Additional data in support of the quadruplicated white-ivory reversion system to test for somatic genotoxicity in Drosophila melanogaster.

Cyclophosphamide, ethyl methanesulfonate, propyleneimine and tritiated water were tested in a new short-term somatic mutation bioassay, previously described by Green and coworkers (1986), to evaluate the suitability of the quadruplicated white-ivory system of Drosophila melanogaster for genotoxicity testing of chemicals. A 2.9-kb tandemly duplicated sequence of w+ within a w+ gene is responsible for the white-ivory phenotype. Reversion of wi to w+ is, in general, associated with the loss of the appended 2.9 kb or other alterations affecting this duplicated region. The appearance of light (white or nearly white) phenotypes could be due to the loss of some wi copies. Thus, in the eyes of adult males resulting from treated larvae, we can detect 2 types of sectors (red and light) on the orange-yellow background. Our results indicate that the genetic system used in this somatic assay is sensitive to the genotoxic effects of the 4 compounds tested. All 4 compounds tested were positive. Tritiated water had weak effects, cyclophosphamide was characterized by small revertant clones and ethyl methanesulfonate and propyleneimine by large clone size. From the available data, we feel that the wi system can be considered of potential value for genotoxicity testing.     

Genetic activities of 4-chloromethylbiphenyl,the 4-hydroxy derivative and benzyl chloride in the soma and germ line of Drosophila melanogaster

The genetic activities of 4CMB, 4HMB and BC were assayed as regards the induction of somatic alterations in gene expression on an unstable w⁺ locus with an intragenic TE and all the simultaneously induced germinal mutations on the X-chromosome carrying this locus. The compounds were applied topically in solution at equimolar doses on late embryos and newly hatched larvae. The somatic events were scored as aberrantly pigmented eye sectors in the emerging adult males and the germinal mutations in their F₂ progeny, according to the Muller-5 technique.The somatic events were expressed as red or white mosaic eye sectors; the former could be an outcome of the repression or deletion of the zeste-regulatory proximal subunits of w⁺ locus, and the latter generally attributable to deletions (w⁻) within its structural part. All 3 compounds were effective in the induction of red sectors at the higher tested doses (0.5–2.0 mM) and the level of this activity was virtually the same for 4CMB and 4HMB, but was 2-fold higher for BC. In contrast, the frequencies of the simultaneously scored white sectors were not raised significantly above the controls with 4CMB, but showed decisive increases above this level with both 4HMB and BC.The germinal X-chromosome mutations (recessive lethals and visibles) were only induced at the highest tested dose (2.0 mM), and their frequencies were virtually the same for all 3 compounds reaching a common level of about 0.6%, which is some 3-fold the normal control level for the test system. Specific-locus mutability at the TE w⁺ was suggestively positive only with BC.     

The w/w SMART assay of Drosophila melanogaster detects the genotoxic effects of reactive oxygen species inducing compounds

The somatic mutation and recombination w/w⁺ eye assay has been used for genotoxic evaluation of a broad number of chemicals with different action mechanisms yielding high values of sensitivity, specificity and accuracy. The aim of this work was to determine the utility of this assay in the evaluation of reactive oxygen species inducers. For this, we have tested eight compounds: diquat, paraquat, menadione, juglone, plumbagin, streptonigrin, tert-butyl hydroperoxide and 4-nitroquinoline 1-oxide, using the Drosophila Oregon K strain which had previously shown advantageous conditions to test this type of compounds. Diquat was the only chemical for which the results were clearly negative, probably because its high toxicity, whereas indications of a marginal genotoxicity rised for menadione. The remaining compounds were evaluated as positives. The conclusion of these experiments is that the w/w⁺ assay is capable to detect genotoxic effects induced by compounds that generate reactive oxygen species through different action mechanisms.     

Effects of artichoke (Cynara scolymus) leaf and bloom head extracts on chemically induced DNA lesions in Drosophila melanogaster

The genotoxicity of bloom head (BHE) and leaf (LE) extracts from artichoke (Cynara scolymus L.), and their ability to modulate the mutagenicity and recombinogenicity of two alkylating agents (ethyl methanesulfonate – EMS and mitomycin C – MMC) and the intercalating agent bleomycin (BLM), were examined using the somatic mutation and recombination test (SMART) in Drosophila melanogaster. Neither the mutagenicity nor the recombinogenicity of BLM or MMC was modified by co- or post-treatment with BHE or LE. In contrast, co-treatment with BHE significantly enhanced the EMS-induced genotoxicity involving mutagenic and/or recombinant events. Co-treatment with LE did not alter the genotoxicity of EMS whereas post-treatment with the highest dose of LE significantly increased this genotoxicity. This enhancement included a synergistic increase restricted to somatic recombination. These results show that artichoke extracts promote homologous recombination in proliferative cells of D. melanogaster.     

Genotoxicity testing of five compounds in three Drosophila short-term somatic assays

To provide further background data for the somatic mutation and/or recombination tests in Drosophila melanogaster, we have evaluated the responses in 3 assyas (zeste-white, white-ivory and wing spot) of 5 chemicals classified by the U.S. National Toxicology Program (NTP) as genotoxic non-carcinogens (or ambiguous). The selected compounds were 2-chloromethylpyridine, 1-nitronaphthalene, 4-nitro-o-phenylenediamine, 3-nitropropionic acid and p-phenylenediamine. Our results show that all the compounds tested produce significant increases in the frequency of mutant clones, in at least one of the assays, p-phenylenediamine being the compound which present a clearer mutagenic activity, and the wing spot test, the assay the detects more genotoxic compounds (4/5).     

Conservation and DNA sequence arrangement of the DNA polymerase I gene region from Klebsiella aerogenes, Klebsiella pneumoniae and Escherichia coli

Linked multiple mutation is observed after treatment of Escherichia coli with methyl methanesulfonate, N-methyl-N′-nitro-N-nitrosoguanidine, ethyl methanesulfonate, and N-ethyl-N-nitro-N-nitrosoguanidine but not ultraviolet light. Induction of linked multiple mutations requires the uvrE⁺ gene product indicating the involvement of the mismatch repair system. The observation of linked multiple mutations is not due to mutagenesis occurring in a subpopulation of cells. Growing point mutagenesis also occurs after treatment with these mutagens but not with ultraviolet light. It is likely that the excess of mutations observed with these mutagens at growing points is at least partly a relative effect, rather than one due to an absolute increase of reactivity at the DNA growing point region. This relative effect may result from the operation of an inducible repair mechanism which removes O⁶-alkylguanine residues from the DNA distal to the bacterial growing point. The adaptive response, first described by Robins &; Cairns (1979) prefers O⁶-methylguanine over O⁶-ethylguanine.     

Inactivation of Bacteriophage T4 by Ethyl Methanesulfonate: Influence of Host and Viral Genotypes

Inactivation of bacteriophage T4 by ethyl methanesulfonate (EMS) is a complex process which depends critically upon the conditions of treatment and upon both the viral and the host genotypes. EMS-inactivated particles are capable of multiplicity and cross-reactivation, indicating the need for caution in using EMS in certain types of mutation studies. The pyrimidine dimer excision systems of the phage and the host do not affect the EMS sensitivity of T4, but the T4x⁺y⁺ system does. Mutational defects in the deoxyribonucleic acid (DNA) ligase and the DNA polymerase systems both of the virus and of its host also affect viral EMS sensitivity.     

Peripheral Blood Invariant Natural Killer T Cells of Pig-Tailed Macaques

In humans, invariant natural killer T (iNKT) cells represent a small but significant population of peripheral blood mononuclear cells (PBMCs) with a high degree of variability. In this study, pursuant to our goal of identifying an appropriate non-human primate model suitable for pre-clinical glycolipid testing, we evaluated the percentage and function of iNKT cells in the peripheral blood of pig-tailed macaques. First, using a human CD1d-tetramer loaded with α-GalCer (α-GalCer-CD1d-Tet), we found that α-GalCer-CD1d-Tet⁺ CD3⁺ iNKT cells make up 0.13% to 0.4% of pig-tailed macaque PBMCs, which are comparable to the percentage of iNKT cells found in human PBMCs. Second, we observed that a large proportion of Vα24⁺CD3⁺ cells are α-GalCer-CD1d-Tet⁺CD3⁺ iNKT cells, which primarily consist of either the CD4⁺ or CD8⁺ subpopulation. Third, we found that pig-tailed macaque iNKT cells produce IFN-γ in response to α-GalCer, as shown by ELISpot assay and intracellular cytokine staining (ICCS), as well as TNF-α, as shown by ICCS, indicating that these iNKT cells are fully functional. Interestingly, the majority of pig-tailed macaque iNKT cells that secrete IFN-γ are CD8⁺ iNKT cells. Based on these findings, we conclude that the pig-tailed macaques exhibit potential as a non-human animal model for the pre-clinical testing of iNKT-stimulating glycolipids.     

Genotoxic potency of monofunctional alkylating agents in E. coli: comparison with carcinogenic potency in rodents

A quantitative correlation between carcinogenicity and genotoxicity was investigated by a comparison between the carcinogenic potency in rodents and the mutagenic (M), recombinogenic (R) and SOS-inducing (I) potencies in a bacterial test (E. coli multitest) for 9 monofunctional alkylating agents: N-nitroso-N-methylurethane, N-nitroso-N-ethylurea, epichlorohydrin, N-nitroso-N-methylurea, N-nitroso-N-methyl-N'-nitroguanidine, methyl methanesulfonate, diethylsulfate, dimethylsulfate, ethyl methanesulfonate. A significant positive correlation between the carcinogenic potency and the product of the mutagenic and recombinogenic potencies was found for all tested compounds. Thus, the E. coli multitest may be used as a simple test to search for correlations between carcinogenicity and genotoxicity of DNA-damaging agents.     

Enhancement of transport in Micrococcus lysodeikticus by sodium ions

Na⁺ (at a concentration of 10 mM) increased the uptake of succinate, glucose and l-valine by Micrococcus lysodeikucus cells considerably. The effect of Na⁺ could be duplicated by Li⁺ only, which, however, was less active. The other cations tested (K⁺, NH₄⁺, Cs⁺, Mg²⁺, Ca²⁺ and Mn²⁺ were ineffective at concentrations up to 100 mM. Addition of Na⁺ increased the affinities of the uptake system for the substrate studied, while uptake capacity remained unaltered.     

Induction of homologous recombination following in utero exposure to DNA-damaging agents

Much of our understanding of homologous recombination, as well as the development of the working models for these processes, has been derived from extensive work in model organisms, such as yeast and fruit flies, and mammalian systems by studying the repair of induced double strand breaks or repair following exposure to genotoxic agents in vitro. We therefore set out to expand this in vitro work to ask whether DNA-damaging agents with varying modes of action could induce somatic change in an in vivo mouse model of homologous recombination. We exposed pregnant dams to DNA-damaging agents, conferring a variety of lesions at a specific time in embryo development. To monitor homologous recombination frequency, we used the well-established retinal pigment epithelium pink-eyed unstable assay. Homologous recombination resulting in the deletion of a duplicated 70 kb fragment in the coding region of the Oca2 gene renders this gene functional and can be visualized as a pigmented eyespot in the retinal pigment epithelium. We observed an increased frequency of pigmented eyespots in resultant litters following exposure to cisplatin, methyl methanesulfonate, ethyl methanesulfonate, 3-aminobenzamide, bleomycin, and etoposide with a contrasting decrease in the frequency of detectable reversion events following camptothecin and hydroxyurea exposure. The somatic genomic rearrangements that result from such a wide variety of differently acting damaging agents implies long-term potential effects from even short-term in utero exposures.     

Comparisons of mutation induction in reversion systems of Saccharomyces cerevisiae and Salmonella typhimurium

The principle of the treatment condition routinely used in Salmonella typhimurium is to allow the cells to divide in the presence of the chemical being tested; only the revertants will be able to form visible colonies (softagar procedure). In Saccharomyces cerevisiae, the routinely used procedure is to treat the cells in liquid non-nutrient medium under non-growing conditions (non-nutrient procedure). We compared mutation induction under both experimental conditions using S. cerevisiae; we also compared the mutagenic response of the two microorganisms to six compounds; two nitrofuran derivatives, AF-2 and SQ18,506, three hair dye components, 1,2-diamino-4-nitrobenzene, 1,4-diaminoanthraquinone, and methyl violet, as well as ethyl methanesulfonate. Of the six compounds tested in S. cerevisiae strain XV185-14C, only ethyl methanesulfonate was mutagenic under both experimental conditions. The two nitrofuran derivatives, AF-2 and SQ18,506, induced mutations in S. cerevisiae when the non-nutrient procedure was employed. None of the three hair dyes tested was mutagenic in S. cerevisiae. However, the results obtained with Salmonella typhimurium indicate that the hair dye 1,2-diamino-4-nitrobenzene is a mutagen, confirming the earlier study by Ames et al. [2]. Among the other five compounds tested in Salmonella typhimurium, the base-substitution-detecting strain TA100 responded to one concentration of AF-2, and EMS was mutagenic in strains TA1535, TA100 and TA1537.     

Construction of an Unstable Ring-X Chromosome Bearing the Autosomal Dopa Decarboxylase Gene in Drosophila melanogaster and Analysis of Ddc Mosaics

An unstable Ring-X chromosome, Ddc⁺- Ring-X carrying a cloned Dopa decarboxylase (Ddc) encoding segment was constructed. The construction involved a double recombination event between the unstable Ring-X, R(1)w^vC and a Rod-X chromosome which contained a P-element mediated Ddc ⁺ insert. The resulting Ddc⁺-Ring-X chromosome behaves similarly to the parent chromosome with respect to somatic instability. The Ddc⁺-Ring-X chromosome was used to generate Ddc mosaics. Analyses of Ddc mosaics revealed that while there was no absolute requirement for the Ddc ⁺ expression in either the epidermis or the nervous system, very large mutant clones did affect the viability of the mosaic.     

Genetic instability in Drosophila melanogaster

Summary An unstable long tandem duplication which includes the white locus twice, marked with w ^sp in the left and w ^17G in the right locus, when kept in males has been found to produce red-eyed sons which have lost the long duplication and with it the w ^sp and w ^17G mutants. Such exceptions were produced also when w ^17G had been exchanged for w ^a.Stocks originating from these exceptions are unstable, producing: 1) zeste males, also unstable, 2) w ^- deletions, stable, 3) transpositions of the white locus to sites in other chromosomes.The instability is interpreted as the effect of an IS element, within or adjacent to the white locus, which is supposed to retain a duplication of the proximal zeste interacting part of this locus. According to the orientation of the IS element the duplicated part can be active or inactive, giving a zeste or red eye phenotype.The frequency of exceptional offspring after X-ray treatment of the red and zeste unstable stocks have been compared to stable stocks with corresponding genotypes.     

Podophyllotoxin resistance: A codominant selection system for quantitative mutagenesis studies in mammalian cells

Mutants resistant to the microtubule inhibitor podophyllotoxin (Pod^R), a codominant marker, can be readily selected in various mammalian cell lines such as, CHO, HeLa, mouse L cells, Syrian hamster cells (BHK₂₁) and a mouse teratocarcinoma cell line OC15. In CHO cells, the recovery of Pod^R mutants is not affected by cell density (up to 1 × 10⁶ cells per 100-mm diameter dish), and after treatment with the mutagen ethyl methanesulfonate maximum mutagenic effect is achieved after a relatively short expression time (40–48 h). The frequency of Pod^R mutants in various cell lines increased in a dose-dependent manner in response to treatment with the mutagens ethyl methanesulfonate and N-methyl-N′-nitro-N-nitrosoguanidine. The Pod^R selection system thus provides a new genetic marker which should prove useful in studies of quantitative mutagenesis in mammalian cells.     

Inorganic phosphate uptake in Trypanosoma cruzi is coupled to K cycling and to active Na extrusion

Background

Orthophosphate (P_i) is a central compound in the metabolism of all organisms, including parasites. There are no reports regarding the mechanisms of P_i acquisition by Trypanosoma cruzi.

Methods

³²P_i influx was measured in T. cruzi epimastigotes. The expression of P_i transporter genes and the coupling of the uptake to Na⁺, H⁺ and K⁺ fluxes were also investigated. The transport capacities of different evolutive forms were compared.

Results

Epimastigotes grew significantly more slowly in 2 mM than in 50 mM P_i. Influx of P_i into parasites grown under low P_i conditions took place in the absence and presence of Na⁺. We found that the parasites express TcPho84, a H⁺:P_i-symporter, and TcPho89, a Na⁺:P_i-symporter. Both P_i influx mechanisms showed Michaelis–Menten kinetics, with a one-order of magnitude higher affinity for the Na⁺-dependent system. Collapsing the membrane potential with carbonylcyanide-p-trifluoromethoxyphenylhydrazone strongly impaired the influx of P_i. Valinomycin (K⁺ ionophore) or SCH28028 (inhibitor of (H⁺ + K⁺)ATPase) significantly inhibited P_i uptake, indicating that an inwardly-directed H⁺ gradient energizes uphill P_i entry and that K⁺ recycling plays a key role in P_i influx. Furosemide, an inhibitor of the ouabain-insensitive Na⁺-ATPase, decreased only the Na⁺-dependent P_i uptake, indicating that this Na⁺ pump generates the Na⁺ gradient utilized by the symporter. Trypomastigote forms take up P_i inefficiently.

Conclusions

P_i starvation stimulates membrane potential-sensitive P_i uptake through different pathways coupled to Na⁺ or H⁺/K⁺ fluxes.

General significance

This study unravels the mechanisms of P_i acquisition by T. cruzi, a key process in epimastigote development and differentiation to trypomastigote forms.     

Effect of Reduced H+-ATPase Activity on Acid Tolerance in Streptococcus bovis Mutants

In order to confirm that H⁺-ATPase plays an important role in the acid tolerance ofStreptococcus bovis , two mutants with low activities of H⁺-ATPase were isolated by use of ethyl methanesulfonate and neomycin resistance. The activity of H⁺-ATPase per cellular nitrogen was related to the lowest culture pH permitting growth. A mutant with little H⁺-ATPase activity (Mutant 2) was unable to grow below pH 5.5, which suggests that the intracellular pH should be maintained above 5.5 in S. bovis. Since lactate dehydrogenase activity, which is important for acid tolerance, was similar in parent and mutant strains, H⁺-ATPase activity is likely to affect acid tolerance. The amount of H⁺-ATPase protein as determined by Western-blot analysis with polyclonal antibody, was similar in Mutant 2 and its parent, indicating that H⁺-ATPase activity per enzyme protein is reduced by mutation. Probably, H⁺-ATPase synthesis was not changed by mutation. The gene encoding H⁺-ATPase of Mutant 2 had mutations at positions close to the ATP-binding motif A sequence in the β-subunit, which probably explains the reduced activity of H⁺-ATPase in this mutant. These results strongly support the assumption that H⁺-ATPase has a key role in the acid tolerance of S. bovis.     

Isolation of a hybrid plasmid with homologous sequences to a transposing element of Drosophila melanogaster

In Drosophila several transposing elements that contain the white locus are known. Transpositions of one such element, which carries both the white-apricot (w^a) and the neighboring roughest (rst⁺) genes, have been isolated at more than 120 sites scattered over the entire genome (Ising and Ramel 1976). We have isolated a recombinant plasmid (61F4) containing sequences that appear to be present on this transposing element (TE). In nontransposed stocks, 61F4 hybridizes to approximately 40 sites in the polytene chromosomes including the nucleolus, the chromocenter and chromosome section 3C (that is, the white-apricot roughest region). In six different tranpositions tested, the genetic map position of the TE corresponds to one site of in situ hybridization of 61F4, indicating that the TE contains homologous sequences. The sites of in situ hybridization correlate with the w^a allele or alleles derived from w^a but not with w⁺ and other w alleles tested, nor with an X-ray-induced revertant of w^a. Thus w^a strains appear to carry additional DNA sequences homologous to 61F4, close to or within the w gene. The recombinant plasmid 61F4 carries 7.3 kb of Drosophila DNA inserted into pSF2124. It contains a segment homologous to a member of the copia gene family (Finnegan et al. 1978). Since copia appears to be a highly mobile element (Strobel, Dunsmuir and Rubin 1979), the association of copia sequences with the w^a-rst⁺ transposing element suggests that copia sequences may be responsible for the transposition of this element.     

Contents Volume 93 (1982)

A thymidine kinase heterozygote was isolated from a diploid human lymphoblast line which forms colonies with high efficiency in microtiter dishes. We show that this cell line, called TK6, can be mutated from a tK^+/? to TK^?/? state by diverse mutagens, including ethyl methanesulfonate, butyl methanesulfonate, nitrosomethylurea, UV light, ICR-191, 4-nitroquinoline oxide, fluorodeoxyuridine, benzo[a]pyrene and aflatoxin B₁. We report here the experiments required to demonstrate the applicability of this new line in quantitative assays of mutation in human cells.Mitotic recombination between the centromere and the tk locus could not be induced by either dimethylsulfoxide or phorbol-12-myristate-13-acetate.