A balanced translocation in mice with a neurological defect.

A semisterile male translocation heterozygote [t(2; 14) 1Gso] that exhibited neurological symptoms and an inability to swim (diver) was found among the offspring of male mice treated with triethylenemelamine. All breeding and cytogenetic data showed a complete concordance between translocation heterozygosity and the neurological disorders. Homozygosity for the translocation seemed to be lethal at an early embryonic stage. Despite the distinctive neurologic symptoms, no anatomic or histological defects in either the ear or in the central nervous system were observed. Thus, a balanced chromosomal translocation can produce disease with an inheritance pattern that mimics a single dominant gene defect.

Appearance of virulent bacteriophages in lysogenic E. coli cultures after prolonged growth in the presence of triethylenemelamine.

Continuous culture of coli 12lambda, P22, 600-434, 600-21, and 600-299 in the presence of triethylenemelamine (TEM) results in the appearance of a new virulent virus which attacks the parent culture. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) is effective with 600-21 and ultraviolet light with 12lambda and 600-21. The cultures which produce the virulent virus continue to do so indefinitely in the absence of the mutagen, but are not lysogenic for the virus. Most of the cells in such cultures are resistant to the virus and do not produce any, but there are a few mutant cells sensitive to the virus and the virus multiplies by infection of these sensitive mutants.

Effects of dose on the induction of dominant-lethal mutations with triethylenemelamine in male mice.

Dose effects of triethylenemelamine (TEM) in the induction of dominant-lethal 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.

Delayed formation of chromosome aberrations in mouse pachytene spermatocytes treated with triethylenemelamine (TEM).

Induction of chromosome aberrations in pachytene spermatocytes of mice by 2 mg/kg TEM was compared with induction by 400 R X rays. These doses induced comparably high dominant lethal effects in pachytene spermatocytes of mice. Cytological analysis at diakinesis-metaphase I stage showed that whereas 76.4% of the cells treated with X rays at pachytene stage had aberrations, the frequencies observed in two TEM experiments were only 0.8 and 2.2%. On the other hand, 5% of the progeny from TEM-treated pachytene spermatocytes were found to be translocation heterozygotes. This is the first report on the recovery of heritable translocations from treated spermatocytes of mice. The aberration frequencies observed for TEM in diakinesis-metaphase I were much too low to account for all the lethal mutations and heritable translocations. Thus, the formation of the bulk of aberrations induced by TEM in pachytene spermatocytes was delayed--a marked contrast to the more immediate formation of X-ray-induced aberrations. It is postulated that the formation of the bulk of TEM-induced aberrations in pachytene spermatocytes and in certain postmeiotic stages occurs sometime during spermiogenesis, and not through the operation of postfertilization pronuclear DNA synthesis.

The production of chromosome aberrations in various mammalian cells by triethylenemelamine.

The cytogenetic effects of triethylenemelamine (TEM) were studied using five different mammalian tissues. Treatments of 0.1 and 0.2 mg/kg TEM on differentiating mouse spermatogonia and bone marrow cells showed no significant differences in the frequency of chromosomal aberrations produced in these two tissues. At higher doses, however, the sensitivites of the two tissues appear to be different. The frequency of aberrations varies with time after treatment, with the greatest amount occurring at the latter fixation times. Results of an experiment on primary spermatocytes indicated a correlation between the frequency of chromosome aberrations and DNA replication. Human peripheral leukocytes were utilized in an attempt to clarify the cell-stage specificity of TEM-induced chromosome aberrations. Cultures were treated with TEM prior to PHA stimulation (G0), as well as various time intervals after stimulation (late G,1 S, and G2). The most sensitive stages of the cell cycle to aberration induction were later G1 and S, with chromatid aberrations the predominant type. A very low yield of chromosome damage was observed with the G0 and G1 treated stages. The experiments described tend to support the view that TEM is most effective at inducing aberrations when an intervening round of DNA replication has occurred.

Nineteen paracentric chromosomal inversions in mice.

Using mutagens on sperm and spermatids we have produced nineteen chromosomal inversions in mice. The levels of radiation and chemical mutagen we used induced inversions in about one per cent of the animals screened. Nine inversions have been transmitted through successive generations, and the particular frequency of first meiotic anaphase bridges manifested by each inversion remained constant. The cytological properties of first meiotic anaphases varied considerably among the inversions. Chromosomal locations of five inversions are known. Four of the five are fully viable in homozygous condition.

Relative rates at which dominant-lethal mutations and heritable translocations are induced by alkylating chemicals in postmeiotic male germ cells of mice.

There is a close relationship between the rates at which dominant lethal mutations and heritable translocations are induced by ethyl methanesulfonate (EMS) or triethylenemelamine (TEM) in male postmeiotic germ cells. This relationship does not hold for isopropyl methanesulfonate (IMS), which induced only negligible frequencies of heritable translocations at doses that induced high levels of dominant lethal mutations. Nor does IMS behave like EMS and TEM in the degree to which eggs of different stocks of females repair premutational lesions that are carried in the sperm-large differences between stocks for IMS treatment and small differences for EMS or TEM treatment. These dissimilarities between IMS and the other two alkylating chemicals are postulated to be attributable to differences in the types of lesions present at the time of repair activity and to whether or not chromosomal aberrations are already fixed prior to postfertilization pronuclear DNA synthesis.

Difference in the ratio of dominant-lethal mutations to heritable translocations produced in mouse spermatids and fully mature sperm after treatment with triethylenemelamine (TEM).

The relative induction of dominant-lethal mutations and heritable translocations in triethylenemelamine-treated postmeiotic germ cells of mice was determined depending on the stage treated. Males were mated either 11.5-14.5 days after treatment (middle spermatids) or less than 2.5 hours after treatment (fully mature sperm). Results clearly showed that, even through similar levels of dominant-lethal mutations were induced in fully mature sperm and in middle spermatids, the frequency of heritable translocations induced in mature sperm was markedly lower than that induced in middle spermatids. This observation was used, together with earlier ones, to suggest a mechanism by which dominant-lethal mutations and heritable translocations are produced following chemical treatment of male postmeiotic germ cels.

Long-term effects of triethylenemelamine exposure on mouse testis cells and sperm chromatin structure assayed by flow cytometry.

The toxic and potentially mutagenic actions of triethylenemelamine (TEM) on mouse body and testis weights, testicular cell kinetics, sperm production, sperm head morphology, and sperm chromatin structure were assessed in two experiments. The first experiment examined effects of four dose levels of TEM, assayed 1, 4, and 10 wk after toxic exposure. In the second study, effects from five dosage levels were measured at 1, 4, and 10 wk, and the highest dosage level was evaluated over 44 wk. TEM produced an expected dose related loss of spermatogenic activity and subsequent recovery as determined by dual-parameter (DNA, RNA) flow cytometry (FCM) measurements of testicular cells. Both testicular weights and caudal sperm reserves remained generally below controls after 44 wk recovery following exposure to the highest (1.0 mg/kg daily x 5) dosage. Chromatin structure alterations, defined as increased susceptibility to DNA denaturation in situ, and sperm head morphology were highly correlated (.87-.93, P less than .001) with dose and with each other. Data obtained from the sperm chromatin structure essay (SCSA) on fresh sperm was highly correlated with measurements of aliquots of the same sample collected over 44 wk, frozen, and then measured on the same day. Sperm head morphology and sperm chromatin structure remained abnormal at 44 wk for the 1.0 mg/kg TEM dosage, suggesting that the abnormalities, present long after the initial toxic response, may be a result of mutation. This study demonstrates that flow cytometry provides a unique, rapid, and efficient means to measure effects of reproductive toxins and potential mutagens.

A collaborative cytogenetics study to measure and minimize interlaboratory variation.

Six cytogenetics laboratories joined in a collaborative study of rat chromosome aberrations to measure interlaboratory variation in results of standardized procedures and to devise methods to minimize interlaboratory differences. A preliminary workshop was held to resolve scoring differences, to develop a joint protocol and common glossary, and to reach agreement on uniform reporting methods. Osborne-Mendel rats from a common source were sent to each laboratory. Triethylenemelamine (TEM) was used at doses of 100, 200, 300 and 400 microgram/kg to induce clastogenic effects; results were compared to those of a control group of untreated animals. Femoral bone marrow cells were evaluated with the scorers unaware of the dosage. Final results showed highly significant dose effects with the test compound, and most laboratories showed a similar pattern of dose response. This study illustrates that rat cytogenetic analysis can be an effective test system for evaluation of a compound for mutagenic potential, particularly for the index reflecting the proportion of abnormal cells, but that results should be interpreted cautiously when arbitrary values are assigned for some of the categories being analyzed, as was done in this project for the category of severely damaged cells.

A comparison of diameters of micronuclei induced by clastogens and by spindle poisons.

To compare the size of micronuclei induced by clastogens and by spindle poisons, bone-marrow smears were prepared 24 h after a single intraperitoneal injection of triethylenemelamine (TEM) (0.3 mg/kg) or vincristine (VCR) (0.125 mg/kg) into male mice. 100 micronucleated erythrocytes (MNEs) were randomly selected from each group and photomicrographed. Diameters of the cytoplasm (D) and the micronucleus (d) of each MNE were measured on the photographs. Relatively large micronuclei (d greater than or equal to D/4) were frequent (74%) in the VCR-treated group, and infrequent (2%) in the TEM-treated group. The frequencies of MNEs resulting in d greater than or equal to D/4 were determined for several other mutagens. All clastogens tested could be classified as TEM type, and all spindle poisons as VCR type. These findings indicate that it is possible to analyze the action site of micronucleus-inducing agents on the basis of the relative sizes of micronuclei.

Chromosome aberrations and dominant lethality of mouse embryos after paternal treatment with triethylenemelamine.

Adult male mice were injected intraperitoneally with triethylenemelamine (TEM) at 0.3 mg/kg. These males were mated with untreated females twice a week during 24 days after the treatment. On day 3 of gestation, embryos were flushed out from uteri and examined cytologically as well as cytogenetically. A dominant-lethal test was conducted using the male treated in the same way. Paternal treatment of TEM caused developmental retardation of the embryos obtained from the matings on 20 post-injection days. These embryos frequently showed micronuclei in interphase cells and structural chromosome aberrations in methaphases. Most of these aberrations were chromosome types, such as breaks and exchanges, and premature chromosome condensations. The developmental retardation as well as the frequency of chromosome aberrations was most marked in the matings on post-injection day 13. Similarly, the highest dominant-lethal effect was shown in the group mated on post-injection days 11--13. It was concluded that TEM induces chromosome damage in the post-meiotic germ cells and then this damage in turn produces chromosome aberrations in the embryos, resulting in high incidence of dominant lethality.

The influence of multiple scorers on cytogenetics study results.

Cytogenetics data resulting from one laboratory in a multiple-laboratory study were analyzed to determine if 5 well-trained scorers produced significantly different results using metaphase scoring procedures. Although the scorers reached the same general conclusion, results show that scorer differences exists (p less than 0.01). Consequently, all participating scorers in a laboratory should be used equally in all treatment groups and the results should be analyzed accordingly to account for scorer variations. This is easily accomplished in controlled prospective experiments; however, it is often difficult in retrospective studies using data which exists. In such studies, every effort should be made to analyze and interpret the data so that scorer differences are taken into account. For severely damaged cells not only were there scorer differences but the difference were greater at higher doses. This phenomenon may be related to the operational definition of a severely damaged cell, since scorers who identify more damage than other scorers would logically tend to classify more cells as severely damaged both overall and at lower doses.

Dominant lethal effects of triethylenemelamine in the guppy Poecilia reticulata.

The need to screen potential chemical pollutants for mutagenicity has increased with the increasing volume of such materials being introduced into natural water. The present study demonstrates the utility of a fish, the guppy (Poecilia reticulata), as a model test system in which water-borne chemical mutagens may be assayed for dominant lethal effects. Mature male guppies were injected with three doses of triethylenemelamine (0.1, 0.2 and 0.4 mg/kg) in addition to a control sham treatment. Each male was subsequently mated to virgin females. In an alternative test, male fish were allowed to swim in triethylenemelamine solutions of known concentration for a period of 24 h prior to being mated to virgin female guppies. 10 days following matings, females were dissected, and numbers of live and dead embryos were recorded. Significant dose effects were demonstrated by analysis of variance techniques in both the injection and the emersion tests with the results showing higher percentages of dead embryos and lower total number of embryos with increasing doses of TEM.

Effects of novobiocin on heat shock protection against chromatid aberration induction by triethylenemelamine (TEM) and maleic hydrazide (MH) in Vicia faba.

Heat shock (10 min 40 degrees C) prior to challenge treatment with triethylenemelamine (TEM) or maleic hydrazide (MH) significantly reduced the frequency of induced chromatid aberrations in Vicia faba main root meristems. Novobiocin treatment before heat shock did not prevent heat shock protection against both clastogens; novobiocin application after heat shock prevented protective effects. These results and those obtained earlier for heat shock protection against X-ray challenge are used to discuss possible causes underlying the protective effects triggered by heat shock.

Sister-chromatid exchanges induced by triethylenemelamine: in vivo and in vivo/in vitro studies in mouse and Chinese hamster bone marrow and spleen cells.

This study was designed to obtain sister-chromatid exchange (SCE) frequencies in bone marrow and spleen cells of mice and Chinese hamsters under in vivo and in vivo/in vitro systems following treatment of animals with varying doses (15-405 micrograms/kg) of triethylenemelamine (TEM). A dose-related SCE response was found in both species, tissues, and systems analyzed following TEM treatment. In vivo, similar responses were noted for both tissues in both species. However, in vivo/in vitro, the response was lower than in vivo and it varied with the tissue. The spleen cells were more sensitive and gave higher numbers of SCEs than bone marrow of both species at the two highest doses tested (135 and 405 micrograms/kg). These differences may be attributed to cell-culturing effects, type of cells analyzed, species and tissue specificities, and pharmacokinetic properties of the chemical. This study lends support to recently established in vivo/in vitro cell culture methodologies employing mice and Chinese hamsters for comparative cytogenetic analysis.

Heat shock protection against induction of chromatid aberrations is dependent on the time span between heat shock and clastogen treatment of Vicia faba root tip meristem cells.

Variation of the time span between heat shock (hs) and clastogen treatment of V. faba root tip meristems showed that hs protection is a very quick response (effective after less than 10 min) and lasting for up to 240 min in the case of induction of chromatid aberrations by maleic hydrazide (MH). Analogous protective responses are significantly slower and shorter when TEM is used for aberration induction. This, together with absence of 'clastogenic cross-adaptation' to these agents and differential effects of benzamide (BA, an inhibitor of poly-ADP-ribosylation) pretreatment before hs on hs protection, suggests that hs before clastogen treatment triggers at least 2 clastogen-specific, protective functions which eventually result in protection against these 2 clastogens.

Reproductive capacity and dominant lethal mutations in female guinea-pigs and Djungarian hamsters following X-rays or chemical mutagens.

The reproductive capacity and induction of dominant lethal mutations in adult female guinea-pigs and Djungarian hamsters were tested following treatment with 400 rad X-rays, 1.6 mg/kg triethylenemelamine (TEM) or 75 mg/kg isopropylmethanesulphonate (IPMS). A fairly high level of dominant lethals were observed in female guinea-pigs mated at the first oestrus after irradiation (23.4 +/- 6.4%) with a lower yield at 3 months (9.6 +/- 8.2%). Neither of the chemicals caused any significant induction of dominant lethals at either mating time. In the reproductive capacity experiments, the mean litter size of irradiated female guinea-pigs was reduced for about 12 months and this was especially marked in the first 6 months following treatment. Neither of the chemicals caused any significant differences in early litter sizes but there was a noticeable reduction in the litter sizes of TEM-treated females in the 18--24 month interval. With Djungarian hamsters a marked effect of X-rays on reproductive capacity was apparent. After 400 rad a smaller proportion of irradiated females littered in the first 25-day interval than after the other treatments, and no irradiated females produced more than one litter. Neither of the chemicals caused such a drastic reduction in fertility but TEM-treated females produced fewer litters and became sterile at an earlier age than control or IPMS-treated females. With IPMS, the number of litters produced was similar to the controls. Both chemicals caused a significant reduction in litter-size but further work is needed to establish whether this was due to induction of dominant lethals. No translocations were observed in the sons of treated female guinea-pigs or hamsters, but the numbers of animals studied were too small for any conclusions to be drawn.

Nonrandom distribution of chromosomal aberrations induced by three chemicals.

The G-band locations of 3244 breakpoints induced by cis-platinum (II) diamminedichloride (PDD), 1460 breakpoints induced by cytosine arabinoside (ara-C), and 1257 breakpoints induced by triethylenemelamine (TEM) in human lymphocyte chromosomes were identified. The breakpoints induced by each of these chemicals demonstrated a significantly nonrandom distribution within the human karyotype. The overall pattern of the interarm distribution was dependent upon the chemical used, but certain chromosomes arms exhibited similar responses to all 3 chemicals. Comparison of the frequencies of breakpoints within individual G-bands indicated that (1) certain bands were susceptible to damage induced by all 3 chemicals; (2) certain bands were resistant to damage by all 3 chemicals; (3) certain bands demonstrated variable susceptibility to induced damage dependent upon the chemical agent; and (4) other bands demonstrated near expected frequencies of damage (by length) to all 3 agents.

Heritable translocation test on random-bred mice after prolonged triethylenemelamine treatment.

Heritable translocation and dominant lethal tests were conducted with random-bred Swiss albino male mice. The animals were provided drinking water containing triethylenemelamine (TEM) for 4 weeks, and were then mated for 3 successive weeks for analysis of dominant lethality and production of F1 progeny. Potential translocation carriers among F1 males were selected after two breedings and confirmed by cytogenetic analysis. Translocation heterozygotes were obtained in offspring of the TEM-treated groups, but not in the control groups. In F1 males produced from the first week of mating, the frequencies of translocations were 0, 1.78 6.2 and 10.0% for the control group and groups receiving TEM at 0.0125, 0.025 and 0.050 mg/kg/day, respectively, and in those produced from the third week of mating, the values were 0 and 2.1%, respectively, for the control group and the group receiving TEM at 0.050 mg/kg/day. F1 males from the second week of mating were not studied for the induction of heritable translocations. TEM-induced dominant lethality and heritable translocations were most prominent in the first week of mating after 4 weeks of treatment. In addition, heritable translocations appeared to be a more sensitive endpoint than dominant lethal mutations for the measurement of mutagenic effects of TEM.