EFFECTS OF DOSE ON THE INDUCTION OF DOMINANT-LETHAL MUTATIONS WITH TRIETHYLENEMELAMINE IN MALE MICE1

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1 EFFECTS OF DOSE ON THE INDUCTION OF DOMINANT-LETHAL MUTATIONS WITH TRIETHYLENEMELAMINE IN MALE MICE1 B. E. MATTER2 AND W. M. GENEROSO Biology Division, Ouk Ridge National Laboratory, Oak Ridge, Tennessee Manuscript received January 9, 1974 Revised copy received April 1, 1974 ABSTRACT Dose effects of triethylenemelamine (TEM) in the induction of dominantlethal mutations were studied at the early spermatozoon, midspermatid and spermatocyte stages. The pattern of effects on spermatocytes, unlike midspermatids and early spermatozoa, indicated possible cytotoxic damage, so for the determination of TEM dose-response curves in the induction of genetic damage only the data for midspermatids and early spermatozoa were used. The TEM dose-effect curves for those two stages differ markedly from ethyl methanesulfonate (EMS) dose-effect curves. Beginning with the lowest doses at which significant effects are observed, there is a considerably more rapid increase in dominant-lethal effects with dose of EMS than TEM. Another marked difference between the two compounds is in the ratio of the genetically effective dose (as measured by dominant-lethal mutations) to the lethal dose. The ratio is 1: 100 for TEM and only 1:3.5 for EMS; thus, TEM is mutagenic far below its toxic level. Obviously, these results have important implications not only for our understanding of the nature of chemical induction and recovery of chromosomal aberrations but also for the practical problems of evaluating the mutagenic effects of chemicals. ESTING for mutagenic effects of chemicals in mammals usually involves administration of very high doses of the test compounds. This type of treatment often does not represent the human situation, where the exposure may occur at much lower levels. Unfortunately, very little is known about the relative effects of low and high doses of most chemicals or the extent to which dose-effect curves of various chemicals differ. Thus, for a proper evaluation of the genetic hazards of chemicals to man, it is necessary to study the dose effects of a wide variety of mutagenic compounds in laboratory mammals. Certainly it cannot be expected that the shapes of the curves will be the same for all chemicals. The present study was started after the dose effects of the compound ethyl methanesulfonate (EMS) on induction of dominant-lethal mutations and heritable translocations became clear (GENEROSO et al. 1974). The EMS dominantlethal dose-response curve, plotted on the basis of living embryos as a percentage of the control value, deviates clearly from linearity being markedly concave downward. Also, the translocation dose-response curve shows a more rapid increase in the number of translocations with injected dose than would be expected Research jointly sponsored by the National Center for Toxicological Research and by the United States Atomic Energy Cmmissim under contract with the Union Carbide Corporation. Present address: Sandoz Ltd., Biological and Medical Research Division, Basel, Switzerland. Genetics 77: August, 1974.

2 ~ ~~~ ~ 754 B. E. MATTER AND W. M. GENEROSO that level it is questionable whether the dominant-lethal effect is real. At 0.2 mg/kg the dominant-lethal effect was also revealed by increase in dead implantations and reduction in living embryos. In addition, a significant reduction in the average number of total implantations was detected for the first time. At the next higher dose (0.3 mg/kg) the same three end points again showed significant effects. At that dose level the reduction in the frequency of fertile matings was not statistically significant, but there seemed to be an indication of an effect for this end point. Marked effects of TEM on all four end points were found only with doses of 0.4 mg/kg and higher. Thus, for early spermatozoa there appears to be a pattern of effect caused by TEM dose-i.e., the first indication of dominant-lethal effects is shown by an increase in dead implantations, and as the dose increases a progressive increase in dominant-lethal effects is shown by a significant reduction in the number of living embryos, followed by a reduction in the number of total implantations and then by a reduction in the frequency of fertile matings. It is interesting to point out that the dominant-lethal effects of EMS follow the same pattern (GENEROSO et al. 1974). The same situation was found for the midspermatid stage (Table 3). The important difference between the two stages was the fact that in the midspermatid stage appreciable increases in induced dominant-lethals were detectable at a lower dose (0.05 mg/kg as compared to 0.1 mg/kg for early spermatozoa). Results on spermatocytes were grouped into 2-day intervals within the mating period of days after tratinent (Table 4). According to OAKBERG S 1960) cytological timing of spermatocytes, days correspond to diplotene, diakinesis, and metaphase I stages, with some admixture from pachytene; days correspond to pachytene; days to zygotene and leptotene; and days to early leptotene and preleptotene spermatocytes. In all mating intervals during the mating period of days, the lowest doses TABLE 3 Dose effect of TEM in the induction of dominant-lethal mutations in mouse spermatids* Total Living Living embryos as penent of controls Implants embryos No of No of among fertile among fertile Dead Among Among all Dose mated pregnant females females implants fertile mated Treatment (mghg) females females (avg) (avg) (Percent) females females Control (HBSS) loo TEM TEM t TEM TEM M TEM f TEM TEM o * All matings occurred between 11.5 and 15.5 days after treatment. p < 0.05 us. controls

3 TEM DOSE EFFECTS ON DOMINANT LETHALS 755 Dominant-lethal mutations may be indicated by any ol the following criteria: reduction in the average number of living embryos, increase in the frequency of dead implantations, reduction in the average number of total implantations, or reduction in the frequency of fertile matings. The dominant-lethal effects in the experimental groups were calculated from the average number of live embryos obtained, either from all mated females or from pregnant females only, as percentage of control. Tests of significance for differences in the average numbers of implantation sites and live embryos per female were made with DUNNETT S (1955) method for comparing a control group with each treated group. RESULTS The relative sensitivities of the different postspermatogonial stages to dominant-lethal induction with TEM were studied using the dose of 0.2 mg/kg. This dose was chosen on the basis of earlier results with mice (CATTANACH 1959). Days , which correspond to the midspermatid stage, proved to be the most sensitive period, followed by days , which correspond to most of the spermatozoon stages and the late spermatid stage. There was no apparent induction of dominant-lethal mutations at this dose in the meiotic stages studied (days ). For dose-effect studies, matings were restricted to days , and The first two periods were selected so that the doseeffect curves for spermatozoa and spermatids could be compared, and the third was included so that any effects of higher doses of TEM on meiotic stages could be detected and compared with the effects on the most sensitive stages. Effects of TEM dose on dominant-lethal induction in early spermatozoon, midspermatid, and spermatocyte stages are shown in Tables 2-4. In the early spermatozoon stage (Table 2) appreciable increases in induced dominant-lethal mutations were detectable beginning with the 0.1 mg/kg dose. At that dose, the dominant-lethal effect was detectable in terms of an obvious increase in dead implantations, accompanied by a significant reduction in living embryos. It is possible that an increase in dominant lethals was also induced with the dose of 0.05 mg/kg, as indicated by a slight increase in dead implants, but at TABLE 2 Dose effect of TEM in the induction of dominant-lethal mutations in mouse spermdoma* Living embryos Total Living as percent of controls implants embryos No. of No. of among fertile among fertile Dead Among Among all Dose mated pregnant females females implants fertile mated Treatment (mg/kg) females females (avg) (avg) (Percent) females females Control (HBSS) TEM TEM i TEM t TEM t TEM 0.3 M TEM t TEM * All matings occurred between 4.5 and 7.5 days after treatment. p < 0.05 us. controls.

4 756 B. E. MATTER AND W. M. GENEROSO on the basis of dose-square kinetics. Thus, for both end points the effectiveness of EMS is proportionally much lower at low than at high doses. The compound triethylenemelamine (TEM) was selected for comparison with EMS because in many respects its effects in the mouse are similar to those of radiatioa. Furthermore, TEM has been shown to induce dominant-lethal mutations (CATTANACH and EDWARDS 1958) and translocations (CATTANACH 1957,1959) in post-spermatogonial stages in the mouse at doses that are low relative to the approximate lethal dose. Thus, there were previous indications that TEM and EMS might differ markedly in their dose-effect curves and in their relative effectiveness in inducing genetic damage at low doses. MATERIALS AND METHODS Before studying the dose effects of TEM (Lederle Laboratories, Pearl River, N. Y.), we undertook two preliminary experiments. First, we determined the toxicity of TEM by treating male mice (10-12 weeks old) with doses ranging from 2.0 to 10.0 mg/kg and observing them for survival for a period of 30 days (Table 1). Second, the pattern of sensitivity to induction of dominant-lethal mutations in post-spermatogonial stages was determined. This we did by injecting TEM at 0.2 mg/kg into 40 (101 x C3H)F, inales and mating them serially with (101 X C3H)F, females (about 12 weeks old) for a period of 31 days. Matings on days , , and represented germ cells exposed to TEM as spermatozoa, spermatids and primary spermatocytes, respectively (OAKBERG 1960). The next series of experiments was on dose effects and was based on the results of the two preliminary studies. TEM doses ranging from to 4.0 mg/kg were used. Control males received 1 ml of Hanks balanced salt solution (HBSS). Dose effects of TEM in inducing dominant-lethal mutations were studied in early spermatozoa, midspermatids, and primary spermatocytes (mating periods , , and days after treatment, respectively). Each dose was prepared in HBSS and administered intraperitoneally in a single injection. The amount of TEM injected was corrected for the body weight of each mouse. The maximum volume of TEM solution injected was 1 ml. Control mice received 1 ml of HBSS. Experimental and control males were mated to (C3H x C57BL)F, virgin females (7-14 weeks old). In a small study to determine the most suitable age of the females of this strain for use in dominant-lethal experiments, it was found that the numbers of total implantations and living and dead embryos and the frequency of plugged females that became pregnant did not differ appreciably between the ages of 5 and 24 weeks. On the other hand, females that were 28 weeks or older appeared to be less suitable for dominant-lethal studies, primarily because of the higher frequency of dead implantations. Matings were detected by daily examination of females for vaginal plugs. Mated females were immediately replaced with fresh ones, and this procedure was continued up to the day before the last day of each mating period. Mated females were killed for uterine analysis days after the plug was found. TABLE 1 Mortality of TEM-treated (101 x C3H)F, male mice 30 days after treatment Dose No. of No. of Percent (mg/kd males injected dead animals mortality ) la

5 TEM DOSE EFFECTS ON DOMINANT LETHALS 75 7 TABLE 4 Effects of TEM on mouse spermatocytes Total Living Stage No. of No. of implants embryos Percent (days after Dose mated pregnant per fertile per fertile dead Grouv treatment) (maha) females females females females implants Control I I1 I11 IV V VI * p < 0.05 us. controls ' ' * * * 3 5' ' 5.3* ' 6.0* * 6.4* o * ' * ' 1.7 I that induced detectable effects were much higher than the lowest doses that induced detectable effects in either spermatids or spermatozoa. Two other interesting points about the fertility effects of TEM in spermatocytes were noted. First, with the exception of the preleptotene stage there was an increasing degree of sensitivity to the fertility effects of TEM as meiosis progressed. For instance, the lowest dose that induced a significant effect on matings at days was 0.8 mg/kg, whereas in matings at intervals and days the doses were 1.6 and 2.0 mg/kg, respectively. The effects of TEM on fertility were generally the same for the three intervals within days. Second, the amount of reduction in the number of living embryos that could be attributed to death during implantation is markedly lower in the interval days than in the interval The relationship between total implants, living, and dead embryos on days was generally similar to that in spermatozoa and spermatids. Thus, there is no doubt that the TEM effect on matings in this interval was due primarily to induced dominant lethality. On the

6 758 B. E. MATTER AND W. M. GENEROSO other hand, in earlier meiotic stages, although dominant-lethal effects may also be apparent, the major cause for the reduction in the numbers of total implants 2nd living embryos and for the increase in the frequency of sterile matings may be different. At these stages the primary fertility effects of TEM may be attributable to dominant lethality expressed prior to implantation, or to cytotoxicity of the chemical to spermatocytes, or to a combination of the two. Experiments to study this problem have been initiated and will be reported elsewhere. It is interesting to note, in this regard, that preleptotene spermatocytes have the lowest X-ray LD,, of all spermatocyte stages (OAKBERG and DIMINNO 1960). Dominant-lethal effects induced in early spermatozoa and midspermatids by different TEM doses were calculated from the average number of living embryos as a percentage of control (columns 8 and 9, Tables 2 and 3). For the purposes of plotting the dose-effect curves, it was necessary to calculate the average number of living embryos in two different ways-on the basis of fertile matings only, or on the basis of all mated females (fertile and sterile). Dominant-lethal effects calculated from fertile matings only are underestimates for high doses, since matings in which all embryos are lost before implantation are omitted, and it has been shown that TEM-induced pre-implantation embryonic death, which leads to reduced numbers of live embryos and a high frequency of sterile matings in 01, \ I I I TEM (mg/kg) DOSE FIGURE l.--effects of TEM dose on induction of dominant-lethal mutations in mouse spermatazoa and spermatids. The curves represent least-squares fits of the numbers of living embryos us. the doses. Experimental points are taken from Tables 2 and 3, columns 8 and 9; 95% confidence intervals are indicated by vertical bars.

7 TEM DOSE EFFECTS O N DOMINANT LETHALS 759 these two spermatogenic stages, is attributable to genetic causes ( CATTANACH and EDWARDS 1958). The calculation based on the total number of matings (fertile and sterile) generally gave higher dominant-lethal effects in the upper dose range. The true dominant-lethal effects for the upper dose range, therefore, may be closer to the values calculated on the basis of all mated females, whereas for the lower dose range the calculation based on fertile matings is a more accurate measure of dominant lethals. Hence, in describing the dose-response relationships, values in column 8 of Tables 2 and 3 were used when the frequency of fertile matings was not significantly lower than control; otherwise, values in column 9 were used. For the dose-effect curves (Figure 1) values in column 8 were used, except for doses of 0.4 mg/kg and 0.3 mg/kg for spermatozoa and spermatids, respectively; higher doses were not included, as the saturation point appeared to have been reached already. The dose-effect curves were tested for linearity by the method of least squares. The slope parameter in the model is P, = bd,, where Pi is the average number of living embryos at dose Di, divided by the average number of living embryos in the control group times 100. For both spermatozoa and spermatids the deviations from this model were significant (p < 0.01 in both cases), indicating that the linear model gave a poor fit. The TEM dose-effect curve plotted by BATEMAN (1960) for the rat is generally similar to those of the mouse, but whether this is the true situation is debatable since the rat dose-effect curve was drawn using pooled data from the first 4 weeks of mating. DISCUSSION CATTANACH and EDWARDS (1958) and CATTANACH (1959) have already described the effects of TEM on the fertility of male mice. They found that spermatids are more susceptible to the induction of dominant-lethal mutations than are spermatozoa, and by the use of artificial insemination they showed that embryonic mortality results from direct damage to the germ cells and not from damage to the eggs at fertilization as a result of accumulation of the drug in the seminal plasma. Differences in fertility levels after TEM treatment thus reflect the differential sensitivities of the post-spermatogonial stages to the induction of dominant-lethal damage by the drug. The results presented here generally agree with those earlier data. Damage induced by lower doses leads mainly to postimplantation embryonic loss, and as the dose increases the number of implantations also decreases, presumably due to pre-implantation embryonic loss. At high doses, presumed pre-implantation embryonic loss apparently leads to matings that are classified as sterile. The effects on fertility induced by relatively high doses of TEM to spermatocytes (with the exception of late primary spermatocytes) are probably not entirely due to dominant lethality. The frequency of fertile matings is reduced, as are the average numbers of living and total implantations, but the frequency of dead implantations is only slightly increased. This pattern of effects is markedly different from that found in spermatozoa and spermatids. From the dose-response studies two interesting comparisons, which have basic as well as practical significance, can be made between TEM and EMS. First,

8 760 B. E. MATTER AND W. M. GENEROSO there is a marked difference between the shapes of the dose-response curves for EMS and TEM (Figure 2). (The EMS [GENEROSO et al and X-ray [EHLING dose-effect curves shown in Figure 2 were drawn along with the TEM spermatid curve, using as the common point of reference doses which induced 50% reduction from control level in the average number of living embryos. The TEM curve for spermatozoa was not included because, when drawn on the basis of 50% survival, the curves for spermatozoa and spermatids were superimposeable.) Beginning with the dose points at which a significant effect was first observed, there is a considerably more rapid increase in dominant-lethal effects with dose of EMS than of TEM. For both spermatozoa and spermatids there is a slight shoulder at the lowest dose levels of TEM, but this shoulder is not as pronounced as that observed for EMS. It may be mentioned again that in both spermatids and spermatozoa there are no significant reductions in the number of living embryos at the lowest doses. It is interesting to note that, if one considers only the doses at which significant effects were observed, the TEM and X-ray dose-effect curves are generally alike. It is conceivable that lower doses of X-rays would produce a shoulder similar to that seen for TEM. X-RAY DOSE ( R) \ POSTTREATMENT - TEM EMS \ 0', I I I TEM DOSE (mg/ kg I I I EMS DOSE (mg/kg) FIGURE 2.-Comparison of TEM, EMS, and X-ray dose-effect curves for the induction of dominant-lethal mutations in mice. The X-ray curve was drawn from data of EHLING (1971) and the EMS curve from data of GENEROSO et al. (1974). A 501% effect was used as the reference point.

9 TEM DOSE EFFECTS ON DOMINANT LETHALS 761 At present there is very little information available to help explain the large difference in the shapes of the dose-effect curves for EMS and TEM. This is a very important fundamental problem, since it touches on the basis mechanisms involved in the production of chromosome breaks by chemicals. Several questions can be raised in this regard, but the most important ones pertain to dosimetry and mechanism differences. Thus, it is essential to determine (a) the rates of alkylation of chromosomal materials as a function of injected EMS and TEM doses, (b) the molecular consequences of EMS and TEM alkylations that are important in the production of chromosome breaks, and (c) whether there are factors that affect the transmission of the molecular lesions through the germ cells. Studies with tritium-labeled EMS have shown that at the dose range ( mg/kg) used in our EMS genetic study (GENEROSO et ae. 1974) the degree of ethylation of mouse sperm heads or sperm DNA increases only slightly faster with dose than would be expected for a linear relationship (G. A. SEGA, R. B. CUMMING and M. F. WALTON, in preparation). Although the chromosomal consequences of DNA ethylation are not well understood, it seems clear that the relatively much lower chromosomal aberration effect at low EMS doses is not due to the inability of EMS to reach the germ cells. Similar information is not available for TEM. Another interesting difference between EMS and TEM that has practical importance is revealed by a comparison based on the ratio between the genetically effective dose (as measured by dominant lethals) and the lethal dose. For EMS, the lowest dose that kills 100% of the animals is approximately 525 mg/kg (EHLING, CUMMING and MALLING 1968; CUMMING and WALTON 1973) ; for TEM this dose is 5 mg/kg (Table 1). The lowest genetically effective doses for EMS and TEM are 150 and 0.05 mg/kg, respectively, which yield comparable frequencies of dominant-lethal mutations. Thus, the ratio of genetically effective dose to lethal dose for TEM is 1 : 100, whereas for EMS it is only 1 : 3.5. If the two compounds are compared on the basis of the ratio between the dose that yields approximately 90 % dominant-lethal mutations (saturation point) and the lowest effective dose, a large difference still exists; the ratios are 1: 1.7 for EMS and 1 : 6 and 1 :4 for TEM in spermatids and spermatozoa, respectively. The ratios change when translocations are used as the genetic end point, since genetic damage can be detected at lower doses. A comparative dose-response study on the induction of reciprocal translations with TEM is currently in progress. In any case, it is already clear that TEM, in contrast to EMS, is mutagenic far below the toxic level. Obviously, these results have important implications for the practical problems of evaluating mutagenic effects of chemicals. The teratogenic effects of various doses of TEM have been studied by JURAND (1959), who found that relatively high doses had to be administered in female mice of two different strains before any retardation of fetal development or teratological effects could be detected. For instance, a dose of 0.4 mg/kg given daily for 3 consecutive days (total of 1.2 mg/kg), beginning on either the 5th or 7th day of pregnancy, did not induce any detectable effects on the fetuses. In contrast, only 0.05 mg/kg was required for a detectable dominant-lethal effect. Although the strains used in that teratogenicity study were not the same as the one used

10 762 B. E. MATTER AND W. M. GENEROSO in the present dominant-lethal study, the results indicate a marked difference in sensitivity between genetic and teratological endpoints in educing biological damage by TEM. It is not yet clear whether the human population is indeed at risk to the possible genetic hazards of chemicals that are either already in the environment or still to be introduced. The present results strengthen the argument that chemicals in the human environment constitute a potential genetic hazard. The types of genetic damage referred to are chromosome breakage and all classes of aberrations that may arise from initial breaks. The most important findings in this regard are (a) that TEM is mutagenic at very low dose levels and (b) that postspermatogonial stages are uniquely very sensitive to mutagenic chemicals. On the first point, it may be recalled that TEM is mutagenic, as indicated by dominantlethal mutations, at doses as low as 1/100 of the lethal dose. Our early results with reciprocal translocations indicate clearly that "EM is mutagenic even at doses as low as 1/400 of the lethal dose. Even though alkylating chemicals like TEM are considered as a unique class of compounds in terms of their mutagenic potential, it cannot be overlooked that human exposure to other chemicals can often be at relatively much &her doses and that some of these chemicals may be transformed in vivo into mutagenic forms. Thus, it is not unreasonable to think that the dose levels of the activated forms may be large enough to constitute a hazard. On the second point, it is now clear that, in addition to the spermatogonial stem cells, the post-spermatogonial stages are also very important as far as chemical hazard is concerned, because of their high sensitivity to mutagenic chemicals and because of the nature of human exposure to chemicals, which is generally either continuous or repetitive. The senior author wishes to thank DRS. W. L. RUSSELL and H. I. ADLER for the laboratory facilities and generous hospitality extended to him during his stay in the Biology Division of Oak Ridge National Laboratory. He was supported by a Sandoz Ltd., Basel, Switzerland, postdoctoral fellowship. We would like to thank DR. J. BEAUCHAMP and DR. D. G. GOSSLEE of Computer Sciences Division for the statistical analysis, DRS. E. F. OAKBERG and R. F. KIMBALL for reviewing the manuscript, and DR. IRA RINGLER of Lederle Laboratories for supplying US with TEM. LITERATURE CITED BATEMAN, A. J., 1960 Induction of dominant-lethal mutations in rats and mice with triethylenemelamine (TEM). Genet. Res. 1: CATTANACH, B. M., 1957 Induction of translocations in mice by triethylenemelamine. Nature 180: 134& , 1959 The sensitivity of the mouse testis to the mutagenic action of triethylenemelamine. A. Vererbungsl. 90 : 1-6. CATTANACH, B. M. and R. G. EDWARDS, 1958 The effects of triethylenemelamine on the fertility of male mice. Proc. Roy. Soc. Edinburgh Sect. B 67: CUMMING, R. B. and MARVA WALTON, 1973 Modification of the acute toxicity of mutagenic and carcinogenic chemicals in the mouse by prefeeding with antioxidants. Food Cosmet. Toxicol. 11: DUNNETT, C. W., 1955 A multiple comparison procedure for comparing several treatments with control. J. Am. Statist. Ass. 50: EHLING, U. H., 1971 Comparison of radiation- and chemically-induced dominant lethal mutations in male mice. Mutation Res. 11 :

11 TEM DOSE EFFECTS O N DOMINANT LETHALS 763 EHLING, U. H., R. B. CUMMING and H. V. MALLING, 1968 Induction of dominant lethal mutations by alkylating agents in male mice. Mutation Res. 5: GENEROSO, W. M., W. L. RUSSELL, S. W. HUFF, S. K. STOUT and D. G. GOSSLEE, 1974 Effect of dose on the induction of chromosome aberrations with ethylmethanesulfonate (EMS) in male mice. Genetics 77: -. JURAND, A., 1959 Action of triethanomelamine (TEM) on early and later stages of mouse embryos. J. Embryol. Exptl. Morphol. 7 : OAKBERG, E. F., 1960 Irradiation damage to animals and its effect on their reproductive capacity. J. Dairy Sci. (Suppl.) 43: OAKBERG, E. F. and R. L. DIMINNO, 1960 X-ray sensitivity of primary spermatocytes of the mouse. Intern. J. Radiation Biol. 2: Corresponding editor: E. H. Y. CHU