C.W. Sheu

Heritable translocation and dominant-lethal mutation induction with ethylene oxide in mice

Ethylene oxide was studied for induction of dominant-lethal mutations and heritable translocations in male mice. The chemical was prepared in water and injected intraperitoneally. The dominant-lethal study was conducted using a single injection of 150 mg/kg (maximum tolerated dose); in the heritable translocation study males were injected daily on weekdays for 5 weeks with 60 or 30 mg/kg dose per day. Results clearly showed that ethylene oxide is effective in inducing dominant-lethal mutations and that the 4 stocks of untreated females used do not differ or may differ only slightly in the ability of their eggs to repair ethylene oxide-induced lesions in male germ cells. Increases in the frequencies of heritable translocations were also observed at the 2-dose levels. These frequencies did not deviate significantly from those expected on the basis of dose-square kinetics.

A guide for mutagenicity testing using the dominant lethal assay

The dominant lethal assay has been used and continues to be used to provide information about the effects of chemicals on the gonadal cells of male animals. Guidelines for conducting this test are useful but as with any guideline scientists should avoid interpreting them as protocols. Thus this document is a general approach to dominant lethal testing and should be used in conjunction with other available protocols and procedures.

Comparison of two methods for detecting translocation heterozygotes in mice

An accurate estimate of the error of misclassifying male translocation heterozygotes as normals is essential for the proper evaluation of results of the heritable translocation test in mice. The size of this error may vary from one laboratory to another depending, primarily, on the method or variation of the method used in screening for translocation heterozygotes. This report shows a way to estimate for misclassification errors involved in two methods, sequential and direct cytological analysis, of screening for translocation heterozygotes. A positive correlation was found between the degree of partial sterility of a male and the frequency of cells with multivalent configurations among his diakinesis-metaphase I cells. We interpreted this to confirm that the length of translocated chromosome segments has some influence on the proportion of unbalanced gametes in the ejaculate, presumably reflecting the frequency with which adjacent-1 and adjacent-1 segregations and 3-1 misdivisions occur.

Effects of prolonged chemical treatment with cyclophosphamide and 6-mercaptopurine in the dominant lethal test system

Abstract The effects of prolonged chemical treatment with cyclophosphamide and 6-mercaptopurine were investigated in the dominant lethal test system. Groups of 15 male Holtzman rats were given 5 daily i.p. injections of the compounds per week for 10 weeks; dose levels were 0 (water), 5.0, 10.0 and 20.0 mg/kg in the cyclophosphamide study and 0 (dimethylsulfoxide), 12.5, 25.0 and 50.0 mg/kg in the 6-mercaptopurine study. Each study also contained a group given i.p. injections of triethylenemelamine (0.025 mg/kg), the positive control compound, which was diluted in water. After the final injection, each male rat was housed with 2 virgin females per week for 2 weeks. Females were killed 14 days from the midweek of cohousing and uterine contents were examined to evaluate the numbers of total implants, dead implants, dead implants per total implants and females with 1 or more, or 2 or more, dead implants. Results from the cyclophosphamide study indicated a positive dominant lethal effect in both weeks of mating. Negative results were obtained for all levels of 6-mercaptopurine treatment for both weeks, with the exception of 1 aberrant value at the 25.0-mg/kg level for week 1.

Proliferation and morphological transformation of BALB/3T3 cells by a prolonged treatment with sodium orthovanadate

BALB/3T3 mouse embryo cells were used to study the effect of sodium orthovanadate on cell proliferation and morphological transformation. In the presence of the chemical (0.25-1.0 micrograms/ml), the cells continued to proliferate after the cultures were confluent. However, contact-inhibited growth was resumed after removal of the chemical from the culture medium. Continued exposure of the cells to the chemical for 4 wk led to the production of numerous foci consisting of morphologically transformed cells. In contrast, as in vitro transformation assay with a 48-hr treatment protocol followed by 4 wk of incubation without the chemical produced negative results. To test the stability of the transformed foci that were produced on prolonged exposure, we isolated 20 foci with distinctly transformed characteristics from treated cultures and grew them in medium without orthovanadate. 15 isolates gradually reverted to contact-inhibited growth and five maintained the transformed phenotype through ten serial subcultures. The results show that the majority of the transformed foci from the orthovanadate-treated culture failed to maintain transformed characteristics in the absence of the chemical. However, a small fraction of the foci appeared to be altered permanently and exhibited a transformed phenotype in the absence of the chemical.

Dominant lethal assay of some hair-dye components in random-bred male rats

Male rats were exposed to maximally tolerated doses of 5 hair-dye components in a dominant lethal test. Each component was tested at 3 dosage levels with 15 random-bred male rats per level. The highest dose, selected on the basis of subacute toxicity testing, generally reduced weight gains without being lethal. Freshly prepared solutions were injected i.p. at 1 ml/kg 3 times a week for 10 weeks. Rats injected with dimethylsulfoxide and triethylenemelamine served as solvent and positive controls, respectively. A majority of rats survived the treatment at the levels tested and were mated to two virgin females each per week for 2 weeks. The females were sacrificed at midterm of pregnancy and examined for live and dead implants. Dominant lethality was evaluated on the basis of 4 criteria: dead implants per pregnant female, dead implants per total implants, proportion of females with one or more dead implants, and proportion of females with two or more dead implants. 2-Nitro-p-phenylenediamine, 2,4-diaminoanisole sulfate and 2,5-diaminoanisole sulfate produced negative responses, whereas m-phenylenediamine and 4-nitro-o-phenylenediamine induced weak dominant lethality in the first trial. On retesting these weakly positive components, both m-phenylenediamine and 4-nitro-o-phenylenediamine produced negative responses.

Transforming activity of selected polycyclic aromatic hydrocarbons and their nitro-derivatives in BALB/3T3 A31-1-1 cells

The transforming activities of four polycyclic aromatic hydrocarbons and six of their nitro-derivatives were studied using BALB/3T3 clone A31-1-1 cells in the absence of exogenous metabolic activation. Each compound was assayed two to four times to its maximal level of solubility. A transformation response was induced by 1-nitropyrene, 2-nitropyrene, 4-nitropyrene and benzo[a]pyrene in the BALB/3T3 mouse embryo cells. Pyrene and 7-nitrobenz[a]anthracene produced questionable responses, and benz[a]anthracene, chrysene, 6-nitrobenzo[a]pyrene and 6-nitrochrysene produced negative responses. The capacity of the assay system to indicate tumorigenicity of the test compounds is discussed.

Morphological transformation of BALB/3T3 mouse embryo cells in vitro by vomitoxin.

The transforming potential of vomitoxin, a trichothecene mycotoxin produced on cereal grains by fungi of the genus Fusarium, was assessed using mouse embryo BALB/3T3 A31-1-1 cells. Cells grown in Eagles basal medium with Earles salts supplemented with 7.5% foetal bovine serum were treated with highly purified vomitoxin, which was dissolved in distilled water and filter-sterilized. Assays were conducted using cells from three different passages at dose levels ranging from 0.1 to 1.6 microgram/ml. The treatment time was 48 hr and the highest dose levels tested produced approximately 10% survival as determined by in situ cell counts. Distilled water and 3-methylcholanthrene (5.0 micrograms/ml) were used as the vehicle and positive controls, respectively. Of the 20 dishes examined per dose group, the numbers of type III foci were 0-1 in the solvent control, 12-15 in the positive control and 0-9 in the treated groups. Comparison of the three assays showed that the level of response varied with passage number. Of the three passages of cells tested-passage numbers 6, 8 and 9 (p6, p8 and p9)--passage-9 cells produced the strongest positive effect.

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.

Detection of Dominant Lethal Mutation in Mice After Repeated Low-Dose Administration of 6-Mercaptopurine

Detection of dominant lethality after repeated low-dose administration was investigated, using the base analog 6-mercaptopurine (6-MP). The compound was administered to groups of 30 outbred CD-1 male mice by i.p. injection at dosage levels of 12.5, 25.0, or 50.0 mg/kg/day 5 days a week for 8 weeks. At the end of treatment, each male was cohoused for 1 week with two untreated females of different strains: one CD-1 and one (C3H x C57BL/10)F1. Negative (solvent) and positive (triethylenemelamine, TEM) control groups were included. Implant data were analyzed statistically. Exposure of male mice to 6-MP at 50 mg/kg/day resulted in 93% mortality and severe weight loss of the survivors. Body weights were also reduced in the group given 25 mg/kg/day. At the lowest dose level of 12.5 mg/kg/day, 6-MP had no noticeable toxic effect on the treated males. Dominant lethal analysis of the implant data showed that a statistically significant increase in dead implantations was induced in CD-1 but not in (C3H x C57BL/10)F1 females. The dominant lethal effect of TEM, the positive control, was detected in both strains of females tested.