High-resolution human chromosomes were obtained from lymphocytes after thymidine synchronization. The block was released either with thymidine to produce GTG (G-bands by trypsin using Giemsa) and RHG (R-bands by heating using Giemsa) banding or with BrdU (5-bromo-2'-deoxyuridine) for RBG (R-bands by BrdU using Giemsa) banding. RHG and RBG band patterns are only 75 to 85% congruent. The dissimilarities increase with the band number per genome and vary from one chromosome region to another. After high-resolution RBG banding, the BrdU-substituted bands show an unequal condensation delay, which can be, according to the bands involved, very important, minimal, or even absent. The bands showing the highest degree of condensation delay are the bands replicating the latest. The GTG- and RHG-band patterns show complementary matching for about 90% of the bands. It was found that two third of the chromosome surface appears positively stained after R-banding. This suggests that more DNA is replicated during early S-phase than during late S-phase. To obtain a fully developed RBG-band pattern in 90 to 95% of harvested mitoses, a period of 4.5 hours after the removal of the blocking agent is optimal. Such a brief release period also implies that late S-phase is much shorter than early S-phase.

Cytogenetic studies by in situ hybridization (ISH) have proven to be valuable for gene mapping on banded chromosomes when combined with fluorescence microscopy (FISH). However, even under the best conditions, FISH technology has a resolving power inherent to light of just 0.2 µm. Its utilization is further limited by the diffusion of light coming from the fluorescent signal which covers an area considerably larger than the target DNA sequence. The development of new ISH protocols applied to electron microscopy (EMISH) should increase the resolution for cytogenetic mapping and fine chromosomal structure studies. Despite these advances, few attempts have been made which exploit this increased resolution. Here we present a detailed analysis of ISH signals obtained by fluorescence and electron microscopy methodologies to demonstrate and define the higher sensitivity obtainable by electron microscopy. This comparative study was conducted with probes of different origins: telomeric, classical satellite, alpha satellite, and single-copy DNA sequences, which provide a good reference point for later studies. We were also able to map a 200-bp cDNA probe by EMISH. This study assesses the nature of the resolution and the better definition of the EMISH signal, which confirms the greater resolution of electron microscopy as compared with that achieved with light microscopy. It also indicates that better delineation of two closely linked sequences is achieved at the electron microscopy level.Key words: In situ hybridization, electron microscopy, fluorescence microscopy, localization, repetitive and small single-copy probes.

We report on two sets of monozygotic twins (MZTs) discordant for phenotypic sex ascertained at birth when the female twin was noted to have signs of the Ullrich-Turner syndrome. Cytogenetic investigations on the female of the first pair showed 45,X/46,XY mosaicism in lymphocytes but fibroblasts grown from two skin biopsies at separate sites and from gonadal tissue showed only 45,X cells. The male showed mosaicism in both blood lymphocytes and skin fibroblasts. In the second family, both twins also showed mosaicism in lymphocytes. The female had a 45,X karyotype in fibroblasts from skin and gonadal tissue, but in contrast to the first family, the male twin had a normal male karyotype in fibroblasts from skin biopsy and from connective tissue adjacent to the vas deferens. Discordant phenotypic sex in monozygotic twins is rare. As in our cases, the nine previously reported sets of MZTs all had mosaicism for sex chromosome abnormalities. A mitotic error leading to the loss of a Y chromosome prior to, accompanying, or following the twinning process would account for the reported combinations of karyotypes.

Styrene-7,8-oxide, an intermediate of styrene, is a known alkylating mutagen. The present study was carried out to investigate the influence of duration of exposure to styrene-7,8-oxide (styrene oxide) on induction of sister chromatid exchanges (SCEs) and inhibition of cell-cycle kinetics using cultured human blood lymphocytes in vitro. Phytohemagglutinin-stimulated whole-blood lymphocyte cultures obtained from heparinized whole blood from healthy donors were exposed to 100 μM styrene oxide for 22, 36, 48 and 72 h. A reduction of SCEs induction with increase in duration of exposure to styrene oxide was observed, i.e. a clear significant inverse relationship between exposure time and frequencies of SCEs induction due to styrene oxide was obtained. Styrene oxide induces significant elevations in unscheduled DNA synthesis DNA repair as well as S-phase synthesis in human blood lymphocytes in vitro, depending on the duration of exposure. The decrease in the induction of SCEs due to styrene oxide with increasing duration of its exposure may be principally due to an increased DNA repair and partly due to an increasing metabolic transformation to styrene glycol with increasing duration of its exposure as well as to some extent due to cell death at the maximum period of exposure, i.e. 72 h. Although the proliferations of lymphocytes exposed to 100 μM styrene oxide were significantly inhibited at different durations of exposure, no linear relationship between the replication index and the duration of exposure was noticed (r=0.47, p>0.05). Similarly, there was no relationship between replication index and SCE frequency (r=−0.36, p>0.05), suggesting that these two parameters may reflect two different endpoints for the cytogenotoxic effects of styrene oxide.

The most prominent class of centromeric DNA sequences belongs to the alpha satellite family of tandemly repeated DNA. The human chromosome 7 has been shown to contain two distinct alpha satellite arrays: D7Z1 and D7Z2, separated by 1 Mb. The order of these arrays was analyzed in normal blood cells and in the melanoma cell line IPC182 with two approaches using in situ hybridization: (1) Relative mapping on high-resolution chromosomes in fluorescence and electron microscopy (EM); and (2) simultaneous visualization of the two sequences using fluorochromes of different colors or gold particles of different sizes. The location within the centromeric area of chromosome 7, on the side of the short arm for D7Z2 and near the long arm for D7Z1 is confirmed. In addition, the hybridization signal of D7Z2 is confined to two small areas of the centromeric region in external positions, whereas the D7Z1 signal covers the entire width of the primary constriction. In situ hybridization with D7Z1 and D7Z2, performed on the melanoma cell line IPC 182, allowed characterization of two isochromosomes, i(7)(q10) and idic(7)(q11), as well as the der(7)t(7;12) observed in this cell line. The three-derived chromosomes appeared to result from different breakpoints, but only D7Z1 was conserved in all cases, suggesting the importance of this sequence for the centromeric function.

The characteristic band patterns of replication banding (dynamic banding) were analyzed. High-resolution (550-1,250 bands per haploid genome) G- and R-band patterns were obtained after 5-bromo-2'-deoxyuridine (BrdU) incorporation during early or late S phase. Thymidine-BrdU permutation culture methods, which arrest DNA synthesis at the R-band/G-band transition, allow complementary BrdU substitution. The RBI (R bands by BrdU using immunological staining) and GBI (G bands by BrdU using immunological staining) band patterns were complementary for all chromosomes. There was no overlapping, and every part of each chromosome was positively stained by one of the two banding procedures. Comparative analysis of RBG (R bands by BrdU using Giemsa staining) and RBI band patterns, as well as GBG (G bands by BrdU using Giemsa staining) and GBI band patterns, showed good congruency, displaying a very good band-for-band match. The congruency and complementarity found for these band patterns show that high concentrations of both thymidine and BrdU blocked S-phase progression near the R-band to G-band replication transition within the S phase. They also prove that BrdU incorporation is complementary and, therefore, demonstrate the existence of the R/G transition: a possible regulatory checkpoint within the S phase of the cell cycle.

A patient with mixed gonadal dysgenesis and Y isochromosomes i(Y) is described. Lymphocyte cultures from peripheral blood contained a high proportion of 45,X cells and several other cell lines with two different marker chromosomes (mars). These markers had either a monocentric (mar1) or a dicentric appearance (mar2). Following high-resolution GTG, RBG, QFQ, and CBG bandings, five cell lines were identified; 45,X/46,X, + mar1/46,X, + mar2/47,X, + mar1x2/47,X, + mar2x2. The percentages were 66/6/26/1/1%, respectively. Chromosome banding analyses were insufficient for characterization of the markers. In situ hybridization of specific probes for the Y centromere and its short arm showed, both in fluorescence and electron microscopy (EM), two different Y rearrangements. Mar1 is an isochromosome for the short arm i(Yp) and mar2 is a dicentric which was shown by EM to be a double isochromosome Yp, inv dup i(Yp). The breakpoint producing mar1 is within the centromere and the one producing mar2 is within one of the short arms of the Y isochromosome. The findings of different cell populations in peripheral blood lymphocytes indicate the postzygotic instability of this i(Yp). © 1995 Wiley-Liss, Inc.

Analysis of the retinoblastoma locus (RB1) by in situ hybridization at the infragenic level was done using human chromosomes. A high level of resolution was attained with nonisotopic in situ hybridization on banded chromosomes in both fluorescence and electron microscopy. DNA sequences less than 100 kb apart could be positioned on band 13q14 in the order expected from molecular mapping. These observations suggest that the DNA target of hybridized probes on metaphase chromosomes may correspond to nucleoprotein loops giving a resolution comparable to that obtained in interphase nuclei.

5 metabolites of styrene were tested in vitro for their cytotoxic effects, induction of SCEs and changes in cell-cycle progression in cultured human blood lymphocytes. Fresh heparinized peripheral blood (0.3 ml) from normal volunteers was cultured for a total of 72 h in 5 ml of RPMI 1640 medium containing 10% fetal calf serum, 0.1% garamycine, 1% glutamine and 1% phytohaemagglutinin. Styrene-7,8-oxide (SO), styrene glycol (SG), phenylglyoxylic acid (PGA), S-1,2-phenyl-2-hydroxyethyl-glutathione (PEG) (a glutathione conjugate of styrene oxide), N-acetyl-S-[1,2-phenyl-2-hydroxyethyl]-cysteine (NAPEC) in dimethyl sulfoxide (DMSO) were injected into the cultures 36 h after initial culture, so that the exposure time for these metabolites was 36 h. The final concentration of SO was 100 μM and those of the other metabolites were 500 μM. 24 h before harvest, BrdU (10 μg/ml) was added into the cultures for assessing cytogenetic endpoints. SO showed significant induction of SCEs and cell-cycle delay as well as a significant decline of cell survival. The same phenomena, but of less magnitude, were also observed with NAPEC, a cysteine derivative of SO. On the other hand, SG, PGA and PEG failed to produce any significant changes of these endpoints compared to the control. Thus, the present results have demonstrated that, in addition to SO, NAPEC possesses some cytogenotoxic potential and hence, these two metabolites together could contribute to the genotoxicity of styrene in human blood lymphocytes.

Although the genotoxic potential of styrene is known, very limited information is available regarding its dose-dependent genotoxic response to human blood lymphocytes and how such response correlates with different metabolic events in whole blood lymphocytes. The present study was therefore carried out to study such a relationship using in vitro human blood lymphocytes from healthy volunteers. To study genotoxic response to styrene, sister chromatid exchanges (SCEs), cell cycle, and cell survival were analyzed. Lymphocytes were cultured for 72 hr in the presence of different concentrations of styrene (0-1,000 microM). Twenty-four hr before harvest, BrdU (5 micrograms/ml) was added to assess the increase in SCEs and cell cycle delay. Both the SCE frequency and the cell cycle length were increased linearly with increasing concentrations of styrene up to 200 microM, without addition of any exogenous metabolizing system. Above 200 microM, no further increase in genotoxic response occurred. The range of concentrations (10-200 microM) at which increase of cell cycle length due to styrene was observed did not impair the viability of the cells, suggesting that such cell cycle delay is a genotoxic-related event and not caused by cytotoxicity. In vitro metabolic transformation of styrene in whole-blood lymphocyte cultures without the presence of any exogenous metabolic activation system showed the formation of a reactive intermediate, styrene 7,8-oxide, to be capacity-limited, as verified from a nonlinear increase in the formation of styrene glycol. The value of such metabolic parameter reached a plateau above 200 microM styrene. The same phenomenon of saturation has also been observed with regard to other metabolic effects due to styrene in whole blood lymphocytes in culture, such as dose-dependent increase in lipid peroxidation and depletion of blood lymphocyte glutathione. Based on the relationship between the formation of different metabolic events and the genotoxicity of styrene, it may be possible that the genotoxic properties of styrene in human blood lymphocytes may be mediated initially not only by the formation of the presumably reactive styrene 7,8-oxide, but also by that of a reactive oxygen species as well. However, the present data are not sufficient enough to definitely identify the role of reactive oxygen species in such toxicity and therefore it warrants further study.

The present study was undertaken to investigate, both in vitro and in vivo, the genotoxic potential of short-term low-level exposure to toluene, xylene, and their mixture, for which information is limited at the present time. Five adult healthy white men were exposed for 7 consecutive hours per day over 3 consecutive days to 50 ppm toluene and 40 ppm xylene either alone or in combination in a controlled exposure chamber. Such an exposure was repeated three times at intervals of 2 weeks. Blood samples were taken before and after the termination of such exposure. Three different cytogenetic end-points were evaluated using peripheral blood lymphocytes: number of sister chromatid exchanges (SCEs), cell cycle delay, and cell mortality. No significant effects on SCEs, cell cycle delay, and cell mortality were observed following such exposure to toluene or xylene or their mixture. Similarly, exposure of human blood lymphocytes in vitro to either toluene (0-2.5 mM) or xylene (0-2 mM) or their mixture for 72 h did not result in any significant cytogenetic effects at lower concentrations, while at higher concentrations, only cell mortality was found to be significantly affected. Thus our present study indicates that simultaneous exposure to low levels (within the admissible limits) of toluene, xylene, or their mixture for a short period does not pose any potential mutagenic threat to humans.

N-Acetyl-S-(1/2-phenyl-2-hydroxyethyl)-cysteine (NAPEC) is a cysteine conjugate derived from the glutathione conjugate of styrene oxide. The dose- and time-dependent effects of NAPEC on SCEs and cell kinetics were studied in cultured human blood lymphocyte in vitro. Different concentrations of NAPEC (0-1500 microM) were added into the lymphocyte cultures. After 36 h of exposure, both the induction of SCEs and cell-cycle delay were increased with increasing concentrations of NAPEC. When the lymphocyte cultures were exposed to 1000 microM NAPEC for 22, 36 and 72 h during a total of 72-h culture period, no significant differences in the induction of SCEs or cell-cycle delay were noticed due to 3 exposure times. These results suggest that NAPEC possesses the potential to induce SCEs and to inhibit cell-cycle progression and such potential seems to be independent of duration of exposure to NAPEC.