Bromodeoxyuridine labelling

Male and female Fischer 344 rats and B6C3Fi mice were fed di(2-ethylhexyl) phthalate for up to 13 weeks (David et al., 1999). In rats fed 12 500 ppm di(2-ethylhexyl) phthalate, there was an increase in hepatocyte replicative DNA s mthesis (measured after continuous bromodeoxyuridine administration (osmotic pump) for three days before sampling) after one week (but not after two or 13 weeks) and an increase in hepatic peroxisomal [3-oxidation activity after one, two and 13 weeks administration. In mice fed 10 000 and 17 500 ppm di(2-ethylhexyl) phthalate, there was no increase in hepatocyte replicative DNA synthesis (measured after continuous bromodeoxyuridine three days before sampling) after one, two or 13 weeks of administration, but there was an increase in hepatic peroxisomal 3-oxidation activity after one, two and 13 weeks administration. In mice fed 1000 ppm di(2-ethylhexyl) phthalate, there was no statistically significant increase in hepatic peroxisomal 13-oxidation activity after one, two or 13 weeks administration (bromodeoxyuridine labelling was not evaluated at this lower dietary concentration of di(2-ethylhexyl) phthalate). 

Immunohistochemical Detection of Bromodeoxyuridine-Labeled Nuclei for In Vivo Cell Kinetic Studies... 

In a p53 null human oral squamous carcinoma cell line curcumin (5 pM and (10 pM) arrested growth in S/G2 which was confirmed with an increased bromodeoxyuridine labelling (Weir and Hague 2002). Treated cells showed increased... 

Dien, B. S. and F. Srienc, Bromodeoxyuridine labeling and flow cytometric identification of replicating Saccharomyces cerevisiae cells lengths of cell cycle phases and population variability at specific cell cycle positions, Biotech. Prog. 1, 292-298 (1991). 

Ormerod, M. G. Kubbies, M. Cell cycle analysis of asynchronous cell populations by flow cytometry using bromodeoxyuridine label and Hoechst-propidium iodide stain. Cytometry 1992, 13, 678-h85. 

Poot, M. Hoehn, H. Kubbies, M. Grossmatm, A. Chen, Y. Rabinovitch, P. S. Cell cycle analysis using continuous bromodeoxyuridine labeling and Hoechst 33258-ethidium bromide bivariate flow C5 ometry. Methods Cell Biol. 1990,55,185-198. 

Tang X, Falls DL, Li X, et al. Antigen-retrieval procedure for bromodeoxyuridine immunolabeling with concurrent labeling of nuclear DNA and antigens damaged by HC1 pretreatment. J. Neurosci. 2007 27 5837-5844. 

Experimental design The authors investigated the ability of chloroform vapors to produce toxicity and regenerative cell proliferation in female B6C3Fj mice and male Fischer 344 rats. Groups of 5 animals were exposed to 0, 1,3, 10, 30, 100, or 300 ppm chloroform via inhalation for 6 hours a day for 7 consecutive days. Actual exposure concentrations measured for mice were 0, 1.2, 3.0, 10.0, 29.5, 101, and 288 ppm and for rats were 0, 1.5, 3.1, 10.4, 29.3, 100, and 271 ppm. Necropsies were performed on day 8. Animals were administered bromodeoxyuridine (BrdU) via implanted osmotic pump for the last 3.5 days. Cell proliferation was quantitated as the percentage of cells in S-phase (labeling index = LI) measured by the immunohistochemical detection of BrdU-labeled nuclei. 

Shinohara, K. Ohara, H. Kobayashi, K. Maezawa, H. Hieda, K. Okada, S. Ito, T. Enhanced killing of HeLa cells pre-labeled with 5-bromodeoxyuridine by monochromatic synchrotron radiation at 0.9 A an evidence for Auger enhancement in mammalian cells. J. Radiat. Res. (Tokyo) 1985, 26 (3), 334-338. 

Accumulated evidence indicates that PCNA also plays a critical role in the initiation of cell proliferation, and its expression is elevated almost exclusively during the S phase of the cell cycle. However, not all studies support this observation. This antigen, as detected by PC 10 antibody, does not accurately reflect the S-phase fraction in gastric mucosa, as determined by bromodeoxyuridine (BrdU) labeling (Lynch et al., 1994). 

Fig. 12.4. Bromodeoxyuridine (BRDU) labeling of cells in S phase. Routine paraffin sections from mice injected 1 h prior to euthanasia with BRDU have brown nuclei when labeled by immunohistochemistry using diaminobenzidine as a chromogen. These cells were synthesizing DNA at the time of necropsy and therefore incorporated the label. Counting the number of positive nuclei in basal cells per 1000 basal cells yields the proliferation rate. The boxed area in A is enlarged in B to illustrate the large number of positive (dark) nuclei in the skin of an adult chronic proliferative dermatitis mutant mouse (Sharpincpdm/Sharpincpdm).

Analysis is best carried out by a fluorescence activated cell sorter (see 10.7.5) but, if the cells are pulse labelled with [3H]-thymidine immediately before harvesting the proportion of cells in S-phase in the various fractions can be estimated by autoradiography (see 12.3). The problem with this procedure is that the machines can become contaminated with radioactivity and the tritium may interfere with subsequent enzyme assays. Labelling of a sample after fractionation is a poor alternative, but prior pulse labelling with bromodeoxyuridine allows S-phase cells to be detected using a fluorescent antibody 12.7.5. 

Two relatively new immunodetectable moieties have recently been developed for nucleic acid probe systems which allow us to illustrate two different methods of incorporating labels into nucleic acids. Both methods have been employed for biotin labeling. Originally nucleic acid probes were labeled by enzymatic incorporation of pre-labeled nucleotides, radiolabeled nucleotides, biotin-UTP/dUTP, 5-bromodeoxyuridine (15), or most recently a steroid hapten linked nucleotide analogue, digoxigenin-dUTP (16). 

Many monoclonal andbodies are commerdally available to detect halo-genated pyrimidines. Most antibodies will detect both iodo- and bromodeoxyuridine. However, Br3 shows extremely high specifidty for BUdR (11), whereas IU4 (both available from Caltag Inc.) shows much higher specificity for lUdR (12). These andbodies have opened the pathway for double labeling experiments on the FCM. The diludon of the monoclonal will depend on its specificity and whether it is a purified, ascitic, or supernatant preparation for instance, we routinely use 1U4 at a diludon of 1 2000 compared to 1 20 for the Sera-Lab antibody. 

Wilson, G. D., McNally, N. J., Dunphy, E., Karcher, H., and Pfragner, R. (1985) The labeling index of human and mouse tumours assessed by bromodeoxyuridine staining in vitro and in vivo and flow cytometry. Cytometry 6, 641-647. 

Christov, K., Chew, K. L., Ljung, B. M., Waldman, F. M., Goodson, W. H. 3rd, Smith, H. S. and Mayall, B. H. (1994). Cell proliferation in hyperplastic and in situ carcinoma lesions of the breast estimated by in vivo labeling with bromodeoxyuridine. J. Cell. Biochem. Suppl. 19, 165-172. 

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