Mari Christensen

Determination of the baseline sister chromatid exchange frequency in human and mouse peripheral lymphocytes using monoclonal antibodies and very low doses of bromodeoxyuridine

We measured the frequency of sister chromatid exchanges (SCEs) in human and mouse peripheral lymphocytes using doses of bromodeoxyuridine (BrdU) ranging from 30 nM to 100 microM (human) and from 10 nM to 10 microM (mouse). Heparinized peripheral blood was obtained from five healthy nonsmokers and from six C57B1/6 male mice. The blood was stimulated with PHA (human) or lipopolysaccharide (LPS, mouse) and grown for the first of two cell cycles in BrdU. Metaphase chromosomes were denatured and exposed to a monoclonal antibody reactive to single-stranded DNA containing BrdU. A second antibody was used to label the first antibody with fluorescein, and propidium iodide was used as a counterstain. Second-division metaphases were thus differentially stained red to indicate DNA content and yellow-green to indicate the presence of BrdU. The results indicate that the baseline SCE frequency in human and mouse peripheral lymphocytes is 3.6 and 2.4 SCEs per cell per generation, and that in the human these frequencies are invariant at the lowest BrdU levels. This suggests that SCEs are an integral part of DNA replication, even in the absence of agents known to induce SCEs. The distribution of SCEs per chromosome was analyzed and found to be Poisson-distributed in all 24 murine cultures and in 25 of 36 human cultures. The distribution of SCEs per chromosome may be due to either species-specific chromosome packaging or to karyotypic differences between the species.

In vivo cytogenetic effects of cooked food mutagens

Using a variety of in vivo cytogenetic endpoints, we have investigated the effects of several compounds formed during the cooking of meat. C57Bl/6 mice were used to test for an increase in the frequency of sister-chromatid exchanges (SCEs), chromosomal aberrations, and micronucleated erythrocytes by 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). MeIQx and DiMeIQx did not induce SCEs in mouse bone marrow cells. PhIP induced sister-chromatid exchanges, but not chromosomal aberrations in bone marrow. In peripheral blood lymphocytes, PhIP did induce aberrations at 100 mg/kg, the highest dose tested. PhIP induced a low but significantly increased frequency of micronuclei in normochromatic but not polychromatic erythrocytes in bone marrow and peripheral blood. However, dose responses were not observed. With the exception of the SCEs induced by PhIP, these results contrast with observations made in vitro, where these compounds were found to have significant genotoxicity in mammalian cells and a very high mutation frequency in prokaryotic systems.

Characterization of somatic cell hybrids by bivariate flow karyotyping and fluorescence in situ hybridization

We report on the use of flow karyotyping and fluorescence in situ hybridization (FISH) to characterize the human chromosomes in somatic cell hybrids. The identity, DNA content, and relative frequency of human chromosomes are derived from flow karyotypes, i.e., measurements of Hoechst and chromomycin fluorescence intensities of chromosomes by dual beam flow cytometry. Chromosome integrity is assessed by comparing the peak position of a human chromosome in the flow karyotypes of a hybrid cell line and its human donor. When human donor cells are unavailable, the peak position of a human chromosome in a hybrid line is compared to the range of peak positions among normal individuals. The relative frequency of human chromosomes in subclones or hybrids grown in culture is monitored using the volumes of peaks in flow karyotypes. FISH with biotinylated human genomic DNA or chromosome-specific repeat sequences as probe is used in conjunction with flow karyotyping to confirm the number of human chromosomes in hybrids. Some small rearrangements are detected by flow karyotyping and not by FISH. On the other hand, translocations between human and rodent chromosomes are detected by FISH and not always by flow karyotyping. Flow karyotyping and FISH were used to characterize over 100 hybrid lines donated by other laboratories. A hybrid set useful for the construction of chromosome-enriched gene libraries is presented. In this set, each of the 24 human chromosome types is present and intact, as judged by these techniques, in a line containing little or no other human material.

Simultaneous identification and banding of human chromosome material in somatic cell hybrids

We have developed a method that identifies human chromosomes in human x hamster somatic cell hybrids and simultaneously bands these same metaphases. Other methods generally require separate slides for banding and detection of human chromosome material, making the precise characterization of human material difficult. Our procedure involves denaturing metaphase chromosomes, followed by in situ hybridization of biotinylated whole human DNA. Fluoresceinated avidin is then bound to the biotinylated DNA, staining the human chromosomes yellow-green when excited with UV light. Chromosome banding is achieved by staining the slides with DAPI and actinomycin D. The fluorescein and DAPI excite maximally at 488 and 355 nm and emit at 520 and 450 nm, respectively. This permits identification of the human material at one excitation wavelength and visualization of the banding patterns at another wavelength. With this procedure, we have successfully identified both intact and broken human chromosomes, as well as human material involved in human x hamster translocations. The results indicate that this procedure is more accurate and considerably more rapid than previous methods and can be routinely employed for the cytogenetic analysis of human x rodent hybrids.

Assignment of the gene encoding DNA ligase I to human chromosome 19q13.2–13.3

The gene encoding DNA ligase I has been mapped on human chromosome 19 by analysis of rodent-human somatic cell hybrids informative for this chromosome and by two-color fluorescence in situ hybridization. The DNA ligase I gene (LIG1) is localized to 19q13.2-13.3 and is distal to ERCC1, the most telomeric of three DNA repair genes on this chromosome.

Cytogenetic response to 1,2-dicarbonyls and hydrogen peroxide in Chinese hamster ovary AUXB1 cells and human peripheral lymphocytes

Mutagenic 1,2-dicarbonyls have been reported to occur in coffee and other beverages and in various foods. We have measured the induction of sister-chromatid exchanges (SCEs) and endoreduplicated cells (ERCs) to determine the genotoxicity of various 1,2-dicarbonyl compounds in Chinese hamster ovary (CHO) AUXB1 cells and human peripheral lymphocytes. The 1,2-dicarbonyls glyoxal, methylglyoxal and kethoxal each induced highly significant increases in both SCEs and ERCs in AUXB1 cells. Glyoxal and kethoxal induced SCEs but not ERCs in human peripheral lymphocytes. In addition, hydrogen peroxide induced highly significant levels of SCEs and ERCs in AUXB1 cells. Bisulfite, which reacts with carbonyl groups to form addition products, significantly reduced the frequency of SCEs and the proportion of ERCs when glyoxal, methylglyoxal, kethoxal and diacetyl were administered to AUXB1 cells. In addition, bisulfite blocked the formation of ERCs, but not SCEs, induced by hydrogen peroxide. These in vitro results suggest that 1,2-dicarbonyls may play an important role in the genotoxicity of some foods and beverages.

Fluorescence in Situ hybridization mapping of human chromosome 19: Mapping and verification of cosmid contigs formed by random restriction enzyme fingerprinting

Automated restriction enzyme fingerprinting of 7900 cosmids from chromosome 19 and calculation of the likelihood of their overlap based on shared fragments have resulted in the assembly of 743 sets of overlapping cosmids (contigs). We have mapped 22% of the formed contigs (n = 165) and all of the contigs with minimal tiling paths exceeding 6 members (n = 50) to chromosomal bands by fluorescence in situ hybridization using DNA from at least one member cosmid. The estimated average size of the formed contigs is 60-70 kb. Thus, members of a correctly formed contig are expected to lie close to each other in metaphase and interphase chromatin. Therefore, we tested the contig assembly process by comparing the band assignment of two or more members selected from each of 97 contigs. Forty-two of these contigs were further characterized for valid assembly by determining the proximity of members in interphase chromatin. Using these tests, we surveyed a total of 431 joins counted along the minimal tiling path (280 in interphase as well as metaphase) and found 6 erroneous joins, one in each of 6 contigs (6% of tested).

Effects of anticoagulants upon sister-chromatid exchanges, cell-cycle kinetics, and mitotic index in human peripheral lymphocytes

The effects of 3 blood anticoagulants, heparin, acid citrate dextrose (ACD), and ethylenediaminetetraacetic acid (EDTA) were investigated using human peripheral lymphocytes. Three different endpoints were examined: sister-chromatid exchange (SCE), cell kinetics index (CKI), and mitotic index (MI). SCEs were significantly increased in cells treated with EDTA, while the CKI and MI were significantly decreased in cultures treated with either ACD or EDTA when compared to cultures treated with heparin. These results suggest that anticoagulants may produce undesired effects upon cultured cells and indicate that the type of anticoagulant should be considered carefully prior to commencing cytogenetic studies using human peripheral lymphocytes.

Assignment of the human lens fiber cell MP19 gene (LIM2) to chromosome 19q13.4, and adjacent to ETFB

The LIM2 gene may play a major role in lens fiber cell structure or communication, and thus cataractogenesis. A human cDNA encoding the corresponding lens fiber cell intrinsic protein MP19 has been previously isolated and characterized. This cDNA had been mapped to human chromosome 19. We have independently confirmed this assignment and fine mapped it to 19q13.4. The position of the LIM2 gene appears to be within 40 kb of the electron transport flavoprotein gene (ETFB) as a cosmid containing sequences from both genes has been identified.