptibility to insulin protease changed to B26 greater than A14 greater than A19 greater than B16. This change in apparent isomer susceptibility was prevented by including in the incubation mixture a rat renal peptidase, which did not degrade the intact isomers, suggesting that insulin protease converted the isomers to trichloroacetic acid-soluble products via trichloroacetic acid-precipitable intermediates. Using the immunoprecipitation assay, the susceptibility of isomers to hydrolysis did not change during incubation, but remained highest with the A14 isomer, lowest with the A19 isomer, and intermediate with the two B-chain-labeled isomers, of which the B16 isomer was degraded more rapidly. Each isomer was converted more rapidly to nonimmunoprecipitable products than to trichloroacetic acid-soluble products, implying that insulin protease converted the isomers to trichloroacetic acid-precipitable, nonimmunoprecipitable intermediates, which it then converted to trichloroacetic acid-soluble form. Using Sephadex G-50 chromatography (SGC) assay, the susceptibility of isomers to hydrolysis did not change during incubation, but remained highest with the A14 isomer, lowest with the A19 isomer, and intermediate with the two B-chain-labeled isomers, of which the B16 isomer was hydrolyzed more rapidly. With the exception of the A19 isomer, isomer hydrolysis appeared faster with SGC assay than with either of the other two assays.(ABSTRACT TRUNCATED AT 400 WORDS)

Chemical Precipitation↗

PubMed1985

Species differences in response to trichloroethylene. II. Biotransformation in rats and mice.

Detailed analysis of urine from two strains of rats and mice dosed po with trichloroethylene at four doses from 10 to 2000 mg/kg failed to detect any major species or strain differences in the metabolism of trichloroethylene. Although a greater proportion of the dose was metabolized in mice than in rats, the relative proportions of the major metabolites were very similar in both strains and were unaffected by the dose amount. Analysis of the same urine samples for minor metabolites failed to establish a major species difference. Small amounts of dichloroacetic acid (less than 1% of the dose) were present in both rat and mouse urine and were not considered significant. Monochloroacetic acid accounted for less than 0.1% of the dose. Daily dosing of trichloroethylene (1000 mg/kg po) for 180 days did not induce the overall metabolism of trichloroethylene but did double the urinary excretion of trichloroacetic acid. This finding was accompanied by an equivalent percentage decrease in the concentration of trichloroethanol. CO2 has been shown to be a major metabolite of trichloroacetic acid, suggesting that this is the source of trichloroethylene-derived CO2. Trichloroacetic acid was also excreted in bile in both rats and mice suggesting possible conjugation of this metabolite in the liver. Very little evidence was found for the formation of chemically reactive species from trichloroethylene in either rats or mice and none that could be the basis of a major species difference. The increased rate of metabolism in the mouse, the resulting high blood concentrations of trichloroacetic acid, and stimulation of hepatic peroxisome proliferation in this species appears to be the major species difference possibly related to tumor formation in the liver. The conjugation of trichloroacetic acid and its metabolism to CO2 may be related to peroxisome proliferation.

Animals↗

PubMed1967

Analysis of phosphate esters in plant material. Extraction and purification.

1. A critical study was made of the quantitative extraction of nucleotide and sugar phosphates from plant tissue by either boiling aqueous ethanol or cold trichloroacetic acid. The effect of the extraction technique on the inactivation of the enzymes in the plant tissue and the possibility of adsorption of the phosphate esters on the cell wall were especially considered. 2. In the recommended method the plant tissue was frozen in liquid nitrogen, ground to a powder and then blended with cold aqueous trichloroacetic acid containing 8-hydroxyquinoline to prevent adsorption. 3. The extract contained large amounts of trichloroacetic acid, cations, chloride, sugars, amino acids, hydroxy organic acids, phytic acid, orthophosphoric acid and high-molecular-weight material including some phosphorus-containing compounds. All of these were removed as they were liable to interfere with the chromatographic or enzymic assay of the individual nucleotide or sugar phosphates. 4. The procedure was as follows: the last traces of trichloroacetic acid were extracted with ether after the solution had been passed through a column of Dowex AG 50 in the hydrogen form to remove all cations. High-molecular-weight compounds were removed by ultrafiltration and low-molecular-weight solutes by a two-stage chromatography on cellulose columns with organic solvents. In the first stage, sugars, amino acids, chloride and phytic acid were separated by using a basic solvent (propan-1-ol-water-aqueous ammonia) and, in the second stage, the organic acids and orthophosphoric acid were separated by using an acidic solvent (di-isopropyl ether-formic acid-2-methylpropan-2-ol-water). The final solution of nucleotide and sugar phosphates was substantially free from other solutes and was suitable for the detection of individual phosphate esters by either chromatography or enzymic assay. 5. The recovery of d-glucose 6-phosphate or adenosine 5'-triphosphate added to a trichloroacetic acid extract simulating that from peas and potatoes, and isolated according to the standard procedures, was better than 95%. Estimation of naturally occurring d-glucose 6-phosphate and adenosine 5'-triphosphate in the initial extract of peas and potatoes and in the final purified extract also indicated a recovery of about 95%. A similar estimation of uridine diphosphate glucose in potatoes showed that little or no breakdown occurred.

Animals↗

PubMed1979

Reversed-phase high-performance liquid chromatography of catecholamines and their congeners with simple acids as ion-pairing reagents.

We have investigated the value of various common acids as ion-pairing reagents for high-efficiency separations of catecholamines and their metabolites in reversed-phase high-performance liquid chromatography. The retention of norepinephrine, alpha-methylnorepinephrine, dopamine, alpha-methyldopamine, L-dopa, alpha-methyldopa, dihydroxybenzylamine, epinephrine, carbidopa and DOPAC was measured in mobile phases composed of nitric, sulfuric, acetic and trichloroacetic acids at pH 2-5 and anion concentrations ranging from 5-500 mM. The solute capacity ratios were dependent on the hydrophobicity and concentration of the ion-pairing reagent and the pH of the mobile phase. Good retention, peak symmetry and high efficiency (3000 theoretical plates for 300 mm) was found for mobile phases composed of the strong inorganic acids and trichloroacetic acid. Chromatography was compared to that seen using the detergent sodium octylsulfate. Trichloroacetic acid gave retention and efficiency similar to sodium octylsulfate. These experiments show that simple acids can replace alkylsulfates as ion-pairing reagents for the separation of the catecholamines and their metabolites.

Catecholamines↗

PubMed1985

Species differences in response to trichloroethylene. I. Pharmacokinetics in rats and mice.

The elimination of radioactivity in two strains of rats and mice following a single po dose of trichloro[14C]ethylene at dose levels from 10 to 2000 mg/kg has shown a marked dose dependence in rats but not in mice. The metabolism of trichloroethylene in the mouse was linear over the range of doses used, whereas in the rat it became constant and independent of dose at 1000 mg/kg and above. At the 10-mg/kg dosage, both species metabolized trichloroethylene almost completely, 60% of the dose being excreted in urine with only 1 to 4% being eliminated unchanged in expired air in the first 24 hr. At 2000 mg/kg, 78% of the dose was eliminated unchanged in the rat, but only 14% in the mouse. Consequently at high dosages, the mouse was exposed to significantly higher concentrations of trichloroethylene metabolites than the rat. Blood level kinetics of trichloroethylene and its metabolites confirmed a faster rate of metabolism in the mouse than in the rat. Peak concentrations of the metabolites were reached within 2 hr of dosing in the mouse compared to 10 to 12 hr in the rat. The concentrations of both trichloroethanol (4X) and trichloroacetic acid (7X) were significantly higher in the mouse than in the rat. Whereas trichloroethanol was rapidly eliminated from blood, the higher concentrations of trichloroacetic acid were maintained for over 30 hr. The high blood quantities of trichloroethylene-derived trichloroacetic acid are known to induce hepatic peroxisome proliferation in mice but are insufficient to induce this response in rats. These data suggest that trichloroacetic acid blood amounts, peroxisome proliferation, and the link between peroxisomes and liver cancer are the basis of species difference in response to trichloroethylene.

Animals↗

PubMed2002

Simultaneous analysis of oxidized and reduced glutathione in cell extracts by capillary zone electrophoresis.

Glutathione (GSH) and glutathione disulfide (GSSG) levels in cells constitute a thiol redox system. They can be used as an indicator of oxidative stress of the cell. In this study, a capillary zone electrophoresis (CZE) method is described that enables quantitation of GSH and GSSG from cellular extracts. The CZE buffer used was 20 mM ammonium acetate containing 5% (v/v) acetic acid at pH 3.1 in conjunction with a polybrene coated capillary operated in reverse polarity mode. Effects of different acids used to prepare cell samples were investigated on CZE performance. The acids include meta phosphoric acid (MPA), trichloroacetic acid (TCA), phosphoric acid (PA) and sulfosalicylic acid (SSA) and are used to stabilize GSH and GSSG before performing CZE analysis. The method features a limit of detection of 4 microM and a limit of quantitation of 12 microM for both GSSG and GSH and recoveries of 94% for GSH and 100% for GSSG. Quantitative analysis of GSSG and GSH in HaCaT cell extracts (5% SSA, w/v) was performed with this method and changes in the ratio of GSH to GSSG in N-ethylmaleimide treated cell sample was observed by comparing with control cell samples.

Cell Extracts↗

PubMed1995

Destaining of Coomassie Brilliant Blue R-250-stained polyacrylamide gels with sodium chloride solutions.

A novel method for destaining of polyacrylamide gels, stained with Coomassie Brilliant Blue R-250, is described, based on the use of 0.5 M NaCl in water as the destainer, requiring only 2-3 h. Concentrated (> 2 M) or dilute (< 0.1 M) salt solutions were unsuitable. The method affords the advantage that expensive organic solvents, such as methanol, acetic acid or trichloroacetic acid, are not needed. Furthermore, salt destaining results in darker purple-blue protein bands as compared to the pale blue color with e.g. methanol/acetic acid destaining.

Acrylic Resins↗

PubMed1996

Preparative electrophoresis on linear polyacrylamide-agarose composite gels.

A preparative method for isolating centigram quantities of high molecular weight polypeptide chains with high resolution and recovery uses linear polyacrylamide/agarose composite (LPAC) gels as electrophoretic media from which the polypeptides can be easily extracted. The composites are prepared in a manner yielding linear copolymers of acrylamide and 1-allyloxy-2,3-propanediol within 2% agarose gels. After electrophoresis in sodium dodecyl sulfate (SDS), protein bands were rapidly visualized for excision by briefly immersing the gel in cold 0.1 M KCl which precipitates the protein-associated SDS. The gel slices are then freeze-thawed to disrupt the agarose matrix and promote syneresis of fluid upon centrifugation. The polypeptides are then separated from the polyacrylamide in the supernatant solution by precipitating with either acidic isopropanol, trichloroacetic acid, ammonium sulfate or other general protein precipitants. As determined with polypeptide chains of fibrinogen and its cross-linked derivatives, recoveries were virtually complete (95.4% +/- 2.2%), and were independent of molecular weights over the range tested (10(4) --10(6)).

Electrophoresis, Agar Gel↗

PubMed2002

Mammalian cell cytotoxicity and genotoxicity analysis of drinking water disinfection by-products.

Cytotoxicity and genotoxicity assays were used to analyze drinking water disinfection by-products (DBPs) in Chinese hamster ovary (CHO) AS52 cells. The DBPs were chosen because they are common in drinking water, resulting from conventional disinfection using chlorination and chloramination. Data were also available to compare these results with cytotoxicity and mutagenicity studies in Salmonella typhimurium. The rank order in decreasing chronic cytotoxicity measured in a microplate-based assay was bromoacetic acid (BA) >> 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) > dibromoacetic acid (DBA) > chloroacetic acid (CA) > KBrO(3) > tribromoacetic acid (TBA) > EMS (ethylmethanesulfonate, positive control) > dichloroacetic acid (DCA) > trichloroacetic acid (TCA). The induction of DNA strand breaks by these agents was measured by alkaline single-cell gel electrophoresis (SCGE, comet assay) and the rank order in decreasing genotoxicity was BA >> MX > CA > DBA > TBA > EMS > KBrO(3), while DCA and TCA were refractory. BA was more cytotoxic (31x) and genotoxic (14x) than MX in CHO cells. BA was over 400x more genotoxic than potassium bromate. The brominated haloacetic acids (HAAs) were more cytotoxic and genotoxic than their chlorinated analogs. The HAAs expressed a statistically significant inverse relationship in CHO cell cytotoxicity and genotoxicity as a function of increased numbers of halogen atoms per molecule. A quantitative comparison was conducted with results from a previous study with cytotoxicity and mutagenicity in S. typhimurium. There was no correlation between chronic CHO cell and bacterial cell cytotoxicity. DBP-induced CHO cell cytotoxicity was not related to mutagenic potency in S. typhimurium. Cytotoxicity in CHO cells was statistically significant and highly correlated to CHO cell genotoxicity. Finally, we determined that the DBP genotoxic potency in CHO cells and the mutagenic potency in S. typhimurium were not related. This suggests that toxicity data in S. typhimurium did not quantitatively predict the toxic effects of DBPs in mammalian cell systems. The microplate CHO cell cytotoxicity and genotoxicity assays were well suited for the analysis of DBPs, especially when the quantity of test material is limited.

Animals↗

PubMed1978

Enhancement of ampicillin partition behavior.

Several ion-pair or adduct forming additives tha" enhanced ampicillin partition behavior were identified and evaluated. At pH 3, picric acid and trichloroacetic acid increased the ampicillin aqueous-octanol partition coefficient 250 and 30 times, respectively. At pH 7, quaternary compounds gave the most significant increases in the partition coefficient. Values for an aqueous pH 7 chloroform system increased from zero in the absence of additives to 2.28, 1.86, 1.82, and 1.70 for equimolar amounts of benzalkonium, tetraheptylammonium, benzethonium, and cetalkonium chlorides, respectively. Extraction of ampicillin from aqueous pH 7 solution was possible by adding a quaternary agent in an equimolar amount. However, extraction of ampicillin from plasma required large molar excesses. Tetraheptylammonium chloride was added at a molar concentration 10(3) times greater than that of the ampicillin. Plasma samples spiked at the 3-microgram/ml level gave 93% recovery (CV 6.7%, n = 16) when extracted three times. The extracts were quantitated by TLC.

Ampicillin↗

PubMed2002

Analysis of the cytotoxicity and mutagenicity of drinking water disinfection by-products in Salmonella typhimurium.

We analyzed the cytotoxicity and mutagenicity of the drinking water disinfection by-products (DBPs) bromoform (BF), bromoacetic acid (BA), dibromoacetic acid (DBA), tribromoacetic acid (TCA), chloroform (CF), chloroacetic acid (CA), dichloroacetic acid (DCA), trichloroacetic acid (TCA), 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX), and potassium bromate (KBrO3) in Salmonella typhimurium strains TA98, TA100, and RSJ100 +/- S9. Solvent controls of DMSO and ethanol and a positive control of ethylmethanesulfonate (EMS) were also analyzed. We developed a rapid microplate-based method to determine the cytotoxicity of the DBPs and we determined their mutagenic potencies. The distributions of the rank order for the cytotoxicity and mutagenicity of these DBPs were compared and the structure-function relationships were identified. TA100 -S9 was the most sensitive strain for these DBPs. The rank order of the mutagenic potency adjusted with a cytoxicity factor was MX > BA > EMS > DBA > DCA > CA with TBA, TCA, BF, and CF not mutagenic. From a structure-function perspective, the brominated acetic acids were more cytotoxic and mutagenic than their chlorinated analogs. BA was 150x more mutagenic than CA. The mutagenic potency of the haloacetic acids was inversely related to the number of halogen atoms of the molecule. BA was 36x more mutagenic than DBA. The differential cytotoxicity expressed by the DBPs indicated that a cytotoxicity analysis enhanced the sensitivity of the mutagenicity data, which resulted in an enhanced precision for comparing their relative mutagenic strengths. This information is critical when conducting quantitative structure-function analysis of these hazardous agents.

Disinfectants↗

PubMed1992

Comparison of an endogenous protein kinase C substrate in rat aorta with rat brain MARCKS.

We have compared the properties of a rat aorta-derived protein kinase C substrate (p75) with those of 80 kDa kinase C substrates from rat brain (MARCKS) and rabbit aorta (p80). Rat aortic p75 appeared to be closely related to rat brain MARCKS on the basis of: solubility in perchloric acid and trichloroacetic acid, heat stability, isoelectric point (pI approximately 4.2), overall V8 protease phosphopeptide map, and immunocrossreactivity with an antibody directed against the N-terminal domain of MARCKS. However, p75 could be distinguished from rat brain MARCKS and from the rabbit aorta-derived p80 on the basis of its consistently more rapid electrophoretic mobility in SDS-containing gels, and in terms of a unique proteolytic phosphopeptide found in MARCKS but not in aortic p75. We conclude that p75 probably belongs to the family of protein kinase C substrates represented by MARCKS, and that differences in post-translational processing (glycosylation) or mRNA processing may account for the unique properties of the p75 protein in rat aortic tissue.

Animals↗

PubMed1984

Selective retrograde axonal transport of free glycine in identified neurons of Aplysia.

The specific retrograde axonal transport of free glycine within the identified neurons R3-14 of Aplysia californica was studied. The soma of the R3-14 neurons are located in the parietovisceral ganglion and their axons project down the branchial nerve to end in a large peripheral field. Using a double-chambered apparatus, the peripheral tissue was incubated in medium containing a 3H-amino acid for 4-48 hr, while the nerve and ganglion were isolated and perfused with plain or chemically altered medium. The nerve and ganglion were then either rapidly frozen for scintillation counting or fixed for autoradiography. When 3H-glycine was used, radioactivity entered the nerve rapidly, reached the ganglion in 3 hr, and was transported largely (greater than 80%) in the free amino acid form [trichloroacetic acid (TCA) soluble]. The right parietovisceral hemiganglion accumulated up to nine times more radioactivity than the left hemiganglion, reflecting the presence of the R3-14 axons and soma. Two phases of radioactivity were observed, a fast component moving at about 3 mm/hr and a slower (but larger) component moving at about 0.4 mm/hr. Light microscope autoradiography on nerves containing 3H-glycine revealed that the R3-14 axons accounted for more than 30% of the total label in the nerve but occupied less than 7% of the total cross-sectional area of the axonal core. Electron microscope autoradiography showed a close association of silver grains and dense core vesicles in the R3-14 axons. Retrograde axonal transport of free glycine was inhibited by (in decreasing order of effectiveness) mercuric chloride, vinblastine, colchicine, Nocodazole, and 2,4-dinitrophenol (2,4-DNP). Comparative studies of other amino acids [3H-leucine, 3H-serine, 3H-glutamic acid, 3H-gamma-aminobutyric acid (3H-GABA), and 3H-alanine] showed that 3H-glycine is the only amino acid that is rapidly axonally transported in large quantities within the R3-14 axons. This work demonstrates, for the first time, that a free amino acid, glycine, is transported in the retrograde direction within a select group of axons. The significance of this transport of glycine is discussed in relation to its use as a neural messenger by neurons R3-14.

2,4-Dinitrophenol↗