The development of painting probes for dual-color and multiple chromosome analysis in the mouse.

The recent development of mouse chromosome painting probes for fluorescence in situ hybridization has extended the use of this common laboratory mammal in cytogenetics. We now report the development of additional painting probes by degenerate-oligonucleotide-primed PCR on chromosomes from mouse lung fibroblast cultures, each homozygous for a single Robertsonian translocation chromosome. These probes are for Rb(1.2), Rb(1.3), Rb(4.6), and Rb(6.7). Probes were also made for the sex chromosomes by isolating shoulders from larger peaks (X) or small, clearly resolved peaks (Y) in the flow karyotype. Combinations of probes were used to paint four chromosomes simultaneously in a single color. Multicolor painting was achieved with a biotinylated Rb(1.2) probe and a digoxigenin-labeled Rb(2.8) probe. Each of the three different homologous pairs was uniquely colored by avidin-Texas Red, anti-digoxigenin-FITC, or both simultaneously. These results extend the usefulness of the mouse as a model for understanding adverse environmental exposures and genetic diseases in humans.

The development of chromosome-specific composite DNA probes for the mouse and their application to chromosome painting.

The speed and ease of human cytogenetic analysis has been greatly enhanced by the technique of fluorescence in situ hybridization (FISH). Non-radioactive fluorescently tagged complex DNA probes specific for individual chromosomes can be hybridized to conventionally obtained metaphase chromosome spreads. Several chromosomes may be "painted" concurrently by using combinations of different labeled probes. Surveys of chromosome breakage and rearrangement may be performed very quickly by avoiding the time consuming process of GTG-banding. The application of FISH to mouse cytogenetics would allow large scale molecular toxicology studies to be conducted on the effects of such environmental insults as potential carcinogens, mutagens and radiation. Progress has been hampered, however, as the Mus musculus karyotype consists of 40 acrocentric chromosomes of approximately the same size, making the recognition and separation of individual chromosomes very difficult. We now describe the successful production and application of chromosome-specific composite DNA probes for M. musculus chromosomes 2 and 8. Stable Robertsonian translocated chromosomes were isolated on a flow sorter and their DNA subsequently amplified by degenerate oligonucleotide primer (DOP) PCR. Small pools (300 copies) of each chromosome were denatured at 94 degrees C then annealed with the primer at 30 degrees C for 15 cycles. This was followed by 20 cycles at an annealing temperature of 62 degrees C. Additional amplification was performed at an annealing temperature of 62 degrees C. The chromosome-specific DNA was labeled with biotin 11-dUTP by nick translation and used for FISH. The usefulness of the technique for translocation detection is demonstrated by analyzing chromosome exchanges induced in mice irradiated with 137Cs gamma rays.

Validation of chromosome painting as a biodosimeter in human peripheral lymphocytes following acute exposure to ionizing radiation in vitro.

Fluorescence in situ hybridization with chromosome-specific composite DNA probes ('chromosome painting') appears to be a useful tool for quantifying symmetrical cytogenetic damage. However, a thorough comparison between chromosome painting and the conventional methods of GTG-banding and dicentric analysis has not been performed. We have undertaken the validation of chromosome painting using human blood exposed in vitro to 137Cs gamma-rays at doses ranging from 0 to 400 cGy, then cultured according to standard procedures and harvested at 52 h. For painting, bound probes were detected either with fluoresceinated avidin and counterstained with propidium iodide, or with ChromoBlue WCP Probe and Giemsa. The first approach utilizes ultraviolet excitation in which painted chromosomes appear yellow and the remaining chromosomes appear red. The ChromoBlue labelling approach requires ordinary light microscopy in which painted chromosomes appear dark blue and the remaining chromosomes appear light blue. With each method, exchanges between painted and unpainted chromosomes appear bi-coloured. Because only a fraction of all possible exchanges are detected, the number of metaphases examined is adjusted according to the fraction of the genome painted. We have performed painting by several methods, including fluorescence with chromosome 4 probe alone, fluorescence with probes for chromosomes 1, 3 and 4 simultaneously, and chromogenic painting with chromosome 4 probe alone. The results obtained by the various painting methods were compared with GTG-banded cells which were examined for both translocations and dicentrics. In addition, unbanded metaphases stained with Giemsa were scored for dicentrics. Our data show that the frequency of chromosome exchanges detected by painting and banding agree with each other and with the number of dicentrics seen in unbanded cells, at least at doses of < or = 200 cGy.

Biological effectiveness of neutrons from Hiroshima bomb replica: results of a collaborative cytogenetic study.

The effectiveness of neutrons from a facsimile of the Hiroshima bomb was determined cytogenetically. The "Little-Boy" replica (LBR), assembled at Los Alamos as a controlled nuclear reactor for detailed physical dosimetry, was used. Of special interest, the neutron energy characteristics (including lineal energy) measured 0.74 m from the LBR were remarkably similar to those calculated for the 1945 Hiroshima bomb at 1 to 2 km from the hypocenter, as shown in a companion dosimetric paper (Straume, et al., Radiat. Res. 128, 133-142 (1991)). Thus we examine here the effectiveness of neutrons closely resembling those that the A-bomb survivors received at Hiroshima. Chromosome aberration frequencies were determined in human blood lymphocytes exposed in vitro to graded doses of LBR radiation (97% neutrons, 3% gamma rays). Vials of blood suspended in air at distances up to 2.10 m from the center of the LBR uranium core received doses ranging from 0.02 to 2.92 Gy. The LBR neutrons (E approximately 0.2 MeV) produced 1.18 dicentrics and rings per cell per Gy. They were more effective than the higher-energy fission neutrons (E approximately 1 MeV) commonly used in radiobiology. The maximum RBE (RBEM) of LBR neutrons at low doses is estimated to be 60 to 80 compared to 60Co gamma rays and 22 to 30 compared to 250-kVp X rays. These results provide a quantitative measurement of the biological effectiveness of Hiroshima-like neutrons.

Metabolic activation and cytogenetic effects of 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) in Chinese hamster ovary cells expressing murine cytochrome P450 IA2.

We have utilized Chinese hamster ovary cell lines which stably express a murine cytochrome P450IA2 (P(3)450) cDNA to characterize more fully the mechanisms of genotoxicity of heterocyclic amines derived from cooked meats. To verify that these cell lines were capable of converting promutagens into active metabolites, we studied the microsomal metabolism and cytogenetic effects of 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pridine (PhIP). Microsomal preparations derived from excision repair-deficient Chinese hamster ovary cells expressing the mouse cytochrome P(3)450 cDNA (UV5P3) converted PhIP to the genotoxic N-hydroxy-PhIP metabolite. Cytotoxic activity in UV5P3 was observed at concentrations of PhIP as low as 1 microM. Cytotoxicity of PhIP was an order of magnitude lower in a matched repair-proficient cell line (5P3R2) expressing the P(3)450 cDNA. PhIP produced a concentration-dependent increase in sister chromatid exchange (SCE) in UV5P3. N-Hydroxy-PhIP, at concentrations as low as 0.1 microM, produced an increase in SCE in both UV5P3 and in UV5 cells which lack the P(3)450 cDNA. Incubation of PhIP with UV5P3 cells increased the frequency of micronuclei (MN) in cytokinesis-blocked cells. Chromatid gaps, but not aberrations also were induced by treatment with PhIP. N-Hydroxy-PhIP produced increases in MN and chromatid gaps in both UV5 and UV5P3 cell lines; chromosomal aberrations were induced in UV5P3 cells. These results suggested that UV5P3 cells metabolize sufficient quantities of PhIP to produce cytogenetic damage and further indicated that N-hydroxylation of PhIP was requisite for mammalian genotoxicity.

Cytogenetic characterization of the ionizing radiation-sensitive Chinese hamster mutant irs1.

The X-ray-sensitive mutant V79 cell line irs1 was characterized with respect to chromosomal aberrations induced by 137Cs, mitomycin C (MMC), and decarbamoyl mitomycin C (DCMMC). To measure chromosome damage induced at different cell cycle stages, irs1 and the parental V79-4 cell lines were pulse-labeled with bromodeoxyuridine (BrdUrd) at the time of exposure and harvested at various intervals corresponding to exposure in G1, S, and G2 phases of the cell cycle. Metaphase spreads were stained with an anti-BrdUrd antibody, followed by a fluorescein-conjugated second antibody. With propidium iodide as a counter stain, cells were scored for aberrations. Compared to the parental V79 cells, irs1 cells had: (1) greatly increased sensitivity to all 3 agents; (2) a high frequency of chromatid exchanges after exposure in each phase of the cell cycle; and (3) more sensitivity to the agent causing crosslinks (MMC) than its monofunctional analog (DCMMC). The finding of chromatid-type damage in cells exposed to ionizing radiation during G1 is atypical of normal cells, but is similar to observations made in several mutant rodent cell lines and in ataxia telangiectasia cells. Our results suggest that the defect in irs1 cells can manifest itself as misrepair or misreplication during all phases of the cell cycle and leads to a high incidence of chromatid exchanges and deletions.

Identification of nucleotide-excision-repair genes on human chromosomes 2 and 13 by functional complementation in hamster-human hybrids.

The CHO UV-sensitive mutants UV24 and UV135 (complementation groups 3 and 5, respectively) are defective in nucleotide excision repair. After fusing each mutant with human lymphocytes, resistant hybrid clones showing genetic complementation were isolated by repeated exposure to UV radiation. Using a combination of isozyme markers, DNA probes, and cytogenetic methods to analyze the primary hybrids and their subclones, correction of the repair defect was shown to be correlated with the presence of a specific human chromosome in each case. Chromosome 2 corrected UV24, and the gene responsible was designated ERCC3. Line UV135 was corrected by human chromosome 13 and the gene designated ERCC5. The UV-sensitive mouse cell line, Q31, was shown not to complement UV135 and thus appears to be mutated in the same genetic locus (homologous to ERCC5) as UV135. Breakage of complementing chromosomes with retention of the genes correcting repair defects allowed the following provisional assignments: regional localization of ERCC5 to 13q14-q34, exclusion of ERCC3 from the region of chromosome 2 distal to p23, and relief of the ambiguity of ACP1 assignment (2p23 or 2p25) to 2p23 proximal to MDH1.

Incorporated bromodeoxyuridine enhances the sister-chromatid exchange and chromosomal aberration frequencies in an EMS-sensitive Chinese hamster cell line.

The mutant Chinese hamster cell line, EM9, is characterized by a high baseline sister-chromatid exchange (SCE) frequency, increased sensitivity to cell killing, and a defect in DNA strand-break repair. The molecular basis for this pleotrophic phenotype is not known. We examined, at the chromosomal level, the increased sensitivity of this mutant to incorporated BrdUrd. By varying the amount of BrdUrd in template DNA and measuring the frequency of SCEs and chromosomal aberrations, we demonstrated the enhanced sensitivity of EM9 to BrdUrd present in the template strand of DNA. Our results show that a 6-fold increase in SCEs occurs due to DNA replication over a BrdUrd-substituted template relative to a dThd-substituted template. With regard to aberration production in EM9, there is a significant enhancement of aberrations and a specific bias toward damage for the chromatid with Brdurd in the template strand. While these cells share some phenotypic properties with cells from patients with Bloom's syndrome, the genotypic similarities have not yet been established.

DNA-mediated transfer of a human DNA repair gene that controls sister chromatid exchange.

The Chinese hamster cell line mutant EM9, which has a reduced ability to repair DNA strand breaks, is noted for its highly elevated frequency of sister chromatid exchange, a property shared with cells from individuals with Bloom's syndrome. The defect in EM9 cells was corrected by fusion hybridization with normal human fibroblasts and by transfection with DNA from hybrid cells. The transformants showed normalization of sister chromatid exchange frequency but incomplete correction of the repair defect in terms of chromosomal aberrations produced by 5-bromo-2'-deoxyuridine.

In vivo cytogenetic effects of the cooked-food-related mutagens Trp-P-2 and IQ in mouse bone marrow.

Sister-chromatid exchange and chromosomal aberrations were measured in vivo in mouse bone marrow following intraperitoneal injection of the cooked food mutagens, Trp-P-2 and IQ. Trp-P-2 produced a significant positive dose response for both endpoints while IQ produced only a weak but significant sister-chromatid exchange response. The relative potency of these two chemicals is similar to that seen in mammalian cells in vitro but opposite to their potency in Salmonella.

In vivo exposure to plant flavonols. Influence on frequencies of micronuclei in mouse erythrocytes and sister-chromatid exchange in rabbit lymphocytes.

No consistent increases in the micronucleus frequency were observed in bone marrow or peripheral blood erythrocytes from mice treated with quercetin, rhamnetin, neohesperidin dihydrochalcone, or hesperetin dihydrochalcone under various exposure and sampling conditions. Over the dose range of 100-1000 mg/kg, quercetin failed to increase significantly erythrocyte micronucleus frequencies either (1) in bone marrow of male mice at 6 h after the second of 2 i.p. or oral doses given 24 h apart, or at 48, 96 or 192 h after a single i.p. or oral dose, or (2) in peripheral blood of male or female mice sampled for 7 consecutive days following a single i.p. dose. Feeding 5% or 10% quercetin for 8 days also failed to increase the micronucleus frequency in bone marrow erythrocytes of female or male mice. Hesperetin dihydrochalcone and neohesperidin dihydrochalcone, at p.o. doses of 100-1000 mg/kg, did not increase the micronucleus frequency in bone marrow erythrocytes 6 h after the second of 2 doses 24 h apart, nor did rhamnetin at 48 or 96 h after a single i.p. dose of 1000 mg/kg. Galangin, in contrast, did significantly increase the micronucleus frequency in bone marrow and blood erythrocytes under certain conditions, but the largest increases were only between 2 and 3 times control values and these were observed at highly toxic doses. Rabbits given up to 250 mg/kg quercetin i.p. showed no treatment-related increase in the sister-chromatid-exchange frequency in peripheral blood lymphocytes sampled at 1 and 7 days after treatment. These results fail to confirm published data which report a markedly increased frequency of micronuclei in bone marrow erythrocytes from quercetin-treated mice, show no quercetin-related alterations in the sister-chromatid-exchange frequency in rabbit lymphocytes, and indicate that clastogenesis in bone marrow erythroblasts due to oral or i.p. administration of the flavonols studied is at most very weak.

The relationship between sister-chromatid exchange and perturbations in DNA replication in mutant EM9 and normal CHO cells.

The majority of the high (12-fold elevated) baseline sister-chromatid exchanges (SCEs) that occur in the CHO mutant line EM9 appear to be a consequence of incorporated BrdUrd, and they arise during replication of DNA containing BrdUrd in a template strand. In normal CHO cells the alkaline elution patterns of DNA newly replicated on a BrdUrd-containing template are significantly altered compared with those seen during the replication on an unsubstituted template. The nascent DNA synthesized on such an altered template is delayed in reaching mature size, possibly because replication forks are temporarily blocked at sites occurring randomly along the template. Transient blockage of replication forks may be a prerequisite for SCE. The delay in replication on BrdUrd-substituted templates was greater in EM9 cells than in parental AA8 cells and was also greater in AA8 cells treated with benzamide, an inhibitor of poly(ADPR) polymerase, than in untreated AA8 cells. Under these conditions, treatment with benzamide also produced a 7-fold increase in SCEs in AA8. An EM9-derived revertant line that has a low baseline SCE frequency showed less delay in replication on BrdUrd-substituted templates than did EM9. However, under conditions where the template strand contained CldUrd, which was shown to produce 4-fold more SCEs than BrdUrd in AA8 cells, the replication delay in AA8 was not any greater in the CldUrd-substituted cells. Thus, other factors besides the delay appear to be involved in the production of SCEs by the template lesions resulting from incorporation of the halogen-substituted pyrimidine molecules.

A CHO-cell strain having hypersensitivity to mutagens, a defect in DNA strand-break repair, and an extraordinary baseline frequency of sister-chromatid exchange.

A mutant of CHO cells (strain EM9) previously isolated on the basis of hypersensitivity to killing by ethyl methanesulfonate (EMS) is approx. 10-fold more sensitive than the parental line, AA8, to killing by both EMS and MMS. It is also hypersensitive to killing by other alkylating agents (ethyl nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine), X-rays, and ultraviolet radiation. The production and repair of DNA single-strand breaks (SSB) were studied using the technique of alkaline elution of DNA from filters. After exposure to 4 Gy of X-rays at 0 degrees C and subsequent incubation at 25 degrees C, SSB were repaired within 12 min in AA8, but little repair occurred in EM9. Similarly, with doses of EMS or MMS that produced comparable numbers of SSB in AA8 and EM9 at the end of a 10-min exposure, repair of SSB occurred more rapidly in AA8 than in EM9, suggesting that individual SSB are longer lived in EM9. EM9 was found to be hypersensitive also to the induction of mutations and sister-chromatid exchanges (SCE) by EMS; per unit dose the mutant had twice as many mutations to thioguanine resistance, 3 times as many mutations to azaadenine resistance, and a 7-fold enhancement in SCE, compared to AA8. Moreover, the baseline frequency of SCE in the mutant was extraordinarily high, i.e., 8.6 +/- 0.6 vs. 107 +/- 5 SCE/cell for AA8 and EM9, respectively, with 10 microM BrdUrd in the medium. The high SCE frequency in EM9 did not vary significantly with BrdUrd concentration over the range examined from 2.5 to 20 microM, and the percentage of 5-bromouracil substitution in the DNA was the same in EM9 and AA8 under these conditions. These data, however, do not rule out the possibility that the high SCE frequency in EM9 is a consequence of an altered sensitivity to incorporated BrdUrd. Thus, EM9 may carry a pleiotropic mutation affecting some function in DNA replication and/or DNA repair and causing the variety of phenotypic properties described in this study.

Mutagenic and toxic activity of environmental effluents from underground coal gasification experiments.

Using bacterial bioassays, we have screened for the presence of mutagens and toxins in extracts from groundwater, and in tar from product gas, at the sites of two Lawrence Livermore National Laboratory (LLNL) in situ experiments: Hoe Creek II and Hoe Creek III. The sites exhibited different potential biological hazards, suggesting that different gasification processes may represent different human health concerns. We found that mutagens are present in groundwater, persist for at least 2 yr after gasification has been terminated, and show a change in activity with time-possibly in parallel with changes in chemical composition. Preliminary evidence suggests that the mutagens in groundwater are quinoline and aniline derivatives, while the toxins in groundwater may be phenolic compounds. In tar from the product gas, the organic bases and neutrals were found to be genotoxic in both bacterial and mammalian cells; the neutral compounds appear to be the major mutagenic health hazards. Neutral compounds constitute most of the tar (85-97 wt%) and were mutagenic in both the bacterial and mammalian cell assays. Tar in the gas stream may be a problem for the aboveground environment if gas escapes through fractures in the overburden. Because it is mutagenic and induces chromosomal damage to mammalian cells, the tar may represent a disposal problem as well. However, it is difficult to assess tar quantitatively as a health hazard because its mutagenic activity is low, possibly due to contaminants in the neutral fraction that act to suppress mutagenicity.

Variation in the baseline sister chromatid exchange frequency in human lymphocytes.

The utility of the sister chromatid exchange (SCE) assay for human population studies is potentially limited by the variability associated with individual baseline SCE Frequencies. This investigation identifies and quantifies the major sources of preparative and biological variation associated with the determination of baseline SCE frequencies in cultured human lymphocytes. Much of the variation in lymphocyte SCE frequencies is attributable to the amount of bromodeoxyuridine (BrdUrd) available per lymphocyte; the pooled coefficient of variation (CV) over the dose range of 10 to 160 micrometer is about 18%. Other variations in the baseline frequency result from culture-to-culture and slide-to-slide differences. The pooled coefficient of variation among donors is about 10%. The effect of cell-to-cell differences in baseline SCE frequency among donors can be minimized by increasing the number of cells scored per donor. When 20 cells are analyzed per individual the pooled cell-to-cell variation is 9% but when 40 or 80 cells are analyzed it is reduced to 6 and 4% respectively. For a single individual the cell-to-cell coefficient of variation at 100 micrometer BrdUrd is 40.8%. Under our experimental conditions, a 30% increase in SCE frequency between two cohort populations can be detected with a 95% probability at a 5% level of significance when 11 individuals per cohort are studied. For a longitudinal or in vitro dose response study of a single individual, a 50% increase in SCE frequency can be detected with a 95% probability at a 5% level of significance when 25 cells per sample are analyzed. These results indicate the feasibility of applying the SCE bioassay to humans as a measure of environmental stress.

DNA crosslinking, sister-chromatid exchange and specific-locus mutations.

Chinese hamster ovary cells were treated with the DNA-crosslinking chemicals, mitomycin C (MMC) and porfiromycin (POR), and their monofunctional derivative decarbamoyl mitomycin C (DCMMC). After exposure, the cells were studied for the induction of sister-chromatid exchanges (SCEs) and mutations at the hypoxanthine phosphoribosyltransferase and adenine phosphoribosyltransferase loci. The frequency of SCEs varied significantly in successive sampling intervals, requiring the weighting of each interval by the percentage of second-division mitosis in that interval to obtain the mean SCE frequency for each dose. All 3 compounds were potent inducers of SCEs but weakly mutagenic. All 3 chemicals by concentration were approximately equally effective in inducing SCEs or mutations. When the induced SCEs and mutations were compared at equal levels of survival, DCMMC was slightly more effective than MMC or POR in inducing SCEs and somewhat less mutagenic. These results indicate that the DNA interstrand crosslink is not the major lesion responsible for the induction of SCE or mutation by these compounds.

Induction of long-lived chromosome damage, as manifested by sister-chromatid exchange, in lymphocytes of animals exposed to mitomycin-C.

The cytogenetic effects of repeated vs. acute exposure to a chemical mutagen--carcinogen were determined with an in vivo system in which chemicals injected into rabbits induce sister-chromatid exchanges (SCEs). SCE induction can be monitored when the animal's peripheral lymphocytes are cultured in the presence of bromodeoxyuridine (BrdUrd) and then scored for SCE frequency. Mitomycin-C (MMC), 0.5 mg/kg, was injected intraperitoneally once a week for 8 weeks. This treatment initially induced small increases in SCE frequency within one day of injection, followed by a return to control levels within 1 week. After the 4th injection, however, the frequency failed to return to normal. After the 5th injection, however it showed a 4-fold increase over the control which was sustained for the remaining 3 weeks of treatment and for an additional 2 weeks thereafter. The frequency then dropped to twice the control value and remained at this level for more than 4 months. All of the high SCE values after the first 4 weeks were due in part to the appearance and persistence of a population of cells with high SCE frequencies. Exposure to the same total dose given as a single injection resulted in a transient elevation in the SCE frequency and a subsequent return to lower values, with no evidence of a delayed effect such as the increase observed after 4 weeks in repeatedly exposed animals. Overall, repeated exposure is at least as effective as acute exposure in eliciting long-lived SCEs in vivo.