Baldev K. Vig

Genetic toxicology of bleomycin.

Bleomycin (BLM), an antibiotic obtained from Streptomyces verticillus, is of significance as an antineoplastic agent. The compound is actually the mixture of some 200 related forms which differ from each other in the amine moiety. The drug, at low concentrations, can cause elimination of bases, particularly thymine. This causes strand breakage of DNA and inhibition of cell growth. The influence of BLM on cell growth may be unrelated to the effects on DNA. In general, mitotically dividing cells show more DNA damage than non-dividing cells. G2 seems to be the most sensitive phase indicating that cell death may not be related to a direct effect of BLM on DNA replication. The antibiotic shows specific effects on chromatin and causes chromosomal damage in all sub-phases of interphase. It can affect early prophase chromosomes also. Suggestion has been made that BLM-induced breakage and cell death are similar to those induced by densely ionizing radiations. Whereas the antibiotic affects the frequency of somatic crossing over and produces micronuclei, the data on mutation induction and production of sister-chromatid exchanges do not permit classifying BLM as a potent inducer of these phenomena. The genetic effects of BLM can be modified quantitatively by thiol compounds, caffeine, hyperthermia and H2O2. It is concluded that the available data do not permit assessment of genetic damage in the offsprings of BLM-treated patients. Such studies are urgently needed, as are the studies to find out the effects of BLM on meiotic phenomena.

Genetic toxicology of mitomycin C, actinomycins, daunomycin and adriamycin.

Abstract Mitomycin C (MMC), actinomycins (ACMs), daunomycin (DNM) and adriamycin (ADR) are antineoplastic, antibiotics, with the anthracyclines (DNM and ADR) being more promising that the others. All these chemicals interact with DNA. MMC brings about cross-linking of the DNA strands and acts as mono-, as well as, bifunctional alkylating agent. The other three intercalate with the DNA molecule. ACMs, especially ACM-D, inhibits RNA synthesis preferentially and discriminates between different species of RNA. At high concentrations, DNA, RNA and protein synthesis are all inhibited by any of these antibiotics. The agents effect the cell cycle traverse at various stages; MMC being most unspecific of all. Mitotic cells at post-G2 stages are the least effected by any of these chemicals. All these chemicals induce chromosome aberrations of chromatid type. The anthracyclines, in addition, produce chromosome-type aberrations perhaps by affecting the single stranded G1 chromosome. Rejoining occurs, but MMC creates a high frequency of quadriradial configurations by utilizing the analogous nucleotide sequences in the repetitive DNA. At other points, it mostly creates free fragments. The mechanisms of action appear to be different for induction of chromosome aberrations by MMC, ACT and the anthracyclines. Both in vitro and in vivo aberration induction has been reported. The associated phenomenon of sister chromatid exchanges is strongly potentiated by these agents. MMC has been a known mutagen in both akaryotes and eukaryotes, and it also increases the frequency of meiotic recombination. ACM-D does not appear to strongly effect these phenomena while sufficient data with the anthracyclines are not available, and perhaps, will turn out to be generally negative. MMC and ACM-D also induced somatic recombination and somatic mosaicism; the former is a potent recombinogen. Correlation between genetic effect and the information available on the binding of ACM-D to chromatin is discussed and some space has been allocated to the discussion of resistance and cross resistance induced by the four chemicals. The data point to a strong need for testing of chemicals for genetic toxicology; particular attention must be given to the subject of co-mutagenicity.

Sequence of centromere separation: analysis of mitotic chromosomes in man.

SummaryMitotic chromosomes from human peripheral lymphocytes studied at the junction of metaphase and anaphase show that the centromeres of various chromosomes separate in a nonrandom, apparently genetically controlled sequence. It does not depend upon the position of the centromere in the chromosome, the length of the chromosome or total amount of detectable C-chromatin. In man, several chromosomes e.g. 18, 17, 2, separate very early. Such “early” cells do not include nos. 1, 13, 14, 15, and Y and very rarely nos. 21 and 22. The last separating chromosomes are those from group D, G, no. 1, 16, and Y. The possible implication of these findings in evolution, non-disjunction and the control of centromere separation sequences is discussed.

Sequence of centromere separation: Occurrence, possible significance, and control

This review describes the existence of a phenomenon, sequential separation of centromeres, in mitotic cells of various species including both animals and plants. Critical observations at metaanaphase show that the centromeres of chromosomes in a given genome do not separate into two sister units randomly, but that there is a genetically controlled, nonrandom, species-specific sequence which is independent of the length of the chromosome or the position of the centromere. A stricter control appears to exist for late-separating than for early-separating chromosomes. At early stages of metaanaphase several chromosomes initiate onset of separation simultaneously or in rapid succession, but late-separating chromosomes are better defined in their sequential position. The effect of Colcemid on the sequence of separation is minimal. It is proposed that aneuploidy in humans and other organisms may result from out-of-phase separation of a given chromosome. With the exception of chromosome No. 16, it appears that very early- or very late-separating centromeres are involved in human trisomies more often than those in between. Perhaps one function of centromeric heterochromatin is the control of centromere separation. The amount of such chromatin shows a positive correlation with the timing of separation of the centromeres. Superimposed upon this quantitative influence is the qualitative aspect, as discussed for various genomes. This suggestion explains a lack of extremely large quantities of heterochromatin near the centromere. Its existence in the form of homogeneously staining regions distal to the centromere, as in some cancer cells or in sex chromosomes, seemingly has no influence on the separation of centromeres. A brief discussion of centromere separation errors in human disease is provided, and suggestions for further studies are made.

Soybean (Glycine max): A new test system for study of genetic parameters as affected by environmental mutagens☆

Abstract The light green, Y11y11, plants of varieties T219 and L65-1237 of soybean have some dark green, yellow and twin (or double) spots resembling the phenotypes controlled by Y11Y11, y11y11 and Y11Y11–y11y11 genotypes, respectively. The process of somatic crossing over is considered responsible for the origin of double spots. Some of the single spots, undoubtedly, originate by failure of one of the two components of the double spots. Non-disjunction, segmental losses and/or point mutations are also inferred to cause some of the spots seen on the heterozygous Y11y11 plants as well as light green spots seen on the Y11Y11 and y11y11 homozygotes. When seeds are treated with caffeine, mitomycin C or 3H2O the increase in the frequency of all three types of spots on the Y11y11 leaves is parellel. Somatic crossing over is considered as the major, common basis for the origin of spots. The treatment with sodium azide (NaN3) increases, on the Y11Y11 plants, the frequency of dark green and yellow spots equally; doubles increase only slightly or not at all. The y11y11 plants lack any spots. The data suggest that NaN3 causes non-disjunction in this material. γ-Rays, on the other hand, increase the frequency of yellow spots much more than that of dark greens or doubles on Y11y11 leaves. Also light green spots are found on y11y11 as well as Y11Y11 plants. These data may mean that spots result from somatic crossing over, point mutations and segmental losses. A list of many other agents tested is included. Because different mutagens increase the frequency of the three types of spots, either equally or differentially, it is suggested that the pattern of action of a given mutagen can be studied by analyzing the relative increase of different types of spots. This material provides the investigator with a eukaryotic, in vivo test system which is relatively rapid and inexpensive.

Chromosome segregation and aneuploidy

Of all genetic afflictions of man, aneuploidy ranks as the most prevalent. Among liveborn babies aneuploidy exist to the extent of about 0.3%, to about 5% among stillborns and a dramatic 25% among miscarriages. The burden is too heavy to be taken lightly. Whereas cytogeneticists are capable of tracing the origin of the extra or missing chromosome to the contributing parent, it is not certain what factors are responsible for this ‘epidemic’ affecting the human genome. The matter is complicated by the observation that, to the best of our knowledge, all chromosomes do not malsegregate with equal frequency. Chromosome number 16, for example, is the most prevalent among abortuses -one-third of all aneuploid miscarriages are due to trisomy 16 (Chandley, 1987) — yet it never appears in aneuploid constitution among the liveborn. Some chromosomes, number 1, for example, appear only rarely, if at all. In the latter case painstaking efforts have to be made to karyotype very early stages of embryonic development, as early as the 8-cell stage. Even though no convincing data are yet available, it is conceivable that the product of most aneuploid zygotes are lost before implantation.

Sequence of centromere separation: orderly separation of multicentric chromosomes in mouse L cells.

Mouse L cells have many dicentric chromosomes and one with eight centromeres. All eight centromeres behave similarly until midmetaphase when most centromeres split into two units each in apparently quick succession but out-of-phase. This premature separation leaves one or perhaps two closely located centromeres intact, which separate at late metaphase-anaphase, drawing the two chromatids to opposite poles. Such dominance of one centromere over all others, though unexplained, ensures the lack of any mitotic abnormality such as bridges or fragments. These observations show that all the centromeres are retained as functional primary constrictions except for a change in functional regulation when more than one centromere are located on a chromosome.

Sequence of centromere separation another mechanism for the origin of nondisjunction

SummaryThe most commonly accepted view about the origin of aneuploidy is that it is due to errors in meiotic division. However, its rare occurrence makes it difficult to explain recurrent births of trisomic children to some parents. This problem causes more serious concern when one accepts that an abnormal (n+1 or n-1) sperm would enter fertilization by overriding thousands, or even millions, of normal haploid sperms. Also, the failure of aneuploidy to be induced in the offspring of mammals treated with mutagens raises questions about the effectiveness of the accepted mode of origin of errors. Current concepts also do not explain why one observes more errors of meiotic I, than of meiotic II, origin. It is known that most chromosomes separating at meta-anaphase junction in mitosis follow a nonrandom, genetically controlled sequence of separation. The present proposal makes use of out-of-phase separation of a rare chromosome, like premature separation in mitosis of the X in elderly humans or of an 18 in parents of trisomy 18 children. The suggestion is made that such out-of-phase separation results in aneuploid cell lines by total failure of the centromere to separate or by it separating too early, before the spindle is formed. The prematurely separating centromeres, it appears, do not attach to spindle fibers and hence cause nondisjunction. Such nondisjunction in embryonic stages will produce apparently normal individuals with mosaicism in somatic and/or gametic tissue. An individual carrying mosaicism in gonadal tissue will produce a large number of abnormal gametes, one of which may have a reasonable chance of entering fertilization. This mode of origin of aneuploidy takes care of all questions raised above and finds support in the data available in the literature. Several of the suggestions made in the hypothesis are easily testable.

Characterization of kinetochores in multicentric chromosomes

Long-term cultures of certain rat and mouse cell lines carry several dicentric and some multicentric chromosomes. Using antikinetochore antibodies obtainable from serum of scleroderma (var. CREST) patients we studied the number of kinetochores formed along the length of these chromosomes. The rat cells displayed as many kinetochores as there were centromeres. However, mouse cells showed the synthesis of only one kinetochore in dicentric and multicentric chromosomes which had been in the culture for a period of 1 year or more. When translocations were induced by bleomycin in mouse L cells, the newly formed dicentric chromosomes showed the formation of two kinetochores. It is not known when the accessory centromeres lose their capacity to assemble kinetochore proteins. Possibly, in the rat the ‘latent’ kinetochores lack a specific component which renders them ineffective for microtubule binding. The reason for the formation of only one kinetochore in mouse multicentric chromosomes is not clear. It may be due to the accumulation of mutations, modification of the kinetochore protein so that it lacks the antibody binding component, or a more effective regulatory gene than in the rat.

Sequence of centromere separation: Minor satellite DNA does not influence separation of inactive centromeres in transformed cells of mouse

Neoplastic cells may carry inactive centromeres on some multicentric, yet stable, chromosomes. We report that some inactive centromeres in L929 mouse cells do not contain minor satellite DNA, the DNA fraction which has been suggested to constitute the centromere. We compared the sequence of separation of inactive centromeres carrying the minor satellite with those lacking this fraction. The sequence of separation appears to be independent of whether or not the inactive centromeres carry the minor satellite DNA. The timing of replication of the inactive centromeres is also independent of this DNA. Hence, minor satellite of mouse is not a factor in holding together the subunits of inactive centromeres. Extension of these results to active centromeres might suggest that the minor satellite DNA is not a factor responsible for adhesion of the two centromere sub-units up until late meta-anaphase.