CHEMICAL MUTAGENESIS

Summary The general methodological requirements for work with chemical mutagens are the same as for general mutation work, with special emphasis on questions of concentration, penetration, possible indirect or delayed effect, differences in susceptibility between individuals, strains and species. In work with weak mutagens the fluctuations of spontaneous mutability should be reduced to a minimum and should be included in the estimate of error. The manner of application of a chemical substance depends on the type of substance tested and the type of organism and tissue to be treated. Various methods are discussed. Drosophila is still the most suitable object for a genetical analysis of mutagenic effects; for complete cytological analysis plants are preferable. Work on mice or other warm‐blooded animals has theoretical as well as practical importance, but offers difficulties to quantitative treatment. Micro‐organisms have great advantages for the detection of mutagens, especially those species in which genetical methods for the testing of suspected mutations can be applied. Results gained with one organism cannot be transposed to another without test, and quantitative comparison between data gained on different organisms or even different cell types of the same organism is not admissible. A number of experiments in which chemical and physical mutagenic agencies were applied in combination have given interesting results which suggest that this method may prove a useful tool in the analysis of mutagenesis. A group of highly toxic vesicants, of which mustard gas is the best known representative, are powerful mutagens. In Drosophila , up to about 24% sex‐linked lethals have been produced with mustard gas, as well as small and large rearrangements. The mustards have proved efficient mutagens also in barley, fungi and bacteria. Two other potent vesicants, lewisite and chloropicrin, gave negative results in mutation tests. So did osmic acid and picric acid, which resemble mustard gas in being fixatives. A lachrymator, mustard oil or allyl wothiocyanate, is a definite, though weak mutagen. Two other lachrymators, chloracetone and dichloracetone, probably are slightly mutagenic. Analysis of the effects of mustard gas and nitrogen mustard has so far produced the following results: in Drosophila , lethals are scattered along the XT‐chromosome in a similar way as X‐ray lethals. Lethals on the second chromosome are 4–5 times as frequent as sex‐linked ones. The frequency of recessive lethals is highest in sperm which becomes available for fertilization about 6 days after treatment. Production of mutations in females is difficult. Doses which increased mutation rate in treated ova did not induce mutations in untreated sperm which, through fertilization, had been introduced into these ova. About 20% of the sex‐linked lethals from treatment of males are caused by small deficiencies. Large rearrangements are less frequent after mustard treatment than after a dose of X‐rays which produces the same frequency of sex‐linked lethals. The shortage of translocations is more pronounced than that of large deletions. One very abnormal type of segregation in the progeny of a treated male points to a centromere effect of the treatment. Treatment of heterozygous embryos results in a great increase in the frequency of somatic crossing‐over. Visible mutations do not seem to differ from those produced by X‐radiation in either relative frequency or type. About 50% of the visible mutations in the progeny of treated males are mosaics. Apparent semi‐lethals were often found to be caused by gonadic mosaicism for a lethal. Visible mosaics occur also in the progeny of treated females. In several cases the same mutation occurred as mosaic in two successive generations derived from a treated male. These observations are most plausibly interpreted by the assumption that mustard gas can produce delayed localized effects. In Tradescantia the cytological effects of mustard gas seem indistinguishable from those produced by X‐rays of low intensity; but data on Vicia suggest that in plant material, as in Drosophila , the two mutagenic agencies may differ in effect. In barley, nitrogen mustard treatment favours the appearance of certain hereditary types; it is not known whether this is due to specific action on certain loci. Phenol was found to increase the rate of visible mutations in Antirrhinum. Applied to excised ovaries of Drosophila it produced high frequencies of autosomal lethals. It is not yet decided whether phenol itself or some contaminant was responsible for the effect. A striking feature of these experiments is that some lethals occurred repeatedly in different ovaries and different series, suggesting a specific action of the treatment. Many phenols, phenol derivatives and related compounds can produce chromosome breaks in Allium roots. Urethane, especially in combination with potassium chloride, induces translocations in Oenothera and other plants, and mutations in Drosophila. In the progeny of mice which for a number of generations had been treated with methylcholanthrene, variants appeared, many of which were shown to be hereditary. Some were identical with known laboratory mutants, others were reversals to wild type, still others were new aberrations. Several hereditary variants were found in a small population of mice after treatment with dibenzanthracene. The frequency of sex‐linked lethals was increased in the progeny of Drosophila males which had been treated with aerosols of various carcinogens. The number of lethals which were connected with rearrangements was strikingly high. Formalin increases mutation rate when mixed with the food of Drosophila , but does not appear to do so when applied as vapour. In bacteria, treatment of the medium with hydrogen peroxide increases the toll of permanent variants in bacteria, although treatment of the bacteria themselves has no such effect. Sulphonamides have been reported to increase mutation rate in Drosophila , the effect being most marked in sperm which was not mature at the time of treatment. Disturbances of the mineral balance in the nutrient medium increases mutation frequency in Antirrhinum; there are indications that the same may apply to Drosophila. Dinitrophenol mixed with the food of Drosophila did not influence mutation rate. Heavy water and proteolytic enzymes failed to produce mutations in Drosophila. Copper sulphate and low pH decreased mutability of an unstable gene in D. virilis. Mutation rate in Neurospora was increased after treatment with serum from rabbits which had been immunized against the fungus; it is not clear whether the antibodies against Neurospora were the effective mutagen. Specific transformations of bacteria have been produced by means of desoxy‐ribonucleic acid from one strain applied to a second; the genetical basis of these transformations is not known. Unspecific permanent variants in bacteria have been produced by a number of substances, carcinogens as well as non‐carcinogens. Theories concerning the mechanism of chemical mutagenesis cannot be more than working hypotheses at the present state of knowledge. It is possible that the mustards and some other compounds may act like X‐rays by transferring energy to loci on the chromosomes. Some of the peculiarities of mustard gas action could be explained if it were assumed that the amount of transferred energy sometimes is sufficient only for the production of an unstable premutation. Other chemical mutagens may act indirectly by influencing metabolism or by disturbing the physicochemical conditions of the cell. It seems likely that chemical mutagens have played a part in evolution, and that their production and the sensitivity of the genotype to their action have been subject to selection.