Mechanisms of mutation induction in germ cells of the mouse as assessed by the specific locus test.

Mouse germ cell specific locus mutagenesis data and a molecular characterization of mutant alleles have been reviewed to arrive at an understanding of the mechanism of mutation induction in mammals. (a) The spermatogenic stage specificity for the sensitivity to mutation induction by 20 chemical mutagens is considered. (b) The effects of a saturable repair process and its recovery over time are examined for the mutagenic efficiency of ethylnitrosourea. (c) The mutagenic events following methylnitrosourea and chlorambucil are shown to be mainly deletions. In contrast the mutations recovered after ethylnitrosourea treatment are almost exclusively base pair substitutions. (d) It is emphasized that to date very few specific locus experiments have been designed to test for mutagenic events outside the interval stem cell spermatogonia-mature spermatozoa. A specific locus mutation has recently been shown to be due to loss of heterozygosity via mitotic recombination in an early zygote stage and suggests a broader range of possible mechanisms of mutation when these stages are considered. (e) With the cloning of all 7 marker loci mutation analysis at the molecular level will allow a more direct assessment of the mutation process in future studies.     

Assessment of the potential germ cell mutagenicity of industrial and plant protection chemicals as part of an integrated study of genotoxicity in vitro and in vivo

An approach is described that enables the germ cell mutagenicity of chemicals to be assessed as part of an integrated assessment of genotoxic potential. It is recommended, first, that the genotoxicity of a chemical be defined by appropriate studies in vitro. This should involve use of the Salmonella mutation assay and an assay for the induction of chromosomal aberrations, but supplementary assays may be indicated in specific instances. If negative results are obtained from these 2 tests there is no need for the conduct of additional tests. Agents considered to be genotoxic in vitro should then be assessed for genotoxicity to rodents. This will usually involve the conduct of a bone marrow cytogenetic assay, and in the case of negative results, a genotoxicity test in an independent tissue. Agents found to be non-genotoxic in vivo are regarded as having no potential for germ cell mutagenicity. Agents found to be genotoxic in vivo may either be assumed to have potential as germ cell mutagens, or their status in this respect may be defined by appropriate germ cell mutagenicity studies. The basis of the approach, which is supported by the available experimental data, is that germ cell mutagens will be evident as somatic cell genotoxins in vivo, and that these will be detected as genotoxins in vitro given appropriate experimentation. The conduct of appropriate and adequate studies is suggested to be of more value than the conduct of a rigid set of prescribed tests.     

Strange goings-on in the mouse germ line

It is a conventional paradigm that mutagens lead to changes in nucleotide sequence when the cell attempts to repair or replicate lesions in DNA (such as adducts or strand breaks) that have been produced by the mutagens or their metabolites. The resulting changes are located at (or very near) the sites of the initial damage. This is the underlying theory behind mutational spectra work, but how general is it in vivo? Work with ionising radiation has shown that there are interesting things going on in the mouse germ line that do not fall within the conventional paradigm. Mutations occur at certain sites remote from initial DNA damage and in greater than expected number. Bryn Bridges discusses some recent papers on mutational changes in the germ line of mice following exposure to chemical mutagens that suggest that such phenomena may not be confined to radiation.     

The SOS-function-inducing activity of chemical mutagens in Escherichia coli

The SOS-function-inducing activities of 42 chemical mutagens were investigated in Escherichia coli K12. The induction of the SOS function was assayed by monitoring the beta-galactosidase activity in the sulA::lacZ fusion strain PQ37 . To correct for the inhibitory effects of test chemicals on mRNA or protein synthesis, the level of the constitutive alkaline phosphatase was assayed in parallel. Most of the mutagens reported to be mutagenic to the Ames' Salmonella tester strains showed the SOS-function-inducing activity. The inducible SOS repair may be responsible for not only base-change mutations but also frameshift mutations. However, 9-aminoacridine, ethidium bromide and 4-nitro-o-phenylenediamine did not induce the SOS function, suggesting that the mutagenesis induced by these mutagens may occur independently of SOS repair. Present results support the SOS mutagenesis model that error-prone SOS repair plays an important role in mutagenesis induced by most chemical mutagens.     

Mutagenicity of gossypol analyzed by induction of meiotic micronuclei in vitro

Chromosome breakage caused by mutagens in male germ cells can be analyzed by micronucleus induction during meiotic division. This can be followed in vitro by culturing seminiferous tubular segments from stages of the epithelial cycle that contain late pachytene and diakinetic primary spermatocytes. We studied the mutagenic potential of a male contraceptive, gossypol, in this test system using adriamycin (10 ng/ml) as a reference mutagen. A small but significant increase in the frequency of micronuclei was induced with concentrations of 10 and 20 micrograms/ml of gossypol, while cytotoxic effects appeared at concentration of 20 micrograms/ml and were evident at 50 micrograms/ml. Analysis of meiotic micronucleus induction in vitro seems to be a sensitive test system of male germ-cell mutagenesis, but further studies on the possible mutagenic effects of gossypol are needed.     

The present lack of evidence for unique rodent germ-cell mutagens

Six chemicals, diethylhexyl phthalate (DEHP), ethanol, cyclohexylamine (CHA), sodium saccharin (NaS), cadmium chloride (CdCl2) and triflupromazine (TFP), were suggested to be unique germ-cell mutagens (Auletta and Ashby, 1988) by the GeneTox Workgroups of the U.S. Environmental Protection Agency (EPA). If this is a correct classification it would have major consequences when screening for mutagenicity and when labelling genotoxic substances. However, our re-evaluation of the GeneTox literature, including some more recent publications, has failed to find substantive evidence that any of these chemicals have been unequivocally established as having unique mutagenic activity in germ cells. For DEHP, NaS and TFP the evidence for genotoxic/mutagenic effects is questionable, in both germinal and somatic cells. Ethanol and CdCl2 showed clastogenic activity, but it was not restricted to germ cells. Both, ethanol and cadmium salts, appear to induce aneuploidy. The unconfirmed clastogenic effect of CHA was restricted to rats, but it occurred in both bone marrow and spermatogonia. Therefore, the general observation that rodent germ-cell mutagens are also genotoxic in somatic cells in vivo (Brusick, 1980; Holden, 1982) remains valid.     

Mutagenic activity of selected aromatic amines and polycyclic hydrocarbons in Drosophila melanogaster

With the use of a series of wild-type and repair-deficient strains and appropriate application procedures, it is possible to demonstrate that carcinogenic aromatic amines and polycyclic hydrocarbons are mutagens in Drosophila. We have shown evidence that AAF, N-OH-AAF, AcO-AAF, BP, DAS and DMBA produce recessive lethals when fed to or injected into adult males. Mutagenic activity was also observed when male larvae were exposed to AAF, BP, DMBA, 3-MC or NA. DA was not mutagenic in the recessive lethal assay under the conditions of the test. DMBA can now be considered as a potent mutagen for Drosophila, although demonstration of its activity depends upon the choice of the treatment procedure and the strain selected. One of the questions concerning the action of aromatic amines and polycyclic hydrocarbons is how their genetic effectiveness in Drosophila can be enhanced. The observation that none of several enzyme inducers (PB, BF, AC, 3-MC) increased their mutagenicity may be interpreted in terms of a more efficient metabolic activation or deactivation. This assumes that active metabolite(s) did not reach the testis in doses sufficient for mutation induction. It also appears that, since the problems pertaining to mutagenicity in Drosophila of aromatic hydrocarbons are obviously a matter of metabolism, the use of repair-deficient strains is no longer an attractive proposal for their elucidation. The present investigation shows that, with weak mutagens, usage of strains mei-9Li or y mei-9a mei-4lD5 does not improve the sensitivity of the recessive lethal method or the test for chromosomal loss. As an alternative, in our opinion more attention should be devoted to possible differences in metabolism between somatic and gonadal tissue. We feel strongly that somatic assay systems might be particularly valuable as a complement to recessive lethal tests on the germ line.     

Mechanisms and consequences of paternally-transmitted chromosomal abnormalities

Paternally-transmitted chromosomal damage has been associated with pregnancy loss, developmental and morphological defects, infant mortality, infertility, and genetic diseases in the offspring, including cancer. There is epidemiological evidence linking paternal exposure to occupational or environmental agents with an increased risk of abnormal reproductive outcomes. There is also a large body of literature on germ cell mutagenesis in rodents showing that treatment of male germ cells with mutagens has dramatic consequences on reproduction, producing effects such as those observed in human epidemiological studies. However, we know very little about the etiology, transmission, and early embryonic consequences of paternally-derived chromosomal abnormalities. The available evidence suggests that: 1) there are distinct patterns of germ cell-stage differences in the sensitivity of induction of transmissible genetic damage, with male postmeiotic cells being the most sensitive; 2) cytogenetic abnormalities at first metaphase after fertilization are critical intermediates between paternal exposure and abnormal reproductive outcomes; and 3) there are maternal susceptibility factors that may have profound effects on the amount of sperm DNA damage that is converted into chromosomal aberrations in the zygote and that directly affect the risk for abnormal reproductive outcomes.     

DNA damage induced by mutagens in plant and human cell nuclei in acellular comet assay

Higher plant cells have a long tradition of use in the studies on environmental mutagenesis in situ, especially in relation to human health risk determination. The studies on the response of plant and human cells to physical and chemical mutagens showed differences in their sensitivity. The differences in the presence of cell components in plants and humans could influence such response. Additionally, the level of the organization of the employed material could influence DNA-damaging effect: leukocytes are isolated cells and plant--an intact organism. To preclude these obstacles, the effects of direct treatment of isolated nuclei with genotoxic agents were determined to compare the sensitivity of plant and human cells. In the present study, we have determined the DNA-damaging effects of two chemical mutagens: maleic acid hydrazide (MH) and N-methyl-N-nitroso-urea (MNU) applied to isolated nuclei of both plant and human cells. In order to compare the sensitivity of the nuclei of Nicotiana tabacum var. xanthi and the nuclei of leukocytes, the acellular Comet assay was carried out. The results showed higher sensitivity of the nuclei of leukocytes as compared to the nuclei of plant cells to mutagenic treatment with the applied doses of MH and MNU.     

Concomitant lethal mutagenesis of human immunodeficiency virus type 1

RNA virus population dynamics are complex, and sophisticated approaches are needed in many cases for therapeutic intervention. One such approach, termed lethal mutagenesis, is directed at targeting the virus population structure for extinction or error catastrophe. Previous studies have demonstrated the concept of this approach with human immunodeficiency virus type 1 (HIV-1) by use of chemical mutagens [i.e., 5-azacytidine (5-AZC)] as well as by host factors with mutagenic properties (i.e., APOBEC3G). In this study, these two unrelated mutagenic agents were used concomitantly to investigate the interplay of these distinct mutagenic mechanisms. Specifically, an HIV-1 was produced from APOBEC3G (A3G)-expressing cells and used to infect permissive target cells treated with 5-AZC. Reduced viral infectivity and increased viral mutagenesis were observed with both the viral mutagen (i.e., G-to-C mutations) and the host restriction factor (i.e., G-to-A mutations); however, when combined, they had complex interactions. Intriguingly, nucleotide sequence analysis revealed that concomitant HIV-1 exposure to both 5-AZC and A3G resulted in an increase in G-to-A viral mutagenesis at the expense of G-to-C mutagenesis. A3G catalytic activity was required for the diminution in G-to-C mutagenesis. Taken together, our findings provide the first demonstration for potentiation of the mutagenic effect of a cytosine analog by A3G expression, resulting in concomitant HIV-1 lethal mutagenesis.     

High-throughput selection of retrovirus producer cell lines leads to markedly improved efficiency of germ line-transmissible insertions in zebra fish

Vesicular stomatitis virus glycoprotein G-pseudotyped mouse retroviral vectors have been used as mutagens for a large-scale insertional mutagenesis screen in the zebra fish. To reproducibly generate high-titer virus stocks, we devised a method for rapidly selecting cell lines that can yield high-titer viruses and isolated a producer cell line that yields virus at a high titer on zebra fish embryos. Virus produced from this line, designated GT virus, is nontoxic following injection of zebra fish blastulae and efficiently infects embryonic cells that give rise to the future germ line. Using GT virus preparations we generated roughly 500,000 germ line-transmissible proviral insertions in a population of 25,000 founder fish in about 2 months. The GT virus contains a gene trap, and trap events can be detected in the offspring of almost every founder fish. We discuss potential applications of this highly efficient method for generating germ line-transmissible insertions in a vertebrate     

Sensitivity of individual Drosophila to the mutagenic action of ethyl methanesulfonate

The genetic effect of some factors is generally evaluated as an average response of all individuals, without taking into account their potential differences. The presence of individual sensitivity in separate Drosophila organisms to the mutagenic influence of ethyl methanesulfonate was shown when analysing recessive sex-linked lethal mutations in germ cells of males. Different sensitivity of separate individuals to mutagens reflects the existence of cryptic genetic variability in Drosophila strains on a large scale. It is advisable to take into account individual sensitivity of organisms to mutagenic factors, when conducting mutation research and studying genetic consequences of biosphere pollution.     

Studies on the induction of histocompatibility gene mutations in germ cells of mice by chemical mutagens and/or virus-inducing compounds

This work continues earlier studies concerning the use of histocompatibility mutations in mammalian germ cells as a mutagenicity test system (H test). The rate of spontaneous H mutations was re-examined using a new basis for the classification of H mutants. This procedure led to very high frequencies of suspected spontaneous H mutants: among C57Bl/6 mice, 6% and among C3H mice, 9%. F₂ hybrids of a cross between these strains revealed 1% suspected H mutants. Using the same procedure, the sensitivity of the H test was examined with the mutagens ethylnitrosourea, benzo[a]pyrene, 2-acetylaminofluorene (2-AAF), with the solvent dimethyl sulfoxide (DMSO) and with the antibacterial nitrofurantoin. It was possible to demonstrate the mutagenic potential of all mutagens tested as well as their specific action on the different stages of male germ cell development. We succeeded in demonstrating the mutagenicity of 2-AAF for the first time in germ cells of a mammal. In contrast to the negative result with benzopyrene (BP) in the specific locus test, BP induced H mutants even at the very low dose of 2 mg/kg. DMSO was found to induce H mutations in spermatogonia. This extraordinary result is possibly due to the virus-inducing properties of this compound. Nitrofurantoin which is often used in treating bacterial infections of the urinary tract in humans showed a very stage-specific action on maturing spermatids. The value of the H test for mutagenicity testing is discussed with respect to its sensitivity and economy. The very high spontaneous frequency of suspected H mutants and the ease of inducing incraased mutant frequencies by mutagens and by DMSO suggest the possibility that the majority of the histoincompatibilities found in the H test are due to induced antigenic gene products of endogenous viruses. This, however, does not interfere with the applicability of the H test for mutagenicity testing, but rather seems to augment its sensitivity to alkylating mutagens as well as mutagens which probably cause frameshift mutations.Other tests for mutations and/or inherited tumor proneness using mouse germ cells can easily be combined with the H test, because the test animal does not have to be killed, thus reducing the cost of the test.     

Mutagenesis and deoxyribonucleotide pool imbalance

Numerous studies have demonstrated that DNA-precursor pool imbalances are mutagenic and can modulate the lethality and mutagenicity of DNA-damaging agents. In addition, physical and chemical mutagens can induce alterations in DNA-precursor levels. Such findings suggest that regulation of intracellular concentrations of DNA precursors may be an important factor in environmental mutagenesis. In this article, results linking mutation and disturbances in DNA-precursor pools are reviewed.     

Genetic damage by bifunctional agents in repair-active pre-meiotic stages of Drosophila males

Most of our understanding of germline mutagenesis in Drosophila is based on the DNA repair-inactive, haploid post-meiotic stages. The diploid, repair-active pre-meiotic stages are more relevant to the situation encountered in somatic cells. DNA mono-adducts induced by agents like methyl methanesulphonate (MMS) and ethylene oxide (EO) are well repaired in the pre-meiotic cell stages, and these agents show therefore, no or considerable lower mutagenic activity in these stages. In contrast, in this study the two bifunctional nitrogen mustards chlorambucil (CAB) and mechlorethamine (MEC) show significantly elevated mutant frequencies of both post- and pre-meiotic germ cells. Results were similar for the X-chromosomal and the autosomal (2nd) recessive lethal (RL) test. CAB and MEC were also active in stem cells, but in comparison with post-stem cell stages they seem to be better protected. The germ cell specific response in post- and pre-meiotic cell stages was for both nitrogen mustards comparable to mutagenic activity patterns observed in the specific locus test in the mouse.It was reported that for diepoxybutane (DEB), another cross-linking agent, the ratio of the RL frequency for the 2nd- and the X-chromosome was increased from 2.1 for post-meiotic stages to 9.5 for pre-meiotic stages. In own experiments aiming to confirm this observation, a high ratio was indeed found. The induction of large deletions by DEB could be the reason for this difference, since such lesions might include both a sex-linked lethal and a vital gene required for the development of spermatocytes into mature sperm. Similar differences were expected for CAB and MEC since they are also inducers of large deletions. But unexpectedly, no differences in 2nd/X RL ratio between post- and pre-meiotic cell stages were found for the nitrogen mustards. Possible causes such as distinct proportions of multi-locus deletions (MLDs), mitotic recombination and the formation of persistent lesions, are discussed.     

Negative effects of chemical mutagenesis on the adaptive behavior of vesicular stomatitis virus.

Changes in adaptability of vesicular stomatitis virus (VSV) upon treatment with chemical mutagens have been investigated. Results showed no improvement in virus viability or adaptability at any given level of mutagenesis. In fact, increasing inhibition of virus production and adaptability was observed with increasing levels of mutagenesis. This was true for all tested VSV variants replicating either in changing or constant host cell environments. Results also showed that mutagen-treated RNA virus populations which had undergone severe fitness declines were able to recover lost fitness completely after several large-population passages in BHK21, cells. The present findings illustrate the highly optimized states of RNA viruses and their potential to adapt readily. These results are significant for the possible development of specific antiviral agents designed to be mutagenic.     

Radiation- and chemically-induced chromosome aberrations in mouse oocytes: a comparison with effects in males.

Data from studies on radiation- and chemically-induced chromosome aberrations in mouse oocytes have been summarized. An attempt has been made to assess the relative sensitivity to mutagenic agents of female and male germ cells through comparison of observations from mutation studies of female and male mice. No unequivocal evidence of a mutagenic effect limited to a single sex could be found in the cytogenetic data, although differences in relative germ cell sensitivity could be inferred for ionizing radiation and some chemicals. However, the pattern of inter-sex variations was not consistent: for example, irradiation of dictyate oocytes yielded a lower rate of heritable chromosome translocations than the same dose to spermatogonia; in contrast, some chemicals, such as mitomycin C, yielded a larger incidence of chromosome anomalies after treatment of dictyate oocytes than spermatogonia. Overall, the limitations in quality and quantity of cytogenetic data, and the uncertainties associated with comparing information obtained in disparate assays, place severe constraints on the use of observations on induced chromosome aberrations to assess the relative sensitivities of female and male germ cells to environmental mutagens.     

Comparative studies of the induction of somatic eye-color mutations in an unstable strain of Drosophila melanogaster by MMS and X-rays at different developmental stages

An unstable white locus in Drosophila melanogaster originally described by Rasmuson and Green (1974) and further by Rasmuson et al. (1978, 1980) contains an IS element. This constellation interacts with the zeste mutation and forms a mutationally unstable system that is sensitive to a variety of mutagens. Mutational shifts between zeste and wild-type eye color as well as deletions and transpositions of the white locus are frequently occurring in the unstable X-chromosome in germ line and in somatic tissue. Germinal mutations from zeste to wild-type eye color are associated with an insertion of a piece of DNA, proximal to the wsp site, and the shifts from red to zeste are caused by an excision of the same piece (Rasmuson, in preparation). Mutations to pigmentless phenotype are interpreted as deletions of the white locus, while they always are irreversible and show non-complementation with wsp. The somatic system can be used as a screening test for potential mutagens, described by Rasmuson et al. (1984). This survey is an attempt to correlate the size of the mutated area of the eyes with the age of the larvae at mutagen treatment. X-Rays and MMS were used to give an indication of the mechanism of the instability, according to the different kinds of DNA damage induced. The results show that the mean size of red spots decreased with increasing age of larvae at treatment, while the mutation frequencies were increased because of the multiplication of the cells in the eye anlage susceptible to the mutagens. This is contradictory to the hypothesis maintained by Fahmy and Fahmy (1980) that the somatic shifts are not mutagenic but epigenetic events, due to altered regulation of the gene expression. Red spots induced with MMS are smaller in size than X-ray-induced red spots, indicating a delay in the establishment of mutations from chemically-induced lesions compared to irradiation damage. White spots on the other hand were equally large in size, irrespective of inducing agent and about twice the size of the chemically-induced red spots, implying a faster and more direct action for fixation of deletions than for the production of MMS induced shifts in eye color from zeste to red.     

piggyBac transposon somatic mutagenesis with an activated reporter and tracker (PB-SMART) for genetic screens in mice

Somatic forward genetic screens have the power to interrogate thousands of genes in a single animal. Retroviral and transposon mutagenesis systems in mice have been designed and deployed in somatic tissues for surveying hematopoietic and solid tumor formation. In the context of cancer, the ability to visually mark mutant cells would present tremendous advantages for identifying tumor formation, monitoring tumor growth over time, and tracking tumor infiltrations and metastases into wild-type tissues. Furthermore, locating mutant clones is a prerequisite for screening and analyzing most other somatic phenotypes. For this purpose, we developed a system using the piggyBac (PB) transposon for somatic mutagenesis with an activated reporter and tracker, called PB-SMART. The PB-SMART mouse genetic screening system can simultaneously induce somatic mutations and mark mutated cells using bioluminescence or fluorescence. The marking of mutant cells enable analyses that are not possible with current somatic mutagenesis systems, such as tracking cell proliferation and tumor growth, detecting tumor cell infiltrations, and reporting tissue mutagenesis levels by a simple ex vivo visual readout. We demonstrate that PB-SMART is highly mutagenic, capable of tumor induction with low copy transposons, which facilitates the mapping and identification of causative insertions. We further integrated a conditional transposase with the PB-SMART system, permitting tissue-specific mutagenesis with a single cross to any available Cre line. Targeting the germline, the system could also be used to conduct F1 screens. With these features, PB-SMART provides an integrated platform for individual investigators to harness the power of somatic mutagenesis and phenotypic screens to decipher the genetic basis of mammalian biology and disease.     

Experimental work on induced mutations

The detection of changes in mutation rate in human populations remains extremely difficult. Thus estimation of genetic hazards of mutagens to man depends on extrapolation from experimental systems. Germ cells of animals show complex variations in sensitivity to mutagenic effects. Some agents predominantly affect stem cells or other immature germ cells, whereas others mainly affect later germ cell stages. Dose-response relations also vary both with the agent and with the stage or sex of germ cell treated. In man, in addition to single-gene defects and chromosome anomalies, conditions of complex or uncertain inheritance, such as congenital malformations, are clinically important. Genetic theory leaves unclear whether the incidence of these would be affected by a change in mutation rate. Recent research has shown that in mice the incidence of malformations is increased by exposure of the parents to mutagens, but the effect is small. Chromosomal non-disjunction is also clinically important. Again, recent research shows that its frequency can be changed by mutagens, but the effects vary with germ-cell stage. Thus, further research is needed to elucidate the relative contributions of different environmental mutagens to human genetic disease.