Flow cytometric measurement of micronuclei induced in a permanent fish cell line as a possible screening test for the genotoxicity of industrial waste waters.
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An in vitro micronucleus assay using the permanent fish cell line RTG-2 (rainbow trout gonads) was developed to test industrial waste waters for their genotoxic potential. Comparison of flow cytometric measurement and microscopic scoring of micronucleus frequency with the reference chemicals 1,4-butane sultone (0.2-1 mM), ethylmethane sulphonate (2-10 mM), potassium dichromate (20-100 microM) and benzo[a]pyrene (5-25 microM) showed similar dose-effect relationships. Thirty-eight industrial waste waters from 11 different branches of industry obtained from the Bavarian state office for water research were tested using the flow cytometric method (18 from metal processing, 10 from combined waste water, two from synthetic fibre production, one sample each from settlement wastes, non-iron metal manufacturing, leather production, sulphuric acid production, ore processing, graphite film production, cellulose production and flue gas washing). Fourteen of them showed a significant increase in micronucleus frequency. (+info)
Determination of bovine beta2-microglobulin and albumin in urine by a reversed-phase high-performance liquid chromatography.
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Reversed-phase high-performance liquid chromatography (HPLC) was used to analyze beta2-microglobulin and albumin in bovine urine. The urine samples were chromatographed on TSK-gel ODS-120T column with an acetonitrile gradient. Urinary beta2-m and albumin were detected at 220 nm. For the pre-treatment, there were two steps proceeding injection: dialysis of urine with distilled water overnight, followed by concentration by solid-phase extraction method using a Sep-Pak cartridge. The retention times of beta2-microglobulin and albumin were 25.35 +/- 0.85 and 32.20 +/- 0.20 minutes (n=5), respectively. The mean analytical recoveries of beta2-microglobulin and albumin added to 0.1 ml of urine samples were 94.5 and 100.5%, respectively. The within-run coefficients of variation ranged from 1.5 to 5.3% for beta2-microglobulin and from 2.3 to 7.0% for albumin. The sensitivity for quantification of each protein was 0.5 microg in 100 microl injected urine samples. Urine samples from healthy cows and from cows with different types of proteinuria were analyzed by this reversed-phase HPLC. Results revealed albumin was remarkable in the urine from a cow with glomerulonephritis, and beta2-microglobulin was, in the urine from a cow with tubular dysfunction. (+info)
Effect of hexavalent chromium on eukaryotic plasma membrane studied by EPR spectroscopy.
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The effect of Cr(VI) anion on an ergosterol-producing strain of eukaryotic yeast Candida albicans and its mutant with ergosterol-less membrane was studied with EPR spectroscopy. 5- and 14-doxyl stearic acid spin probes were used to label the protoplast membrane after removal of the cell wall. In control experiments, the mutant strain exhibited larger rigidity in the membrane than its parental strain. Addition of Cr(VI), at a minimum inhibitory concentration of 0.6 mM, increased the rotational mobility of the spin labels significantly and decreased the temperature of the structural changes in both strains, in the temperature range between 0 and 30 degrees C. The ergosterol-less mutant, having a membrane composition with increased polyunsaturated fatty acid content, exhibited higher Cr(VI) sensitivity. Treatment of the membrane with Cr(VI) for 10 min already resulted in an increase in membrane fluidity. An EPR signal of Cr(V) was detected which reached maximum amplitude after 120 min of treatment with Cr(VI). Further chemical reduction of Cr(V) in the absence of extracellular Cr(VI) led to a lack of detectable paramagnetic chromium intermediates within 200 min. (+info)
Less than additive interaction between cigarette smoke and chromium(VI) in inducing clastogenic damage in rodents.
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A combination of tobacco smoking with certain agents has been shown to exert synergistic carcinogenic effects. On the other hand, antagonism betweeen smoke and other pulmonary carcinogens has also been documented by both epidemiological and experimental data. In spite of a very large number of studies carried out for decades in workers exposed to hexavalent chromium, the influence of smoking habits on lung carcinogenesis induced by this metal has not been clarified. For this reason, we performed two studies evaluating clastogenic effects in rodents. In the first one, BDF(1) mice were exposed whole-body to mainstream cigarette smoke for 5 days and, on the last day, they received an i.p. injection of potassium dichromate. In the second study, Sprague-Dawley rats were exposed whole-body to environmental cigarette smoke for 18 consecutive days and for the same period of time they received daily intra-tracheal instillations of sodium dichromate. Individually, the two hexavalent chromium salts and cigarette smoke, either mainstream or environmental, enhanced the frequency of micronuclei in bone marrow polychromatic erythrocytes of both mice and rats. Moreover, individual exposure to either environmental cigarette smoke or sodium dichromate enhanced the frequency of micronuclei and multiple nuclei in pulmonary alveolar macrophages of rats. In both studies, combined exposure to cigarette smoke and hexavalent chromium produced less than additive clastogenic effects. These results are consistent with our previous data, showing that hexavalent chromium and either benzo[a]pyrene or cigarette smoke condensate behave antagonistically in in vitro mutagenicity test systems and that the chromium reducing capacity of human pulmonary alveolar macrophages and peripheral lung parenchyma is enhanced in smokers. Taken together, in the absence of any epidemiological evidence, these findings rule out any occurrence of synergism between cigarette smoke and hexavalent chromium, at least in certain stages of the carcinogenesis process. (+info)
Procedure for calibrating the Technicon Colorimeter I.
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We describe a rapid method for calibrating the Technicon AutoAnalyzer colorimeter I. Test solutions of bromphenol blue are recommended for the calibration, in preference to solutions of potassium dichromate, based on considerations of the instrument's working range and of the stray light characteristics of the associated filters. (+info)
Localisation of intracellular calcium stores in the striated muscles of the jellyfish Polyorchis penicillatus: possible involvement in excitation-contraction coupling.
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When jellyfish striated muscles were stimulated directly, the amplitude of contractile tension increased as the stimulation frequency increased. Application of 10 mmol l(-1) caffeine reduced the amplitude of contractile tension and abolished this facilitatory relationship, indicating that calcium stores participate in excitation-contraction coupling. Calcium stores were identified ultrastructurally using enzymatic histochemistry to localize CaATPases, and potassium dichromate to precipitate calcium. Electron energy-loss spectroscopy was used to verify the presence of calcium in precipitates. Both CaATPase and calcium were localised in membrane-bound vesicles beneath the sarcolemma. We concluded that sub-sarcolemmal vesicles could act as calcium stores and participate in excitation-contraction coupling. (+info)
Determination of chromate adulteration of human urine by automated colorimetric and capillary ion electrophoretic analyses.
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Various chemicals can be added to urine specimens collected for drug analysis to abnormally elevate ionic concentrations and/or interfere with either immunoassay urine drug-screening procedures or gas chromatographic-mass spectrometric confirmation techniques. One such adulterant, "Urine Luck" (formula 5.3), has been identified in our previous research to contain potassium dichromate. Screening of suspected adulterated specimens and confirmation of the adulterant are important for forensic drug screening. The application and comparison of automated colorimetric and capillary ion electrophoretic techniques for the detection, confirmation, and quantitation of chromate adulteration of urine specimens were the purpose of this investigation. Thirty-six urine specimens suspected of adulteration were analyzed for chromate by colorimetric analysis with diphenylcarbazide. Duplicate aliquots were analyzed for chromate by capillary ion electrophoresis. Results of the colorimetric chromate analyses revealed a mean chromate concentration of 929 microg/mL with a standard error of 177 microg/mL and a range of 30 to 5634 microg/mL. Results of the capillary ion electrophoresis chromate analyses revealed a mean chromate concentration of 1009 microg/mL with a standard error of 218 microg/mL and a range of 20 to 7501 microg/mL. The correlation coefficient between the capillary ion electrophoretic and colorimetric chromate results was r = 0.9669. Application of the automated diphenylcarbazide colorimetric technique provides rapid determination of chromate adulteration of a urine specimen. Capillary ion electrophoresis offers a separation technique to confirm the presence of chromate in suspected adulterated specimens. The excellent correlation between these methods substantiates their application to forensic testing as screening and/or confirmation techniques. (+info)
Differential effects of nephrotoxic agents on renal transport and metabolism by use of in vitro techniques.
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A number of studies by the author and other investigators are reviewed in which the in vitro kidney slice technique has been used to evaluate the nephrotoxicity of various compounds. The kidney slice technique can be used to determine the effect of prior drug treatment of laboratory animals on renal organic acid (p-aminohippurate) or organic base (N-methylnicotinamide) transport, on glucose synthesis, and on oxygen consumption by renal coritical slices. The nephrotoxic agents uranyl nitrate and potassium dichromate exert inhibitory effects on renal function, althouhg both agents enhance organic base transport at low doses and potassium dichromate enhances organic acid transport at moderate doses. Enhanced PAH transport has been found to be a sensitive indicator of gentamicin induced nephrotoxicity, while inhibition of other parameters has been reported. The tissue slice method is less effective in evaluation chronic nephrotoxicity such as that produced by lead. The inhibitory effect of mercurial diuretics has been shown to be due to the general depression of metabolic activity by mercury. The kidney slice technique has been found to be a sensitive indicator in the assessment of halogenated hydrocarbon-induced nephrotoxicity. Differential effects of compounds on in vitro organic acid and base trasport provides information about the transport of these compounds as well as about their nephrotoxicity. Although it is often desirable to perform in vivo tests or other in vitro renal function tests, the kidney slice technique has proved to be extremely useful in toxicological evaluations. (+info)