Evidence for chromosomal replicons as units of sister chromatid exchanges.

Chromosomal replicons have been described as the cytological counterpart of DNA replicon clusters and have previously been studied in vitro using premature chromosome condensation-sister chromatid differentiation (PCC-SCD) techniques. Chromosomal replicons are visualized as small SCD segments in S-phase cells, and measurement of these segments can provide estimates of relative chromosomal replicon size corresponding to DNA replicon clusters functioning coordinately in S-phase. Current hypotheses of sister chromatid exchange (SCE) formation postulate that sites of SCE induction are associated with active replicons or replicon clusters. We have applied the PCC-SCD technique to in vivo studies of mouse bone marrow cells that have been treated with cyclophosphamide (CP) for two cell cycles. We have been able to visualize chromosomal replicons, as well as SCEs which have been induced in vivo by CP treatment, simultaneously in the same cells. Chromosomal replicons visualized as small SCD segments were measured in PCC cells classified at early or late S-phase based on SCD segment size prevalence. Early S-phase (E/S) PCC cells contained 90% of the SCD segments measured clustered in a segment size range of 0.1 to 0.8 micron with a peak value around 0.3 to 0.6 micron regardless of CP treatment. As the cells progressed through S-phase, late S-phase (L/S) PCC cells were characterized by the appearance of larger SCD segments and even whole SCD chromosomes in addition to small SCD segments.(ABSTRACT TRUNCATED AT 250 WORDS)

Sister chromatid exchange analysis in cultured peripheral blood leukocytes of the coldwater marine fish, Pacific staghorn sculpin (Leptocottus armatus): a feasible system for assessing genotoxic marine pollutants.

The genotoxicity of environmental contaminants and test compounds to aquatic and marine fish has primarily been assessed by in vivo techniques that require sacrifice of the test organism for analysis. The major objective of this research was to develop an in vitro sister chromatid exchange (SCE) assay which would utilize cultured peripheral blood leukocytes (PBLs) of a coldwater marine fish species. Use of PBLs in cytogenetic genotoxicity tests has several advantages, the major one being that the experimental fish need not be sacrificed for sample collection. In addition, this nondestructive method of tissue collection permits the investigator to take multiple samples from a single individual and thereby allows the use of an individual as its own control and to monitor its SCE frequency over time. A suitable in vitro culture method for fish PBLs was a prerequisite for cytogenetic analysis of this tissue. The in vitro culture conditions necessary to provide a sufficient number of dividing cells for performance of the SCE assay were established in our laboratory for the PBLs of the Pacific staghorn sculpin (Leptocottus armatus), a common bottom-dwelling Puget Sound fish. The major components of this culture system are heparinized whole blood, fetal bovine serum-supplemented enriched tissue culture medium (RPMI 1640), purified protein derivative of tuberculin as a mitogen, and an incubation temperature of 13.5 degrees C. This in vitro PBL culture system is unique because it involves cultured blood cells from a coldwater marine fish species. Using this culture method, SCE induction was investigated in Pacific staghorn sculpin PBLs which had been exposed in vitro to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a known direct-acting inducer of SCEs. Cultured cells exposed in vitro responded to MNNG in a dose-related manner in regard to SCE induction, and the frequency of "outlier" cells increased at the higher concentrations of MNNG. With further development, this technique may be adaptable for use with in vivo genotoxicity studies and provide information concerning the induction and persistence of chemically induced SCEs in fish. This PBL/SCE assay may also be a feasible assessment tool for detecting exposure of marine fish to genotoxic environmental contaminants in laboratory and field situations.