Document pBgn5OE7Rb0d92w6bmgj5Q0BE

1L 1- 1I Chromosomal Aberrations and Bone I Marrow Toxicity i 1 I by John A. Heddle* and Michael F. Salarnone* i The importance of chromosomal aberrations as a proximate cause of bone marrow toxicity is i discussed. Since ch3micah that can cause nondisjunction are rare. numerical aberrations f aneuploid>-. polyploidy I are not ordinarily important. >Ian! structural aberrations. however. can lead directly to cell death and PO are proximate causes of tosicit!. when the! occur. The micronucleus test which utilizes the polychromatic erythrocyte is rapable of detectinp; agents 4 crlastogens 8 that can cause such structural aberrations. Man: carcinogens can be detected b> I this test. and recent changes in t h e protocol ma!- increase the surcess rate. Severtheless only a i small proportion of chemicals are clastopens. The importance of cell division in the expression of chromosomal damage and the stage of the cell cycle at the time of exporurc on t h e amount of darnape is emphasized. . A speculative m e c h a n i m for the relationship Iwlncen rhromoconial aherrat ions and carcinogenicit!- is proposed. There are two broad classes of chromosomal aberrations: numerical abei-rations in which whole chromosomes are either lost or added. (e.g. trisomy-21 or D o \ n ' s syndrome) and structural aberrations in which pieces of chromosome are lost. added. or translocated. for example the Philadelphia chromosome which leads to chronic myeloid leukemia. Obviously either class of abei-ration. if inherited, can have a significant influence on human health. These aberrations arise in fundamentally different ways. Aneuploidy, Le.. numerical aberrations involving other than a full complement of chromosomes. arise as a result of nondisjunction which is a failure of t h e proper distribution of chromosomes to daughter cells. Agents that interact directly with the cell's spindle mechanism are able to cause nondisjunction; there are also scattered reports which suggest that other agents may also be able to cause nondisjunction. perhaps by some indirect mechanism. Similarly. inhibition of the cell's spindle formation can lead to polyploidy. i.e.. the gain or loss of whole haploid sets of chrumosomes. In most tissues polyploid and aneuploid cells are rare 'Depan.nent of Biolop . York Lnn er-it! 1)ou n-\ le\\ Ontaric~MY 1P3. Canada .lune 1981 after treatment by tosic agents. even those agents capable of causing structural chromosomal aberrations. Thus such aberrations are not usually important contributors to the tosic response. Yinciistine and vinblastine are probably exceptions to this. but their irreversible binding to microtubules may cause toxicity by other means as well. Structural abei-rations. in cantrast. can lead directly to cell death and are often major contributors to tosicity. M'ith respect to tosicity. the kinds of chromosomal aberrations (which from this point on in this paper means structural abnormalities escept where otherwise indicated) may be classified in two ways: those that lead io the loss of genetic information at cell division and those that do not. This is shown diagrammatically in Figure 1. Those aberrations (of which only one of many possible types is shown) that involve a rearrangement of gene order rather than a direct loss of a gene are not cell lethal events and. hence, are not contributors to cellular toxicity. In contrast. th0F.e abelrations that lrad directly to the loss of a section of genetic informatiorb are usually cell lethal events and do contribute directly t o cellular toxicity. These aberrations may involve an acentric chromosomal fragment whose r,iovement at anaphase is dependent upon cytoplasmic currents so that the 23 fragment may o r may not be swept into a position tom which it can be incorporated into a daughter nucleus. If the fragment is not incorporated into one of the daughter nuclei it will become a micronucleus, a small body that resembles a r.ormal nucleus except for its size and possibly the presence of nucleoli (see Fig. 2). If the fragment is incorporated into a daughter nucleus it will be replicated so that a double fragment is present at the next cell division. If the fragment was already double there will be two fragments present, either or both cf which may be lost t o become micronuclei. Thus the longer a fragment escapes anaphase loss and micronucleus formation, the more potential micronuclei exist. The actual mechanism by which the loss of a chromosomal fragment causes death in a diploid cell is not known. It may be that recessive lethal genes existing on the homolog are made hemizygous by the loss of the fragment. Alternatively, it may be that the genetic imbalance between those loci represented twice and those loci represented only once (asa result of the fragment loss) is lethal. Both mechanisms are undoubtedly involved to some extent, with their relative importance being influenced by the actual chromosomal region involved and the demands placed upon the genome by the stage of cell differentiation involved. Regardless of the mechanism by which the loss of a chromosomal region causes cell death, the loss itself is dependent upon cell division. According to this idea the induction of chromosomal aberrations should not affect cells that do not divide or have not yet divided. Traditional techniques do not permit a direct test of this idea because the aberrations cannot be detected unless the cells divide. Indirect lines of evidence rather strongly favor this viewpoint, however. For example. numerous tests of 12 3 4 aC 2 1 F I Z L R E1. 1-4)Schematic diagram of a cnromatid deletion antl its fate ( 1 1at metapl?a.;e. 1 2 )at anaphase. IS) at telophase. and (4) the resultant micronucleus at intrq)ha.ie. The genetic information contained in the micronucleL5 is ordinarilJ-unavailable to the cell which may be ri:hrr the ctauph:er cell with the deletion or the daughter cell with the normal chromozomal complement. ~ R -4J cfiromatid tKinslocation (asymmetrical chromatid interchange) at metapha.-t- tt-hich may zepegate either I 1 1 in a t)alanced foim. or (2) in an unbalanced form giving a duplication for one repon antl a tieficienr: for the other in each daughter cell. If) A chromatid inversion !a.;ymrne!rical chromatid ic::actxpe: !: a: rnetaphze and (2, at anaphase. This aberration will not ordinarly be lethal or have any other genetic cunzequence in a somatic cell. 21 Environmental Health Perspectives I 1 I FIC;I.R2E. Bone marrow preparation showing micronuclei tHo\r-ellJolley bntiie 1 11: T U 11 :..chromatic erythrocpes and a mature enthrocyte {central cell of the thmr relkr. the importance of aberrations show that, at doses of ionizing radiation which 10% or more of the cells can survive, t h e majority of cell death in dividing cellular populations is attributable to chromosomal aberrations. At these doses virtually no cell death is observed in nondividing cell populations. However, if such populations, for example liver, are stimulated t o divide, then a wave of cell death and necrosis occurs. Furthermore, aberrations that involve rearrangement without loss of genomic material are often not cell lethal but are maintained through many cell divisions. Although this view is probably broadly correct, new techniques of cell fusion enable one to examine chromosomes in nondividing cells for aberrations and have thereby revealed at least some cells more heavily damaged than those seen at division ( I ) . It may be, therefore, that there is a reduced division potential in heavily damaged cells even before the first division. It remains true, however, that the tissues sensitive to ionizing radiation and t o chemicals that can cause chromosomal damage are normally those undergoing rapid proliferation. In the case of ionizing radiation, the cells are sensitive to chromosomal damage at all stages of June 1981 the cell cycle ( 2 ) . With chemicals the situation is more complex. Although D S A damage may be induced at any stage of the cell cycle, the aberrations are often produced only during S..Thus cells treated with nitrogen mustard in G2appear normal at the fist division after treatment but may be heavily laden with chromosomal aberrations at the second division (3).Cells of nondividing tissues are normally found in the GIphase. Provided that such cells are not stimulated to en:er S and divide for some time after treatmen;. much of the DXA damage may be repaired prior to S and thus much chromosomal damage may be avoided. Possibly this is true for stem cells in the bone marrow while the proliferating compartment suffers extensive dam- ageBecause it is a ready source of dividing cells, the bone marrow has been a favorite tissue for studies of chromosomal aberrations i l l z:izto. Traditional chromosomal analysis has suffered, however, from being a time-consuming process and from being a rather specialized field in which many of the basic concepts arose in nonmainmalian systems. As a consequence, many of the iir rivo experiments have been inadequate in the number of cells sampled. the 25 time of sampling, or both. In addition. inadequate tion of known mutagens and carcinogens. Recent dose levels have often been used. There is a natural advances suggest that a success rate g-reater than reluctance to work at level.; too close to lethal levels 50% in detecting carcinopeihs ran be e x p r ~ t r dn I t h i for fear of nonspecific effects. Unfortunately al- few if any false positives ( 6 ) . though aberrations probably occur even at very low Since micronuclei arise as a consecluencr of doses. too much scoring effort is required at such chromosomal loss during cell division. t h w e is low doses, especially as a noticeable spontaneous inevitably some lag period before they begn to rate does exist. Once aberrations are common appear. I n human lymphocytes i u r i f w this lac is enough to detect easily. cell death is the conse- approximately -10 h r because few division: wcur quence and thus the animal's life is entlangeretl. before this ( 7 ) . The maximum frequency occur> Therefore it is inevitable that aherrations \vi11 be after about three celi divisions in the 1ymphocJ.te easily detected only at doses close to the LD,,.This cultures. but this \vi11 depend upon the frequent!- of does not mean that aberrations are a nonspecitic fragment loss per cell division. I n lymphoc!.te consequence of tosicity nor that they do nut OCCUI. cultures this has been estimated to be W i per cell at lo\\,er doses. Rather the association betu.een division ( 8 ) . In systems in which fragment loss is significant frequencies of aberrations antl tosicity is more frequent the masinium should OCCUI- eadier. a consequence of the statistical problems of detecting In the polychromatic erythrocyte ( P C E )cell assay IOU- levels together with the importance of high there is an additional factor: the time betiveen the aberration frequencies causing toxicity antl death. last division and the expulsion of the main nucleus Nonspecific induction of chromosomal aberrations (9).As a consequence of this. no micronuclei are has never been established as a real mechanism. detectable in PCE for at least 8-10 hr after treat- C)-togeneticists interested in the mechanisms by ment. Treatments given within this interval are which chromosomal aberrations arise place p e a t wasted and may even be haimful by depressing store on details of the types antl kinds of abei.1.a- PCE production or causing artefacts. Cntil recently tions .produced. The classifications are relatively this has not been taken into account so that the complicated antl are not of concern here except to majority of testing has occur~*erul nder less than say that they depend upon analysis at metaphase. optimal conditions. The question of \vhether or not an agent can cause X second factor of importance in this assay. but chroinosonial breakage can be ans\vei.etl niuch more not recognized until recently. has been the time simply. -4s outlined earlier. acentric chromosomal course of microni~cleusproduction. Although some fragments become micronuclei when lost tluiing cell mutagens such as x-rays or mitomycin C result in a division. Khile these micronuclei do not provide the maximum protluction so011 after treatment. Le.. at cytogeneticist with important details of the abei-ra- about :3!; hr. others shoiv masiinum frequencies at tion types from which they arose. they are much later times. Dimethylbenzanthracene. for example, more easily antl rapidly quantified antl so provide a gives a maximum at about i2 hr that is more than simple means of detecting the occurrence of chro- five times the frequency at :36 h r (Fig. 3). The mosomal damage. No agents have yet been found reason for this vaiiation in the time of maximum that can cause aberrations of any kind without response is not clear; we have speculated that it is causing those kinds that have associated acentric related to the uptake antl metabolism of the agent fragments. Jlicronuclei should thus he a reliahle rather than m y fu:i:lhTet1:L: difft.~*eiiLieir th? index of chi.omosome damage. Micronuclei were interaction with the bone marrow (6).The problem used once as an experimental tool in plants antl raised by this finding is that some agents may not noted occazicmlly in other circumstances but ivere produce detectable increases at 86 hr but are not used as an assay until independently proposed detectable a t i 2 hr. Indeed a new protocol devised by Schmitl and co-workers ($1 and Hetltlle f.5). The to take account of this possibility led tu three cell type proposed by Schmid \vas the polychro- carcinogens that were not detectable at early times matic erythrocyte (see Fig. 2). This cell, although being detected at later times (6). Because the not itself a dividing cell is the immediate product of results even at late times were marginal. this result a series of cell divisions. The success of this assay needs to be confirmed. At the moment. hotvever. it can be judged by the fact that it has largely seems likely that the atldition of later sampling replaced traditional metaphaqe analysis in the screen- times to the standard protocol \vi11 increase the ing of chemicals for their ability to produce chromo- proportion of carcinogens detected by t hi.; assay. somal damage i)c i.ii.0. More than 200 chemicals The micronucleus assay seems at the moment to have now been tested, about a third of which \\ere be a robust assay with a moderate success rnte. It found positive. Of course the selection of agents is robust in that few laboratory to laboratory was highly non-random antl included a high p ~ q m r - discrepancies have emerged. The success rate has 26 Environmental Health Perspectives ni dll G t1h 40 from (I relatively small minrIrit> of chemical^. a fc..i. per cent of chemical> at mIlct.The ralii(l atlvai~cc.in genetic te5;tinp a r c rnd.iilg the 0ett.dicm of 5u(.f. chemicals a much mort. ~.eli;~h~leI * u c ~ +I.t. will tlr interesting to see if (1i-ea-e.. othei' than came-:, a birth defects. and pcne!ic. abnormaii!ie+ will 'be found to be associated with pc*ncttosicit>a-ntl \vhether IIA specific genetic event.<caii he sh(t\vn to he the cau-e of somatic diceases srwh as leukemia. m ,-.L 0 // 0 0l0 a- 48 12 96 hours ofter treotrneni usually been in the 405 to 60';; range. Since the protocols have almost always been less than opti- mal. it is likely that the time success rate \vi11 be higher. Severtheless. it is unlikely that any tissue- specific assay \vi11 be 1005 successful. and this assay But measures only one cia:. of genetic. tlamapr. 111 nui' e opinion. and we rnust stress that it is only an iie opinion, the a s a y detects quite a high fi-action of '1 a the most potent human carcinogens. We believe at that this is true not because the aberrations them- at selves are the cause of cancel- (and certainly not -le, those that lead t o micronuclei) but because the cell I an death that these aberrations cause leads to in- he creased cellular proliferation antl hence to an in- ;rn creased likelihood that a quiescent but transformed is cell will be called upon to divide. Once called upon Ant t o divide it is able to espress its cancerous pheno- be type, Le., it does not respond adequately to the .-m normal feedback mechanisms that keep cell produc- .ot tion in balance with cell loss. r e To return from the issue of genetic tosicdogy to +.d bone marrow toxicity. it should be evident from re this discussion that genotosic agents can contribute .?S greatly to tosicity in any dividing cell population. ne In the bone marrow in which there are so many Zlt different proliferating cell populations. large doses - it of such chemicals can be espected to produce a ng pancytopenia: at lower doses some populations may ...ne .. be more or less severely affected than others. Certainly the time course of tosicity arising in to subpopulations will differ according to their rate of I t proliferation and to the fraction of cells in the r y sensitive phases of the cell cycle. ac well as to the as dose they receive. Genetic tosicity is to be expected res June 1981 27