Mutagen Ethyl Methanesulphonate Research Articles

Over-expression of P-glycoprotein and glutathione S-transferase pi in MCF-7 cells selected for vincristine resistance in vitro.

This study has provided evidence that exposure of the wild-type MCF-7 human breast carcinoma cell line to the mutagen ethyl methane sulphonate (EMS), followed by selection in vincristine (VCR), resulted in a stably-resistant subline, designated VCREMS, which expressed an approximately 14-fold level of resistance to VCR. This VCREMS subline showed cross-resistance (3-fold) to adriamycin (ADR) and to etoposide (3-fold), but not to cisplatin. The addition of a non-toxic concentration of verapamil (6.6 microM) significantly enhanced VCR cytotoxicity only in the resistant subline. This resistance was associated with over-expression of P-glycoprotein (Pgp), but without a concomitant increase in Pgp mRNA or gene amplification. In addition, activities of total glutathione S-transferases (GST) and glutathione peroxidase were elevated in this resistant subline, with over-expression of the GST-pi isozyme and its associated mRNA being identified, without gene amplification. This VCR-selected resistant MCF-7 cell line therefore provides another example of a breast carcinoma subline in which there is co-ordinate over-expression of both Pgp and GST-pi, without attributing a causal relationship to either event, and extends the range of anti-tumour drugs known to elicit modifications in glutathione metabolism.

Abnormal spermiogenesis in hybrid mice (C57BL X C3H)F1 treated with the mutagen ethyl methane sulphonate

Ethyl methane sulphonate is an agent that produces a notable elevation in the percentage of morphologically abnormal spermatozooids. Inducement of minimal defects of the hook, bicephalic, biflagellated and amorphous forms and filamentous spermatozzoids have been described. The cytotoxic effect of the drug is manifested by death of primary spermatocytes, which is associated to the low number of epididlmary spermatozooids. The mechanisms that produce teratospermia have not been previously studied exhaustively.Hybrid mice of genotype (C57BL X C3H)F1 of 8 to 12 weeks of age were treated intraperitoneal ly with EMS at a dose of 200 mg/Kg for five consecutive days and killed four weeks later by cervical dislocation. The testicular tissue was fixed by inmersion in 6.5% glutaraldehyde in 0.2 M phosphate buffered saline at pH 7.3 and 4°C, and was included in epoxyresine. Fine sections were stained in leal citrate and uranyl acetate, and were observed in Zeiss EM109 electron microscope.

Ethyl methanesulphonate induced fatty acid mutations in flax

AbstractSeed of the flax cultivar McGregor were treated with the chemical mutagen ethyl methanesulphonate (EMS) with the objective of producing mutations that reduced the α‐linolenic acid content of the seed. From the seed of 2430 M1 plants initially examined by the thiobarbituric test, 53 were classified as having reduced linolenic acid levels. Utilizing half‐seed analysis by gas chromatography, three lines taken through to the M4 generation had reduced linolenic acid levels, but all three appeared to have different mutations. Mutant E67 has a palmitic acid level of 28.4%, three times the level found in McGregor. Mutant E1747 had a high linoleic content of 52.2%, and mutant E1929 had increased oleic and linoleic levels to twice that of McGregor.

In vitro plant regeneration and ethylmethanesulphonate (EMS) uptake in somatic embryos of date palm (Phoenix dactylifera L.)

The effect of the auxins dicamba (3,6-dichloro-2-methoxybenzoic acid) and picloram (4-amino-3,5,6-trichloropicolinic acid) on callus growth and embryogenesis in Phoenix dactylifera L. was investigated. Maximum callus fresh weight was obtained in nutrient medium enriched with 200 µm picloram. Somatic embryogenesis and subsequent plant regeneration was achieved following transfer of such calli to hormone-free medium. Germination of the somatic embryos was influenced by treatment with the chemical mutagen ethylmethanesulphonate (EMS). Uptake of the labelled mutagen ([14C]EMS) by the somatic embryos increased with increased incubation time. Presence of dimethyl sulphoxide (DMSO) as a carrier agent during mutagenic treatment was necessary for efficient mutagen uptake.

Mutation of Host Cell Determinants which Discriminate between Lytic and Persistent Mouse Hepatitis Virus Infection Results in a Fusion-resistant Phenotype

The expression of mouse hepatitis virus (MHV) E2-specific mRNA, the E2 polypeptide and its associated cell fusing activity was monitored in various cell types inoculated with a recombinant vaccinia virus, designated vMS containing the E2 gene. The results suggest that host cell permissiveness to MHV infection correlates with cellular susceptibility to membrane fusion mediated by the MHV E2 glycoprotein. In addition, we utilized a genetic approach to the analysis of host cell functions involved in determining permissiveness to MHV. By using the chemical mutagen ethyl methanesulphonate, mouse fibroblast cell mutants were generated and selected for their resistance to cell killing by MHV. When challenged with MHV, all five mutants examined gave rise to persistent infections, in contrast to wild-type L-2 cells which were rapidly killed by the virus. The results provide genetic evidence in support of a previous correlation proposed between MHV permissiveness and two host determinants, namely susceptibility to MHV infection and to MHV-mediated cell fusion. Fusion resistance was specific to fusion mediated by the MHV E2 glycoprotein as shown in contact fusion assays between uninfected cells and cells infected either with MHV or with an E2-expressing recombinant vaccinia virus. In contrast, mutant cells were not resistant to fusion after treatment with polyethylene glycol. The observed high rate of generation of these mutants suggests that the conversion of a fully MHV-susceptible cell to a semi-resistant one is a fairly common event, possibly involving a single mutation. In this case, resistance to MHV infection and to E2-mediated membrane fusion may depend on a common host function. This result provides prospects for the precise genetic and biochemical characterization of the steps involved in host cell permissiveness to MHV infection.

In vitro action of ethylmethanesulphonate on banana shoot tips

Shoot tips excised from two banana ( Musa acuminata) clones (SH-3362, a diploid AA, and cultivar ‘Grand Nain’ mutant (GN-60Gy/A), a triploid AAA) were treated with various concentrations of the mutagen ethylmethanesulphonate (EMS). The number of newly initiated adventitious buds decreased with increased concentrations of EMS. Presence of dimethylsulphoxide (DMSO) in combination with EMS affected the growth and development of the shoot tips. Uptake of the labelled mutagen ( 14C EMS) was greatly influenced by time of incubation and presence of DMSO as a carrier agent. Microautoradiography of histological sections of shoot tips confirmed the accumulation of the labelled mutagen in the shoot apical meristem, leaf primordia and more extensively in the corm, from which new adventitious buds are initiated.

A phorbol ester tolerant (PET) variant of HL-60 promyelocytes.

The promyelocytic leukaemia cell line HL-60 differentiates to a macrophage-like cell when exposed to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) and other agents which activate protein kinase C. To investigate this phenomenon we developed an HL-60 variant which does not differentiate when exposed to TPA. HL-60 cells were exposed to the mutagen ethyl methanesulphonate and were cloned in soft agar in the presence of a normally lethal concentration of TPA. One colony of cells that proliferated in TPA was obtained. The cells of this phorbol ester tolerant (PET) line have retained their resistance to TPA for several years without selective pressure. They are somewhat larger than their phorbol ester sensitive (S) parent, but they are otherwise morphologically similar. When PET-cells are exposed to TPA their growth is arrested for approximately 48 h. Thereafter, they resume their original rate of replication at all concentrations of TPA tested. S-cells undergo changes typical of HL-60 when exposed to TPA; they aggregate, stop growing, adhere to the flask and die. The PET-cells appeared to be as sensitive as S-cells to other agents which differentiate HL-60 such as retinoic acid, dimethysulphoxide, and 1,25-dihydroxyvitamin D3, as determined by rate of proliferation in culture, Wright's stain, nitroblue tetrazolium reduction, and induction of the ectoenzyme NAD-glycohydrolase. TPA-induced protein phosphorylation was studied using one- and two-dimensional polyacrylamide gel electrophoresis. Several proteins increased their incorporation of 32P when S- and PET-cells were exposed to TPA, the most prominent of which were the two previously described nuclear matrix proteins of 80 kd and 33 kd. There was no difference in the protein phosphorylation pattern in S- and PET-cells, nor in how this pattern changed on TPA exposure. Fluorescent activated cell sorting and karyotypic analysis revealed PET-cells to be a hypotetraploid variant of S-cells, with approximately 80 chromosomes, including a marker chromosome iso(1p) not found in the S-cells. Identification of the biochemical lesion responsible for this TPA resistance in PET cells will provide clues concerning the mechanism of this important pathway for the induction of cell differentiation.

Effect of mutagen on cultured Schistosoma mansoni.

Although lightly homogenized three-week-old Schistosoma mansoni incubated in Mitsuhashi and Maramorosch insect tissue culture medium or the medium of Weller and Wheeldon produced adherent cell layers, continued growth of these cells did not occur. Non-adherent cells obtained by trypsinization also failed to produce long term cell cultures even after the addition of a range of growth factors. The possibility of producing tumour-like schistosome cells by the use of the mutagen ethyl methane sulphonate was therefore examined. Four-hour exposure of three-week-old schistosomes caused in some worms (a) large fluid filled 'ballooning', which also occurred in adult males, (b) enlargement of the gut, (c) increase in numbers of large round cells within the worms and (d) tissue outgrowths. It is suggested that these effects of mutagen offer new approaches to obtaining permanent schistosome cell cultures.

Molecular dosimetry of the chemical mutagen ethyl methanesulphonate Quantitative comparison of mutation induction in Escherichia coli, Neurospora crassa, V79 Chinese hamster cells, L5178Y mouse lymphoma cells and some in vitro and in vivo cytological results

Molecular dosimetry of the chemical mutagen ethyl methanesulphonate Quantitative comparison of mutation induction in Escherichia coli, Neurospora crassa, V79 Chinese hamster cells, L5178Y mouse lymphoma cells and some in vitro and in vivo cytological results

Interferon production by individual L cells.

Using a protected centre technique in which agarose prevents the diffusion of interferon from individual producing cells, we have shown that essentially every cell in a monolayer of mouse L cells can be induced to produce interferon by infection with Newcastle disease virus (NDV). The amount of interferon produced by individual cells appeared to be highly variable, even when cloned cells and viruses were used. U.v.-irradiated virus lost its capacity to induce interferon in L cells and to infect chick embryo fibroblasts at the same rate. A small proportion of cells (1 X 10-6 to 10 X 10-6) appeared to produce interferon constitutively. This fraction was increased threefold by u.v. irradiation of the cells, and up to 10-fold by exposing cells to the mutagen ethyl methane sulphonate.

Production of pollution‐tolerant strains of unicellular algae through mutagenic treatment. I. Ethyl methane sulphonate

AbstractThe unicellular algae Anacystis nidulans and Chlorella vulgaris were collected from effluents of the Indian Oil Refinery Barauni and the Sindri Fertilizer Factory (India), respectively, and isolated in unialgal and bacteria‐free culture.The algae were treated with the mutagen ethyl methane sulphonate and such mutagenically treated algae were compared with untreated controls with respect to growth characteristics and the capacity to absorb the two important components of polluted habitats, viz., phosphate and nitrate.Treated A. nidulans grew faster and showed a consistently higher uptake of phosphate than untreated. Treated C. vulgaris cells grew more slowly than untreated cells and showed lesser phosphate uptake during early phases of growth but greater uptake during the later phases.Nitrate uptake capacity was increased in both algae following treatment.

Production of pollution-tolerant strains of unicellular algae through mutagenic treatment. I. Ethyl methane sulphonate

The unicellular algae Anacystis nidulans and Chlorella vulgaris were collected from effluents of the Indian Oil Refinery Barauni and the Sindri Fertilizer Factory (India), respectively, and isolated in unialgal and bacteria-free culture. The algae were treated with the mutagen ethyl methane sulphonate and such mutagenically treated algae were compared with untreated controls with respect to growth characteristics and the capacity to absorb the two important components of polluted habitats, viz., phosphate and nitrate. Treated A. nidulans grew faster and showed a consistently higher uptake of phosphate than untreated. Treated C. vulgaris cells grew more slowly than untreated cells and showed lesser phosphate uptake during early phases of growth but greater uptake during the later phases. Nitrate uptake capacity was increased in both algae following treatment.

The induction of supersuppressor mutants of Bacillus subtilis by ethyl methanesulphonate and the posttreatment modification of mutation yield.

Supersuppressor mutants have been induced in a strain of Bacillus subtilis with the chemical mutagen ethyl methanesulphonate. The yield of mutants recovered is dependent on the degree of supplementation of the selective plating medium with minute quantities of either nutrient broth or the previously required growth supplements. The optimal quantities of these medial additives have been established and the superiority of nutrient broth described. This “broth effect” has been shown to be due to components of the nutrient broth other than the previously required growth substances.

Mutation research in peas

Dry seeds of the pea variety Pauli were irradiated with 15,000 r X-rays, 18,000 r X-rays, 18,000 r γ rays and with neutrons; the latter treatment for 6 hours. In addition the chemical mutagen ethyl methane sulphonate (EMS) was used, in concentrations of 0.33% and 0.50% respectively.