The testicular regions of male mice were exposed to x-ray doses ranging from 0 to 400 rads. Forty days after exposure the mice were killed and the testes and cauda epididymal sperm removed surgically. Flow cytometric measurements of acridine orange stained testicular samples indicated a repopulation of testicular cell types following x-ray killing of stem cells. Cauda epididymal sperm were analyzed by the sperm chromatin structure assay (SCSA), a flow cytometric measurement of the susceptibility of the sperm nuclear DNA to in situ acid denaturation. The SCSA detected increased susceptibility to DNA denaturation in situ after 12.5 rads of x-ray exposure, with significant increases following 25 rads. Abnormal sperm head morphology was not significantly increased until the testes were exposed to 60 rads of x-rays. These data suggest that the SCSA is currently the most sensitive, non-invasive method of detecting x-ray damage to testicular stem spermatogonia.

This study investigated the relationship between morphologically abnormal sperm, sperm chromatin structure, and fertility. Semen samples were obtained from 13 bulls. The sperm chromatin structure assay (SCSA), a sensitive measure of denaturability of sperm nuclear DNA in situ following acid treatment, was performed on each sample to quantitate abnormal chromatin structure. Feulgen‐stained sperm head morphology was measured by computerized image analysis (ONCOR‐Image): Sixteen parameters were measured for each of 200 nuclei per sample. Fertility estimates were available for nine of the bulls. The SCSA variable SDαt was correlated with fertility ranking (r = 0.617, P < 0.01). No correlations were seen between means of the imaging variables and SCSA variables % COMPαt or SDαt. Significant correlations (P < 0.05) were seen between SDαt and the variation of imaging variables eccentricity, width, and light blobs. Significant correlations (P < 0.05) were seen between % COMPαt and the variation of imaging variables area, perimeter, p2a, bending energy, nmac, sphericity, eccentricity, length, and width. A regression model for fertility rankings incorporating the standard deviation of the imaging variables area, bending energy, nmac, eccentricity, condensity, light blobs, and dark blobs was highly significant (r2 = 0.999, P < 0.05). These results indicate that variation of morphometry measurements is likely a sensitive biomarker related to fertility potential and abnormal chromatin structure. © 1996 Wiley‐Liss, Inc.

SUMMARY Deuterium oxide (D 2 O) increases both the fluorescence lifetime and the fluorescence intensity of the intercalating dyes propidium iodide (PI) and ethidium bromide (EB) when bound to nucleic acid structures. We have used spectroscopic analysis coupled with conventional and phase-sensitive flow cytometry to compare the alterations in intensity and lifetime of various DNA-binding fluorochromes bound to DNA and Chinese hamster ovary (CHO) cells in the presence of D 2 O vs phosphate-buffered saline (PBS). Spectroscopic and flow cytometric studies showed a differential enhancement of intensity and lifetime based on the mode of fluorochrome-DNA interaction. The fluorescence properties of intercalating probes, such as 7-aminoactinomycin D (7-AAD) and ethidium homodimer II (EthD II) were enhanced to the greatest degree, followed by the probes TOTO and YOYO, and the non-intercalating probes Hoechst 33342 (HO) and 4 Ј ,6-diamidino-2-phenylindole (DAPI). The non-intercalating probe mithramycin (MI) gave unexpected results, showing a great enhancement of fluorescence intensity and lifetime in D 2 O, indicating that when staining is performed in PBS, much of the MI fluorescence is quenched by the solvent environment. Apoptotic subpopulations of HL-60 cells had a shorter lifetime compared to nonapoptotic subpopulations when stained with EthD II. These results indicate that accessibility of the dye molecules to the solvent environment, once bound to DNA, leads to the differential enhancement effects of D 2 O on fluorescence intensity and lifetime of these probes. D na-binding fluorochromes have been used extensively in many applications, including flow cytometry and gel and capillary electrophoresis. Fluorochromes utilized in these applications must satisfy several criteria. They must bind specifically to nucleic acids, there must be a low quantum yield as free dye in solution, and there must be enhancement of quantum yield on binding to nucleic acid structures. Several classes of DNA-specific fluorochromes with different modes of base pair ( bp ) binding are available: (a) the DNA intercalators propidium iodide (PI) and ethidium bromide (EB), which lack appreciable bp specificity; (b) DNA intercalators with A-T bp preference, such as ethidium homodimer II (EthD II) or with G-C preference, such as 7-amino actinomycin D (7-AAD); and (c) non-intercalating probes with either A-T bp specificity, such as Hoechst 33342 (HO) or 4 Ј ,6-diamidino-2-phenylindole (DAPI) or with G-C specificity, such as mithramycin (MI). Two high quantum yield cyanine fluorochromes, TOTO and YOYO, are dimers of the dyes thiazole orange and oxazole yellow, respectively (Lee et al. 1986). These fluorochromes are believed to bind to DNA by intercalation, without base specificity (Haugland 1992) but with some base sequence preference (Netzel et al. 1995).Enhancement of the fluorescence intensity of DNAbinding probes improves the signal-to-noise ratio, allowing the use of lower dye concentrations and thereby reducing potential self-quenching between d...

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The increasing use of tellurium compounds in organic synthesis, industrial applications, and as a possible component in pesticides means that its introduction into the environment will increase in the future. Therefore, knowledge of the relative toxicity and mode of toxic action of tellurium-containing compounds is important. The studies detailed here used three model compounds: diphenyl ditelluride, 3,3'-diaminodiphenyl ditelluride, and 4,4'-diisopropyldiphenyl ditelluride. Experiments with human promyelocytic (line HL-60) cells indicate that all of the organotellurium compounds induce an apoptotic form of cell death. The induction of apoptosis occurs in a time- and dose-dependent manner as assayed by three different analytical methods: fluorescence microscopy, gel electrophoresis, and flow cytometry. Apoptotic cells were evident as early as 2 h following treatment with 1x10(-6) M concentrations of the compounds. Based on these results, future care should be afforded these compounds in laboratory as well as industrial settings.