Short-term tests for carcinogens and mutagens

Bleomycins are a family of glycopeptide antibiotics that have potent antitumour activity against a range of lymphomas, head and neck cancers and germ-cell tumours. The therapeutic efficacy of the bleomycins is limited by development of lung fibrosis. The cytotoxic and mutagenic effects of the bleomycins are thought to be related to their ability to mediate both single-stranded and double-stranded DNA damage, which requires the presence of specific cofactors (a transition metal, oxygen and a one-electron reductant). Progress in understanding the mechanisms involved in the therapeutic efficacy of the bleomycins and the unwanted toxicity and elucidation of the biosynthetic pathway of the bleomycins sets the stage for developing a more potent, less toxic therapeutic agent.

Bleomycins: towards better therapeutics

Metaphase chromosome structure. Involvement of topoisomerase II.

Highly differentiated heteromorphic sex chromosomes were found in the karyotypes of Pyxicephalus adspersus (= Rana adspersa) and analysed with the various banding methods. The W chromosome is considerably smaller than the Z chromosome and consists to a great proportion of constitutive heterochromatin. The chromosome lengths and the DNA content of this species have the lowest values determined in the Ranidae to date. The nucleolus organizer regions in the chromosomes reveal a high frequency of inter-individual variations (duplications, triplications) and aberrations. In one female animal, a translocation between the W chromosome and a nucleolus organizer was identified. Preliminary results indicate, that the same chromosome pair which in P. adspersus constitutes the heteromorphic sex chromosomes is still in an initial stage of morphological differentiation in P. delalandii. Although the ZZ/ZW sex chromosomes in Pyxicephalus have developed independently of the ZZ/ZW sex chromosomes in birds and reptiles, they seemed to have passed through identical stages of morphological differentiation. In this process, the heterochromatinization of the W chromosome had a decisive function.

Chromosome banding in Amphibia

In the preceding article we described a polyclonal antibody that recognizes cSc-1, a major polypeptide component of the chicken mitotic chromosome scaffold. This polypeptide was shown to be chicken topoisomerase II. In the experiments described in the present article we use indirect immunofluorescence and immunoelectron microscopy to examine the distribution of topoisomerase II within intact chromosomes. We also describe a simple experimental protocol that differentiates antigens that are interspersed along the chromatin fiber from those that occupy restricted domains within the chromosome. These experiments indicate that the distribution of the enzyme appears to be independent of the bulk chromatin. Our data suggest that topoisomerase II is bound to the bases of the radial loop domains of mitotic chromosomes.

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Localization of topoisomerase II in mitotic chromosomes.

The cytogenetic toxicity of gentian violet in Chinese hamster CHO cells in vitro has been studied by analyzing (1) squash preparations from direct fixation for recording mitotic anomalies and (2) air-dried preparations (with colcemid—hypotonic pretreatments) for recording metaphase chromosome aberrations. It was concluded that this compound is a mitotic poison as well as a clastogen in vitro. Its clastogenic property was confirmed in 5 other different mammalian cel types. 10 samples of different gentian violet and crystal violet were surveyed and all were shown to be clastogenic. Unless in vivo studies prove otherwise, gentian violet and crystal violet should be regarded as biohazardous substances.

Cytogenetic toxicity of gentian violet and crystal violet on mammalian cells in vitro

The genotoxic effects of adriamycin on somatic and germinal cells were studied in mice treated with single injections of 3, 12 or 24 mg/kg of the drug. From 1 to 5 days post-injection, chromosome aberrations were observed in bone-marrow cells and in diakineses-metaphase I cells from the testes. The frequency of chromosome breakages peaked at 5 h or 1 day for the bone marrow and at 3 and 5 days for the testis. Univalent formation was increased overall but did not have a dose- and time-dependent relationship. In long-term follow-up studies, adriamycin was found to induce cell killing of germline cells which resulted in a reduction in the numbers of spermatocytes and sperm from mice treated with the higher doses. There was complete absence of gametogenetic elements and, eventually, testicular atrophy occurred. In mice treated with 3 mg/kg, there was gradual recovery of spermatogenesis from 50 days onward. Chromosome breaks and translocations were again observed in the recovering spermatocytes. It was concluded that some of the chromosome aberrations must have been induced in the spermatogonial cells which had survived.

The genotoxic effects of adriamycin in somatic and germinal cells of the mouse.

Chromosomes were subjected to either prolonged hypotonic solution pretreatment or aging. Both conditions greatly loosened and dispersed the overlying epichrormatin from the central chromosome core structure. This was followed by silver staining and examination with bright-field microscopy. The chromosome core selectively reduced the silver and stained black while the surrounding epichrormatin stained yellow. A single core was seen extending the length of each chromatid. Nucleolus organizer regions appeared to be attached to the core, while kinetochores seemed to be specialized regions of the core itself. Cytochemical tests indicated that the core component(s) responsible for silver staining was non-histone protein(s).

Chromosome core structure revealed by silver staining

Silver staining of mammalian spermatocytes revealed, in light microscopy, synaptonemal complex and structures within the sex vesicle. It is feasible to follow the chromosome pairing phenomenon from zygotene to pachytene by examining the behavior of synaptonemal complexes. Nucleolus organizer regions take heavy silver stain in pachytene but are no longer detectable in later stages of meiosis.

T. C. Hsu

Papers

Cytogenetic toxicity of gentian violet and crystal violet on mammalian cells in vitro

The genotoxic effects of adriamycin in somatic and germinal cells of the mouse.

Chromosome core structure revealed by silver staining

Silver-stained structures in mammalian meiotic prophase

Cytogenetic analysis on eight human breast tumor cell lines: high frequencies of 1q, 11q and HeLa-like marker chromosomes.