Mechanisms for induction of mutations and chromosome alterations.

Genotoxic agents induce chromosomal alterations, such as aberrations, micronuclei, and sister chromatid exchanges as well as mutations both in vivo and in vitro. Ionizing radiation and typical radiomimmetic agents such as bleomycin are very efficient inducers of chromosomal aberrations. The type of aberrations induced by these agents are cell-cycle dependent, i.e., chromosome type in pre-replication stages and chromatid type in post-replication stages of the cell cycle. Under optimal DNA repair conditions, DNA double-strand breaks (DSBs) appear to be the most important lesion responsible for the production of aberrations. In human lymphocytes, fast-repairing DSBs lead to exchange-type aberrations. The fact that the dose-response curves for induction of exchange aberrations induced by ionizing radiation are similar in vitro and in vivo allows one to use the yield of induced aberrations to estimate absorbed radiation dose in the case of accidents. In this respect, frequencies of translocations detected by the chromosome painting technique appear to be more sensitive. Mutations do not express immediately after exposure and require an expression time before they can be detected. In humans, it is estimated that for the mutations induced in bone marrow, it takes about 2 months for them to express and to be detected in peripheral blood lymphocytes. Hence, frequency of mutations is of limited value for estimating radiation doses immediately after an accident. This holds true for chemical exposure as well.(ABSTRACT TRUNCATED AT 250 WORDS)


Introduction
Exposure of the human population to genotoxic agents can be monitored by measuring DNA and protein adducts as well as by assessing biological effects such as chromosomal alterations and gene mutations. All genotoxic agents induce these events in vitro and in vivo. Because both chromosomal alterations and gene mutations are known to be involved in inherited disorders as well as in etiology of human neoplasms, it is important to develop methods to assess these events after human exposure to known genotoxic agents. In this paper the basic mechanisms of induction of these events and methods to detect them in human cells are reviewed. MGC

Chromosomal Alterations
Chromosomal alterations can be assessed by the frequencies of chromosomal aberrations (CA), micronuclei (MN), and sister chromatid exchanges (SCEs). Ionizing radiation and typical radiomimetic agents such as bleomycin are efficient inducers of chromosomal aberrations. The type of aberrations induced by these agents depends on the stage of cell cycle at the time of treatment. They induce chromosome-type aberrations in prereplicative stages and chromatid-type aberrations in S-phase and post-replicative stages. Among the different types of DNA lesions induced by ionizing radiation [namely, singlestrand breaks (SSBs), double-strand breaks (DSBs), base damages, and DNA-protein cross links], DSB appears to be the critical lesion leading to chromosomal aberrations. Biochemical and cytological evidences leading to this conclusion are a) high LET radiation induces more DSBs than low LET radiation and for a given dose induces more chromosomal aberrations; b) when X-irradiated Chinese hamster fibroblasts are post-treated with Neurospora endonuclease, a treatment which converts SSBs into DSBs, the frequencies of DSBs and chromos( tions increase to a similar extent (1,2); and c, endonucleases, which induce exclusively DSB induce chromosomal aberrations (3,4), an dependent pattern of the types of aberrations REs is similar to ionizing radiation (4).
Human peripheral lymphocytes, because of ability, are usually employed to monitor expo; otoxic agents. The lymphocytes are in a dorma and divide in vitro on stimulation. Two majo aberrations are recognized after irradiatior lymphocytes, namely, exchanges (dicentrics, tions, rings) and fragments. The kinetics of rel DSBs induced by ionizing radiation in hun biphasic, with a fast component in which ak breaks are repaired within 10 min and a slow that lasts for some hours (5). X-ray-induce( appear to be formed by misrepair of fast-repa Evidence for this comes from three types of e First, when irradiated lymphocytes are post-t cytosine arabinoside (araC), the frequenciesc increase by a factor of 2, and this increase is the first 15-30 min after irradiation (6,7). Sec( no influence of araC on the repair of DNA st when challenged 1 hr after irradiation (7). frequencies of dicentrics induced are similar cytes that were fused with mitotic CHO cell premature chromosome condensation (PCC)i after irradiation to those that were stimulatec for 48 hr and scored in mitotic metaphases (8 fusion was carried out after different repair ] frequencies of dicentrics remained the same, i frequencies of acentric fragments declined indicating that most of the dicentrics were forn ately after irradiation [(8), Fig. 1].
Similar frequencies of dicentric chrom( induced in human lymphocytes by ionizing ra4 given dose under in vitro and in vivo condit Dme aberraresponse curves for induced frequencies of dicentric chrorestriction mosomes in lymphocytes generated after in vitro irradia-'s efficiently tion (of different qualities) have been successfully used to Id the cellestimate absorbed radiation dose of individuals in the case 3induced by of radiation accidents, based on the frequencies of dicentrics observed in lymphocytes (9). The distribution of rtheir availdicentrics among the lymphocytes can be used to discern sure to genpartial body exposure, high or low LET exposure, acute or rnt Go stage chronic exposure, external or internal exposure, etc. (10).
)r classes of This technique was effectively used in a recent radiation of human accident in Goiania, Brazil (11). , transloca-In case of external exposure, the frequencies of dicenpair of DNA trics decrease with time, whereas in the case of internal nan cells is exposure the frequencies increase with time up to a cer-)out 80% of tain period (11,12). Dicentrics are unstable aberrations and 7 component will be eliminated during cell divisions. Though  When the immunochemical staining ("chromosome painting") to phereas the specifically stain individual chromosomes (12,13). This with time technique offers the possibility to detect chromosomal ned immedi translocations very efficiently. In earlier studies using chromosome banding techniques, it has been reported that Dsomes are the frequencies of radiation-induced dicentrics and diation for a reciprocal translocations are formed with equal frequen-;ions Dose-cies (14). With the chromosome painting technique, it is possible to discern translocations involving very small segments. In addition, types other than reciprocal trans-0.60 locations, such as interstial and terminal translocations, can be identified (15). o In human lymphocytes irradiated in vitro, using specific probes for six different chromosomes, we have esti- past radiation exposures in the victims of Hiroshima and Nagasaki have been reported (17). But the presence of clones containing the same translocation in some individuals may make dose estimation of past exposures difficult.
Many of the chromosome fragments that lag during anaphase movement will form micronuclei, which can be detected in the interphase of the daughter cells. However, for quantitative evaluation of the frequencies of micronuclei, it is essential to estimate the number of cells that have undergone one cell division. This has been made possible by the use of cytochalasin B, which inhibits cytokinesis, giving rise to binucleated cells after division (18). If one restricts scoring to micronuclei in binucleated cells, it is possible to accurately estimate the frequencies.
The dose-response curve for induction of micronuclei in human lymphocytes induced by X-rays is curvilinear, indicating that part of the micronuclei arise from the acentrics associated with two-hit events, i.e., dicentrics (19). A parallel analysis of chromosome aberrations and micronuclei in irradiated lymphocytes indicated that about 70-90% of fragments form micronuclei at low radiation doses (up to 2 Gy), whereas at higher doses about 40% of fragments formed micronuclei (19). This low efficiency of the micronucleus technique at higher radiation doses appears to be due to the facts that a) some of the fragments are included into the main nuclei and b) cells carrying no or a lower number of fragments divide earlier than highly affected cells. The frequencies of spontaneously occurring micronuclei vary between individuals, and this makes it difficult to use frequencies of micronuclei as a biological dosimeter to estimate absorbed radiation dose, especially at low exposure levels.
Micronuclei can be formed by lagging whole chromosomes or acentric fragments. These two types of micronuclei can be distinguished by immunological staining of centromeres by CREST antibodies or by in situ hybridization with centromere-specific probes. It can be shown that most of the micronuclei induced by ionizing radiation originate from acentric fragments, whereas those induced by agents that interfere with chromosome separation (such colchicine, vincristine, etc.) originate from lagging chromosomes.

Chemically Induced Chromosomal Alterations
Genotoxic chemicals induce a wide variety of DNA lesions in different proportions. Unlike ionizing radiationinduced chromosome aberrations, which are formed immediately after exposure irrespective of the treated cell-cycle stage, most of the chemically induced aberrations are formed only during the DNA synthesis phase, probably due to misreplication. In vivo human exposure to chemical mutagens induce lesions in the DNA of lymphocytes, most of which are repaired by cellular repair enzymes. Unrepaired lesions give rise to chromatid-type aberrations during the S phase, when the lymphocytes are stimulated in vitro. However, sometimes chromosome-type aberrations can also be found, which are probably formed before stimulation in the Go stage by a different mechanism, (e.g., apurinic or apyrimidinic sites converted into strand breaks and misrepaired).
A sister chromatid exchange (SCE) is a cytological manifestation of DNA breakage and rejoining at apparently homologous sites on the two chromatids of a single chromosome. SCEs are efficiently induced by those agents that form covalent adducts to DNA or otherwise interfere with DNA metabolism or repair. The baseline frequency of SCEs varies between individuals, and smokers have increased baseline frequencies in their lymphocytes. Elevated frequencies of SCEs have been found in humans exposed to known genotoxins. A subpopulation of lymphocytes with high frequencies of SCEs (high-frequency cells) has been found in many human population studies (20,21). These cells may represent persistent DNA lesions in long-lived lymphocytes or a sensitive subpopulation. In biomonitoring human populations, consideration of only high-frequency cells appears to be a more sensitive index than consideration of overall SCE values, as demonstrated in a recent study on ethylene oxide-exposed workers (21).

Gene Mutations
Mutations can arise due to single base-pair changes (transition, transversion, frame shift) and small or large rearrangements (deletions, translocations, etc.). In humans, mutations can be studied in lymphocytes and erythrocytes. In erythrocytes, hemoglobin mutations and glycophorin mutations can be detected. Hemoglobin mutations arise as single base-pair change in one of the codons (transition, transversion or deletion) in the (x or 1B chain. Many hemoglobin mutations such as sickle cell anemia and others occur in nature. Monospecific polyclonal antibodies have been raised against several of these mutations, which can be used to detect such mutations in a population of erythrocytes (22). Because the frequencies of such muta-tions are very low, millions of cells have to be screened to detect these rare events, and the automated image analysis system has been developed for this purpose (23). A large increase in the frequency of hemoglobin mutants has been detected in radiation accident victims from Goiania (11).
Erythrocytes can also be screened for mutations in glycophorin A locus. This cell-surface glycoprotein occurs in two allelic forms (M and N) and is codominantly expressed. Mutation in this locus can be studied only in heterozygous (M/N) individuals. Monoclonal antibodies for individual allelic forms are conjugated with a different fluorescent dye and used to label fixed erythrocytes. Flow cytometry and sorting are used to estimate the frequency of cells that lack the expression of one of the GPA alleles. Because this system is based on loss of gene expression, single base-pair or gross changes can lead to this mutations. Increases in the frequency of mutants have been found in atom bomb survivors and chemotherapy patients (24).
There are three systems available to screen for mutants in human lymphocytes, namely, HPRT, HLA, TCR-CD3 assays. Of these, HPRT mutations, which are selected as resistance to 6-thioguanine, have been used in many laboratories (25)(26)(27). Using this method, increases in mutations have been found in the lymphocytes of cancer patients treated with cyclophosphamide, adriamycin, isophosphamide, etc. It is feasible to clone T-lymphocytes in vitro by adding growth factors such as interleukin 2 to the growth medium. HPRT mutants isolated by the clonal method can be used to characterize the exact molecular nature of these mutants (28). Spontaneously occurring mutations have been found at all regions of the gene and comprise base-pair changes (transitions, transversions), frame shifts, small deletions, and large deletions involving one or more exons (splice mutations). Though it has been found that smokers have an increased frequency of mutants compared to nonsmokers, the mutation spectrum appears to be similar in both (29).
In a recent biomonitoring study of workers exposed to ethylene oxide, a significant increase in the frequencies of HPRT mutants was found (21). Though there was no difference in the frequencies of different classes of mutants induced, a hot spot for mutation at position 617 of the HPRT coding region (amino acid residue 206), which was not present in the control spectrum, was found. The fact that exclusively GC to AT transitions were observed at this position makes it possible that this hot spot is caused by ethylene oxide adducts, since alkylation of guanine at the 06 position of ethylating agents predominantly gives rise to this type of base substitution (30).