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Article No.
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CLC1011
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Improvements to the Plaque Assay for Antibody Secreting Cells
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RONALD KISSINGER AND A. DAVID MYL
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A capillary glass microslide has been adapted to function as a chamber for the determination of plaque forming cell responses. Comparisons with Cunningham chambers indicate no significant difference between methods. Further, microslides arrive clean and ready to use, thus eliminating the need to assemble chambers as necessitated by the Cunningham method. Used in conjunction with an electronically assisted enumerating device, the microslides provide a rapid and less tedious means for assaying large numbers of animals for PFC responses.
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CLC1012
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Quantification of unscheduled DNA synthesis by a whole cell counting method.
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HILL LE, YOUNT DJ, GARRIOTT ML, TAMURA RN, PROBST GS
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A procedure was developed for the quantification of the autoradiographic assay for unscheduled DNA synthesis. Relative to commonly used practices for grain counting, this procedure provides a more accurate net nuclear grain count by eliminating the subjectivity currently associated with selection of the areas to be counted for the cytoplasmic background count. Briefly, the object area and aperture area modes of an ARTEK 880 colony counter are used to collect values for the total number of silver grains over a particular cell (nuclear and cytoplasmic counts), as well as for the nuclear and cytoplasmic areas. These values are then employed in a short algorithm to determine the net nuclear grain count. This new method provides greater sensitivity for defining weak UDS responses and the data collected readily lends itself to statistical analysis.
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CLC1013
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Limb bud cell culture for in vitro teratogen screening: validation of an improved assessment method using 51 compounds.
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RENAULT JY, MELCION C, CORDIER A
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Rat embryo limb bud cells multiply and undergo chondrogenesis in micromass culture. Teratogenic agents are identified from their inhibition of chondrogenesis, which is quantified by determination of cartilaginous foci number or proteoglycan production. In other in vitro systems, the detection is based on their ability to affect cell proliferation. So far, these methods have failed to distinguish among true inhibition of differentiation, inhibition of cell proliferation, and nonspecific cytotoxicity. The improved technique involves simultaneous measurement of cartilage synthesis and cell multiplication. Differentiation was evaluated by measurement, using an Artek Counter, of nodule areas after Alcian blue staining and proliferation by spectrophotometric quantification of Crystal Violet bound to micromass cells. Using this method, retinoic acid was shown to inhibit chondrogenesis without affecting cell multiplication, whereas 6 aminonicotinamide preferentially inhibited cell multiplication without affecting nodule size. Doxylamine (succinate), a known nonteratogen, induced inhibition of chondrogenesis, but with a parallel inhibition of cell multiplication, reflecting a nonspecific toxic effect. This improvement increases the specificity of the micromass culture test. Validation was performed using 51 compounds. Compounds were classified according to their inhibitory activity and their active concentration. The sensitivity of the test was 61%; the specificity, 100%; and the final accuracy, 75%. The method is fully miniaturised, automated, and computerised, allowing numerous compounds to be rapidly tested at very low cost.
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CLC1014
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Automated autoradiographic grain counting of DNA repair in cultured human fibroblasts after ultraviolet irradiation.
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SATO K, IKENAGA M, YOSHIKAWA K, SANO S. KITAMURA H. KOSAKI G. HAMAOKA T. KONDO S
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Measurement of autoradiographic grains produced by the decay of incorporated radioisotopes is often used for a quantitative assay of the rate of DNA replication and DNA repair in cells or tissues. However, visual grain counting by microscopic observation is time-consuming and tedious process. Recently, Kraemer et al. reported that automated measurement of grains in cultured human cells may be facilitated by using appropriate grain counting instruments. Under their experimental conditions using Kodak NTB-3 emulsion, instrument determined grain number per nucleus was proportional to visual counts up to 30 grains, and then leveled off at much larger visual counts. The saturation phenomenon was due to counting-loss by the instrument caused by overlapping of neighboring grains. To prevent the counting-loss, we have used in the present study Japanese Sakura NR-M2 emulsion which is less sensitive to radiation exposure than Kodak NTB-3, thereby yielding smaller size of grains per radioactive decay. Samples were prepared from cultured skin fibroblasts derived from normal individuals and xeroderma pigmentosum (XP) patients defective in DNA repair. These cells were irradiated with 254 nm UV incubated for 3 h with culture medium containing 3H-thymidine, and autoradiograms were made by dipping in Sakura NR-M2 emulsion. The number of grains as well as grain surface area per nucleus was measured by using ARTEK CYTO TALLY MODEL 900 counting instrument, and compared with visual counts. The results showed that, under our optimum condition, the instrument-determined number of grains was directly proportional to visual counts, at least up to 150 grains per nucleus, with a correlation coefficient of 0.971.
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CLC1015
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Detection, enumeration, and sizing of planktonic bacteria by image analyzed epifluorescence microscopy.
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SIERACKI ME, JOHNSON PW, SIEBURTH JM
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Epifluorescence microscopy is now being widely used to characterize planktonic procaryote populations. The tedium and subjectivity of visual enumeration and sizing have been largely alleviated by our use of an image analysis system consisting of a modified ARTEK 810 image analyzer and an Olympus BHT-F epifluorescence microscope. This system digitizes the video image of autofluorescing or fluorochrome-stained cells in a microscope field. The digitized image can then be stored, edited, and analyzed for total count or individual cell size and shape parameters. Results can be printed as raw data, statistical summaries, or histograms. By using a stain concentration of 5 micrograms of 4'6-diamidino-2-phenylindole per ml of sample and the optimal sensitivity level and mode, counts by image analysis of natural bacterial populations from a variety of habitats were found to be statistically equal to standard visual counts. Although the time required to prepare slides, focus, and change fields is the same for visual and image analysis methods, the time and effort required for counting is eliminated since image analysis is instantaneous. The system has been satisfactorily tested at sea. Histograms of cell silhouette areas indicate that rapid and accurate estimates of bacterial biovolume and biomass will be possible with this system.
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CLC1016
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An in situ procedure for accurately detecting mutations using mammalian cells.
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CATTANACH PI, RIACH CG, MITCHELL A, WILLINGTON SE, ZAJAC WC, COMBES RD, CASPARY WJ
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Previously an in situ protocol to accurately measure mutation rates at the tk locus using l5178y mouse lymphoma cells has been described (genetics 126:435, '90). Rather than allowing cells to express the mutant Phenotype in liquid suspension, the cells were segregated and Immobilized for expression in semi solid medium. This procedure permitted a more accurate recovery of slowly growing mutants. Using the in situ method, spontaneous mutation rates 50 fold higher than those obtained in liquid suspension were observed. We now report the results of investigating the effects of medium composition (rpmi with 10% horse serum versus fischer's with 10% horse serum for growth and 20% for cloning), time of trifluorothymidine (tft) addition for mutant selection, and manual versus automatic colony counting on the yield of spontaneous tft resistant colonies as part of validating the assay in another laboratory. Under all conditions, the maximum number of mutant colonies that could be counted occurred at 48 hrs. Thereafter, the numbers counted declined, presumably because of nutrient depletion. Mutation frequencies (mf) were consistently slightly higher with rpmi medium (376 x 10( 6)) as compared with fischer's (345 x 10(6)). Irrespective of medium used, higher me values were obtained by manual counting at 48 and 60 hrs compared with enumeration using an ARTEK 880 colony counter. Manual counting was backed up where necessary by microscopic examination.
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CLC1017
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Evaluation of the potential of o-nitroaniline to induced unscheduled DNA synthesis in primary rat hepatocyte cultures with cover memo & sheet.
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SRI INTERNATIONAL, INC.
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The effects of ortho-nitroaniline were examined in the rat hepatocyte primary culture repair assay. Based on preliminary cytotoxicity tests o-nitroaniline was tested at concentrations of 0.1, 0.5, ], 5, 10, 50, 75, and 100 microgram/ml in one assay and at concentrations of 5, 10, 50 and 75 microliter/ml in a second assay. The net grain counts measured on autoradiographs by an ARTEK Model 830 or 980 colony counter were negative at each concentration of the test article and the solvent control while the positive control (2-acetylaminofluorene) produced a strong positive response.
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CLC1018
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Alternatives to the Use of Live Vertebrates in Biomedical Research and Testing: Second Annual Annotated Bibliography
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GEORGE J. COSMIDES, DEPUTY DIRECTOR, TOXICOLOGY INFORMATION PROGRAM, NATIONAL LIBRARY OF MEDICINE, NATIONAL INSTITUTES OF HEALTH, BETHESDA, MARYLAND ROBERT S. STAFFORD, INFORMATION ANALYST, OAK RIDGE NATIONAL LABORATORY, OAK RIDGE, TENNESSEE PO-YUNG LU, DIRECTOR, CHEMICAL HAZARD EVALUATION PROGRAM, OAK RIDGE NATIONAL LABORATORY, OAK RIDGE, TENNESSEE
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Because of considerable interest from Congress, the National Institutes of Health (NIH), and the public about animal welfare and alternatives to animal testing, the National Library of Medicine (NLM) searches its online databases and prepares quarterly annotated bibliographies on alternative or in vitro methods for toxicity testing and biomedical research. The objective is to present current literature organized as citations with brief annotations for easy scanning. The Institute of Laboratory Animal Resources (ILAR) has invited NLM to publish in ILAR News this annual supplement, which is an edited, concatenated version of the quarterly bibliographies. The ILAR News Editorial Panel and outside reviewers edit and condense the quarterly bibliographies so the entries are appropriate for the ILAR News audience. The scientific community is concerned about humane animal care and is sensitive to public concerns about how and why animals are used in biomedical research and toxicity testing. The following events reflect the involvement of the public and the U.S. government in this issue: an array of federal legislation related to animal welfare and the use of laboratory animals, U.S. Public Health Service policy on the humane care and use of laboratory animals, and efforts at NIH to promote and support a search for alternative methods to the use of animals in biomedical research and testing. Scientists generally view the use of laboratory animals in biomedical research and toxicity testing as necessary except where valid scientific alternative methods will satisfy all testing requirements. Howevex, when animals must be used, the scientific community supports careful consideration of the number of animals used and encourages reductions when they are scientifically feasible.
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CLC1019
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Antiestrogenicity of environmental polycyclic aromatic hydrocarbons in human breast cancer cells
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KATHLEEN F. ARCARO A,B,*, PATRICK W. O'KEEFE A,B, YI YANG B, WILLIAM CLAYTON B, JOHN F. GIERTHY A,B
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The total concentration of 14 polycyclic aromatic hydrocarbons (PAHs) was determined to be 3400-fold greater in a sediment sample from an industrial site on the St. Lawrence River (SLR), NY, than in a sediment sample from a non-industrial site on the Kinderhook Creek (KC), NY. PAH fractions from extracts of the two environmental samples and two reconstituted mixtures as well as the 14 individual PAHs were examined for their toxic, estrogenic, and antiestrogenic activities using MCF-7 focus, recombinant human estrogen receptor (ER) binding, whole-cell ER binding, and 17b-estradiol (E2) metabolism assays. PAH fractions from the KC and SLR were antiestrogenic; they significantly inhibited the formation of foci elicited in MCF-7 breast cancer cells by 1 nM E2. Eight of the 14 individual PAHs, and the reconstituted mixtures were also antiestrogenic. Results from the whole-cell ER binding assay and the radiometric analysis of E2 metabolism indicate that the PAHs detected in the KC and the SLR environmental samples induce antiestrogenic responses in metabolically intact human breast cancer cells through at least two mechanisms: one involving competition for the ER by a PAH metabolite and the other involving depletion of E2 through induction of metabolism. Published by Elsevier Science Ireland Ltd.t
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CLC1020
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BIOAVAILABILITY OF GENOTOXIC MIXTURES IN SOIL
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N. BORDELON, K. WASHBURN, L.-Y. HE, AND K.C. DONNELLY*, DEPARTMENT OF VETERINARY ANATOMY AND PUBLIC HEALTH, TEXAS A&M UNIVERSITY, COLLEGE STATION, TX, 77843-4458
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Contaminated media at Superfund sites typically consist of complex mixtures of organic and inorganic chemicals which are difficult to characterize, both analytically and toxicologically. The current EPA approach to risk assessment uses solvent extraction to remove chemicals from the soil as a basis for estimating risk to the human population. However, contaminants that can be recovered with a solvent extract may not represent the mixture of chemicals that are available for human exposure. A procedure using an aqueous extraction was investigated to provide a more realistic estimate of what chemicals are bioavailable. A study was conducted with two soil types: creosote-contaminated sandy soil and coal tar-contaminated clay soil spiked with benzo(a)pyrene [B(a)P], and trinitrotoluene (TNT). Samples were extracted with hexane:acetone and water titrated to pH2 and pH7. HPLC analysis demonstrated up to 35% and 29% recovery of contaminants using the aqueous extracts. The estimated cancer risk for the aqueous extract was one order of magnitude less than that for solvent extracts. Analysis using the Salmonella/microsome assay demonstrated that solvent extracts were genotoxic (133 revertants/mg) with metabolic activation while aqueous extracts of clay soil were not genotoxic. Sandy soil showed genotoxicity both with and without metabolic activation. These results suggest that solvent extraction techniques may overestimate the concentration of contaminants that are available for human exposure and, hence, the risk associated with the presence of the contaminants in soil.
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