Enhancement of concurrent yield of spermatogonial, meiotic I, and meiotic II metaphase chromosomes from Chinese hamster testes.

A procedure to enhance the concurrent yield of large numbers of spermatogonial, meiotic I, and meiotic II metaphases from Chinese hamster testes has been developed. Animals were injected intraperitoneally with 5, 10, 20, or 40 mg of colchicine/kg and killed 3 hr after the treatment. Both testes from each animal were excised; one testis was minced, and germ cells were isolated by using 0.1% trypsin solution; the other testis was minced, and germ cells were isolated by using a conventional procedure. Chromosomal preparations were made with a standard air-drying technique. Examination of slides revealed that with the trypsin-isolation procedure the concurrent yields of spermatogonial, meiotic I, and meiotic II metaphases were significantly higher (P less than .05) than the yields obtained with the conventional procedure. Furthermore, 40 mg of colchicine/kg produced maximum numbers of all three types of germ cell metaphases. At this maximum concentration, metaphases were stable with no evidence of structural aberrations, and the frequency of hyperploidy was not significantly different (P greater than .05) from hypoploidy.

In vitro BALB/3T3 cell transformation assay of nonoxynol-9 and 1,4-dioxane.

The spermicidal surfactant nonoxynol-9 (Igepal CO-630, GAF Corp.) and a potential impurity, 1,4-dioxane, were tested in the in vitro cell transformation assay using BALB/3T3 cells. Two treatment periods, 48 hr and 13 days, were used. Nonoxynol-9, tested at levels up to 10 micrograms/ml, did not induce transformation, whereas dioxane was very active in the induction of type III foci in the cultured BALB/3T3 cells.

The feasibility of using Chinese hamsters as an animal model for aneuploidy.

The utility of Chinese hamsters as a test species for aneuploidy analysis was studied using four chemicals--vincristine, methyl 2-benzimidazole carbamate (MBC), nocodazole, and cyclophosphamide. Ten or more male Chinese hamsters were used per dose and bone marrow was removed at intervals of 6-96 hr. Slides were coded and 50-100 metaphases were analyzed per animal. A metaphase with more than 22 chromosomes was classified as a hyperploid cell, and the data were evaluated by using a one-tailed Fisher's exact test. In experiments using vincristine, MBC, and nocodazole, the frequencies of hyperploid cells were 0.43, 1.14, and 0.91%, respectively, for the control groups. In the experiment using cyclophosphamide, the control value frequency was 3.75%. The treated groups showed no significant increase in hyperploid frequencies when compared to concurrent controls at each of the treated times, except the value at 24 hr for the group that had been treated with vincristine at 0.75 mg/kg. However, this increase was not significant when compared to the overall value for pooled controls, with or without the cyclophosphamide control. Therefore, no significant effects due to chemical treatment were obtained in the present study. The results illustrate the extent of animal-to-animal as well as experiment-to-experiment variability in hyperploid frequencies and the importance of incorporating concurrent controls in assays for aneuploidy.

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.

Detection of vincristine-induced hyperploidy in meiotic II metaphases of male Chinese hamsters.

Induction of hyperploidy in germ cells of male Chinese hamsters treated with vincristine at dose levels of 0.25, 0.50 or 0.75 mg/kg of body weight was investigated. Animals were killed at 6, 24, 48, 72 and 96 h after administration of the chemical by a single intraperitoneal injection. The testes were removed and processed for spermatogonial, meiotic I, and meiotic II metaphases. Significantly increased frequencies of hyperploidy were obtained in meiotic II cells harvested 6, 24 and 48 h but not 72 and 96 h after treatment, indicating the importance of multiple sampling times. Analysis of spermatogonial cells shows that the frequencies of hyperploidy in the treated samples were comparable to those of controls. Limited sampling times used in the present study as well as small sample size or possible loss of hyperploid cells may be responsible for the negative findings for spermatogonial cells. Examination of meiotic I cells from 53 animals reveals the presence of one animal with an elevated level of hyperploidy unrelated to the vincristine treatment.

Detection of triethylenemelamine-induced translocation heterozygotes in CD-1 mice: fertility and cytological methods.

A study was conducted to determine the extent to which translocation carriers can be missed in the heritable translocation test. CD-1 male mice were treated with the mutagen triethylenemelamine and bred to untreated females and male progeny were subjected to fertility and cytogenetic analyses. Fertility testing involved mating 1 male to 3 virgin females and subjecting the females to uterine analysis. The males were classified as having normal or reduced fertility according to the number of live implants produced by the females. The cytogenetic analysis involved scoring 25 primary spermatocytes per mouse for translocation multivalents. A total of 103 male progeny were analyzed. Evaluation of fertility and cytogenetic data confirmed the presence of 18 translocation heterozygotes among the male progeny. On the basis of fertility testing, 1 translocation heterozygote was classified as normal (false negative) based on the production of 11 or more live implants by at least 1 mated female. On the basis of cytogenetic analysis of 25 cells per mouse, 1 partially sterile male was classified as normal (false negative); however, analysis of additional cells showed that this mouse was a translocation heterozygote. The study demonstrates the importance of evaluating fertility and cytogenetic criteria to minimize the extent of misclassification in conducting a heritable translocation test.

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.

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.

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.

Induction of morphological transformation in BALB/3T3 mouse embryo cells by okadaic acid.

Okadaic acid is produced by several types of dinoflagellates (marine plankton) and has been implicated as a causative agent of diarrhoetic shellfish poisoning. Okadaic acid, a known tumour promoter in vivo, has been shown to promote morphological transformation of carcinogen-initiated BALB/3T3 cells. This study shows that okadaic acid is capable of inducing morphological transformation of BALB/3T3 cells in the absence of an initiator.

Lack of transforming activity of fumonisin B1 in BALB/3T3 A31-1-1 mouse embryo cells.

The capacity of fumonisin B1 (FB1) to induce morphological transformation of cultured mammalian cells was assessed using BALB/3T3 A31-1-1 mouse embryo cells. FB1 with 90% purity was prepared from Fusarium proliferatum grown on whole corn. Cell growth was not inhibited by 48 hr of exposure at concentrations up to 1000 micrograms/ml. Moderate inhibition was induced by 6 days of exposure. In transformation assays with a 48-hr exposure, increases in transformed foci were observed at some concentrations; however, the responses were not reproducible. Prolonged exposure for up to 4 wk at 10, 100 and 500 micrograms/ml failed to induce increases in transformed foci. Analysis of combined results showed that only the increase induced by a 48-hr exposure at 500 micrograms/ml was significant. A trend test indicated the lack of a dose response for concentrations of 10-1000 micrograms/ml. FB1 seems to lack in vitro transforming activity.

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 Eagle's basal medium with Earle's 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.

Lipopolysaccharide layer protection of gram-negative bacteria against inhibition by long-chain fatty acids.

Growth, amino acid transport, and oxygen consumption of Escherichia coli and Salmonella typhimurium are inhibited by short-chain (C(2)-C(6)) but not by medium or long-chain fatty acids (C(10)-C(18)) at concentrations at which these processes are completely inhibited in Bacillus subtilis. The resistance of gram-negative organisms is not correlated with their ability to metabolize fatty acids, since an E. coli mutant unable to transport oleic acid is still resistant. However, mutants of both E. coli and S. typhimurium in which the lipopolysaccharide layer does not contain the residues beyond the 2-keto-3-deoxyoctonate core are inhibited by medium (C(10)) but not by long-chain (C(18)) fatty acids. Furthermore, removal of a portion of the lipopolysaccharide layer by ethylenediaminetetraacetate treatment renders the organisms sensitive to medium and partially sensitive to long-chain fatty acids. The intact lipopolysaccharide layer of gram-negative organisms apparently screens the cells against medium and long-chain fatty acids and prevents their accumulation on the inner cell membrane (site of amino acid transport) at inhibitory concentrations. These results are relevant to the use of antimicrobial food additives, and they allow the characterization of gram-positive versus gram-negative bacteria and their lipopolysaccharide mutants.

Effects of fatty acids on growth and envelope proteins of Bacillus subtilis.

Fatty acids of different chain lengths were added to cultures of Bacillus subtilis growing in nutrient sporulation medium, and the effects of these fatty acids on growth, oxygen uptake, adenosine triphosphate (ATP) concentration, and membrane protein composition were examined. All fatty acids inhibited growth, the effect being reduced in the presence of glycolytic compounds and reversed by transfer to medium without fatty acids. The inhibition of growth was correlated with a reduction in both the rate of oxygen consumption and the concentration of ATP per cell. The concentration required to obtain a certain degree of inhibition increased with decreasing molecular weight of the fatty acid. However, the reduced nicotinamide adenine dinucleotide oxidation system of cell envelope preparations (i.e., the electron transport system) was not inhibited. Submaximal growth inhibition was accompanied by the relative increase of a membrane protein band revealed by urea-acetic acid gel electrophoresis. This increase was blocked by actinomycin or chloramphenicol. All of the above changes could also be produced by 2,4-dinitrophenol. The inhibition results are best explained by assuming that the fatty acids reversibly react with the cell membrane or proteins in it; they could either alter the membrane structure or uncouple the electron transport chain from two types of proteins, those used for ATP regeneration and others needed for the transport of certain compounds into the cells.

The effect of cell passage on the susceptibility of BALB/3T3 clone A31-1-1 cells to 3-methylcholanthrene-induced morphological transformation.

The response of BALB/3T3 clone A31-1-1 cells to chemically induced morphological transformation was evaluated using 3-methylcholanthrene (MCA). Stock cultures were initiated from cryopreserved cells, grown in T25 flasks containing 5 ml of medium, and replated at subconfluency. Serially transferred cells were then subjected to transformation assay. After 24-hr seeding, cells were incubated 48 hr with MCA in a 5% CO2 incubator. They were then rinsed and incubated for an additional 4 weeks with twice weekly medium change. Type III foci were scored after fixation and staining with Giemsa. With serial passage from the frozen state, cells of passages 3-14 had a low level of spontaneous transformation; zero to 6 type III foci per 20 dishes were counted. In the MCA-treated cultures the number of transformed foci, however, increased with passage. Such passage-related sensitivity to MCA was demonstrated for cells cultured in two batches of sera: one from MA Bioproducts (Lot no. 2E052) and the other from Armour Pharmaceuticals (Lot no. Y65801). The passage-related increase in number of transformed foci was not related to doubling time, cloning efficiency, or MCA-induced growth inhibition.

Effect of mode of administration of methyl methanesulfonate and triethylenemelamine on induction of unscheduled DNA synthesis in mouse germ cells.

The effect of route of administration on induction of unscheduled DNA synthesis (UDS) in mouse germ cells in vivo was studied using two germ cell mutagens, methyl methanesulfonate (MMS) and triethylenemelamine (TEM). The chemicals were administered to male mice (C3Hf X 101)F1 by IP injection or gavage using acute or 5-day subacute regimens. After completion of dosing, methyl-[3H]thymidine [( 3H]TdR) was injected into the testes, and spermatozoa were collected 16 days later. The sperm heads were isolated, and UDS was determined by the amount of [3H]TdR incorporated. Acute administration of MMS (2-100 mg/kg) induced a strong, dose-related UDS response. The response was slightly higher with IP injection than with gavage. The UDS response after five daily doses of 50 mg MMS/kg was 20-30% higher than that induced by a single IP or gavage dose. Acute administration of TEM (0.05-4.0 mg/kg) by IP injection or gavage induced weak and variable responses. Retesting TEM using inbred C3Hf mice produced weak but exposure-related responses with both acute IP and gavage treatments. There was a slight increase in UDS response with subacute IP injection but not with subacute gavage. Acute testicular injection of TEM produced a higher but more variable UDS response. The study showed that gavage, as well as IP injection, can be used for the administration of test chemicals and that the subacute 5-day regimen induced a higher UDS response than the acute regimen. Furthermore, the testicular route may enhance the detection of weak UDS inducers.

The effect of female strain on identification of male mice carrying reciprocal translocations.

Chemicals capable of inducing heritable chromosomal effects may be detected by the mouse heritable translocation test, which is based on the detection of a specific type of transmissible abnormality, namely, reciprocal translocation. Since mice carrying such a chromosomal abnormality usually have reduced fertility, they may be identified on the basis of fertility data. In the present study, the efficiency of two female strains for identifying CD-1 male translocation heterozygotes was examined. Thirty-three 10-wk-old CD-1 male mice were injected IP with triethylenemelamine (0.025 mg/kg/day) 5 days a wk for 5 wk. The treated males were then mated to untreated CD-1 females for 2 wk to produce progeny. The F1 males were raised to maturity, tested for fertility by using two female strains (CD-1 and B6C3F1), and analyzed cytogenetically. The cytogenetic analysis confirmed that 41 males were translocation heterozygotes and 125 were normal. Examination of the fertility data showed that in the test with CD-1 females all translocation heterozygotes were identified but 19 normal mice were identified as potential translocation carriers because of decreased fertility. In the test with B6C3F1 females, five translocation heterozygotes were not identified on the basis of fertility data, and 11 normal mice were misclassified as potential translocation carriers.

Effects of acetate and other short-chain fatty acids on sugar and amino acid uptake of Bacillus subtilis.

Acetate and other short chain n-fatty acids (C(1)-C(6)) inhibit strongly the uptake of l-serine or other l-amino acids but inhibit only weakly that of alpha-methylglucoside or fructose, whether measured in whole cells of Bacillus subtilis or in membrane vesicles that have been energized with reduced nicotinamide adenine dinucleotide (NADH), l-alpha-glycerol phosphate, or ascorbate plus phenazine methosulfate. The acetate inhibition is noncompetitive, as was shown for l-alpha-aminoisobutyric acid uptake by whole cells and for l-serine uptake by membrane vesicles. In membrane preparations, neither NADH oxidation nor the reduction of cytochromes by NADH are affected by fatty acids. All of these effects are similar to those of 2, 4-dinitrophenol. It is concluded that the fatty acids "uncouple" the amino acid carrier proteins from the cytochrome-linked electron transport system (to which they may be coupled via protein interaction or via a cation gradient).