Sex-linked Recessive Lethal Test Research Articles

Evaluating the effect of low dose rate of gamma rays in germ cells of Drosophila melanogaster

Purpose: Evaluation of genetic risk in germ cells is still matter of research, mainly due to their role in the transmission of genetic information from one generation to another. Although numerous experiments have been carried out in Drosophila in order to study the effect of radiation on germ cells, the role of dose rate (DR) has not been fully explored. The purpose of this study was to evaluate the action of DR on the radioprotection induction on male germ cell of D. melanogaster.Material and method: The productivity and the sex-linked recessive lethal (SLRL) tests were used to evaluate the radio-sensitivity of different states of the germ line of males. Two-day-old males of Canton-S wild type strain were pretreated with 0.2 Gy at 5.4 or 34.3 Gy/h of gamma rays from a 60Co source, three hours later, they were irradiated with 20 Gy at 907.7 Gy/h. Thereafter, each single male was crossed with 3 five-day old Basc virgin females, that were replaced every other day by new females. This procedure was conducted three times, to test the whole germ cell stages.Results: Females crossed with males irradiated with 0.2 Gy at both DR tested, laid a higher number of eggs than control, but egg-viability was reduced. On the other hand, in the group of 0.2 Gy + 20 Gy -combined treatments- the total number of eggs laid decreased only when 0.2 Gy were delivered at 34.3 Gy/h however, the egg-viability increased. The dose of 0.2 Gy at both DR did not modify the baseline frequency of SLRL. A tendency to decrease in the frequency of lethals in brood III was found in combined treatments at both DR.Conclusion: The fact that 0.2 Gy at 5.4 or 34.3 Gy/h induced an increase in the egg-viability and a tendency to decrease the genetic damage in pre-meiotic cells provoked by 20 Gy, might indicate the induction of any mechanism that could be interpreted as radioprotection in male germ cells of D. melanogaster. Results emphasize the need to carry out more studies on the effect of the DR on the induction of genetic damage in germ cells.

Coffee mitigates cyclophosphamide-induced genotoxic damage in Drosophila melanogaster germ cells

In the present study, coffee (CF) was evaluated for its protective effects against genotoxic damage and oxidative stress induced by the chemotherapeutic drug, cyclophosphamide (CPH). The sex-linked recessive lethal (SLRL) test was employed to study the induction of mutations in the larvae as well as in all the successive germ cell stages of treated males. Control and treated third instar larvae were used to monitor the biomarkers of oxidative stress response such as glutathione content (GSH), glutathione-S-transferase (GST), catalase (CAT), superoxide dismutase (SOD) and lipid peroxidation (MDA content). Our results demonstrated that co-administration of CF (2%) with CPH (3 mM) has significantly reduced CPH-induced lethal mutations in the germ cells of larvae and adult flies. The reductions observed in mutation frequencies were: 75% in larvae and 62.4% in the adult. Significant enhancement in antioxidant enzymatic levels: CAT (46.6%) > SOD (43.0%) > GST (42.4%) > GSH (31.6%) and reduction in MDA levels (32.05%) in the pretreated third instar larvae demonstrated the antioxidant activity of CF against CPH-induced oxidative stress. The findings from the present study suggest that the Drosophila model is an ideal one for evaluating the antigenotoxic and antioxidant activity of complex mixtures like CF.

Ameliorative effects of gallic acid, quercetin and limonene on urethane-induced genotoxicity and oxidative stress in Drosophila melanogaster

The main objective of our present work was to ascertain the efficacy of Drosophila melanogaster model for assessing antigenotoxic and antioxidant effects of dietary phytochemicals gallic acid (GA), quercetin (QC) and limonene (Lim) against urethane (URE), a genotoxic environmental carcinogen. Oregon-K (ORK) adult male flies were fed GA, QC and Lim in combination with URE (20 mM) in 10% sucrose for 72 h. Third instar larvae were fed instant medium containing the above phytochemicals and URE for 24 h. Sex-linked recessive lethal (SLRL) test and assays for estimating glutathione content (GSH), glutathione S-transferase (GST), catalase (CAT), superoxide dismutase (SOD) and lipid peroxidation (MDA content) were performed. Adult feeding experiments demonstrated that co-treatment of flies with URE and the test phytochemicals has significantly decreased the frequencies of SLRL mutations in all the germ cell stages when compared to that with URE alone. Larval feeding experiments also showed a similar pattern. The above results correlate well with antioxidative potentials of the test agents where we observed the elevated enzymatic levels with a significant reduction in MDA level in Drosophila larvae. The results further suggest that the dietary phytochemicals have an antioxidant and antimutagenic property which can be assessed using D. melanogaster.

Induction of complete and mosaic sex-linked recessive lethal mutations by cigarette smoke filtrate in Drosophila melanogaster

The genotoxic effects of cigarette smoke filtrate (SF) on the germ-line stages were examined in Drosophila melanogaster using the sex-linked recessive lethal test, which detects a broad spectrum of genetic alterations and proved to show correlations between mutagenicity and carcinogenicity of the tested chemicals. SF was extracted from fiberglass filter cartridges; each used in smoking 15 cigarettes. The proper SF concentrations (0.2 µL) in 0.45% NaCl saline were injected intraperitoneally in 2- to 3-day-old wild-type males, alongside with controls injected with 0.2 µL of saline. The genotoxicity effects of SF were examined in all spermatogenesis stages of treated males. Results showed that SF was toxic with an median lethal dose value of approximately 0.2% and induced significant sterility effects. The mutagenicity of SF (0.2%) was significantly stage specific and induced complete sex-linked recessive lethal mutations in the broods representing the spermatocytes and late and early spermatogonia, and induced mosaic mutations in the untreated progeny in the brood representing late spermatogonia. These results indicated, for the first time, that SF induces mosaic mutations, which could result from DNA instabilities and labile permutations that can be replicated and passed to future generations before being fixed into mutations in the untreated progeny of treated males, or originating from mutations that result in increasing hyperplasia of the gonad that subsequently produce the actual mutations in later cell cycles. Such delayed mutagenic effects of SF indicated that SF and, consequently, cigarette smoking have much greater genotoxicity than what was previously predicted.

Combining molecular docking and 3-D pharmacophore generation to enclose the in vivo antigenotoxic activity of naturally occurring aromatic compounds: Myricetin, quercetin, rutin, and rosmarinic acid

Considering the controversial results concerning the antimutagenicity of some phenolic compounds recorded in the literature, the antigenotoxic effects of four selected phenolic compounds, myricetin, quercetin, rutin, and rosmarinic acid, against DNA damage induced by alkylation with ethyl methanesulfonate (EMS), were evaluated in Drosophila melanogaster males using the sex-linked recessive lethal (SLRL) test. To assess the protective effects against DNA damage, D. melanogaster males were exposed to a monofunctional alkylating agent EMS in concentration of 0.75ppm, 24h prior to one of the selected phenolic compounds in the concentration of 100ppm. The possible differences in mechanisms of protection by selected compounds were determined by molecular docking, after which structure-based 3-D pharmacophore models were generated. EMS induced considerable DNA damage as shown by significant increase in the frequency of germinative mutations. The frequency decreased with high significance (p<0.001***) after post-treatments with all selected phenolic compounds. Further, docking analysis revealed EMS pre-bond conformations against guanine and thymine as a necessary condition for alkylation, after which resulting O6-ethylguanine and O4-ethylthimine were docked into the active site of O6-alkylguanine-DNA alkyltransferase to confirm that particular lesions are going to be repaired. Finally, myricetin and quercetin protected dealkylated nucleotides from further EMS alkylation by forming the strong hydrogen bonds with O6-guanine and O4-thymine via B ring hydroxyl group (bond lengths lower than 2.5Å). On the other side, rutin and rosmarinic acid encircled nucleotides and by fulfilling the EMS binding space they made an impermeable barrier for the EMS molecule and prevented further alkylation.

Protective role of methanol extracts of Gentiana asclepiadea L. and G. cruciata L. against genotoxic damage induced by ethyl methanesulfonate

The methanol extracts from the underground and aerial part of the two species of Gentiana genus, Gentiana asclepiadea L. and Gentiana cruciata L. from Serbia, were investigated for their antigenotoxic activity against wellestablished mutagenic agent ethyl methanesulfonate (EMS) using the in vivo sexlinked recessive lethal (SLRL) test on Drosophila melanogaster. For this purpose, three days old Canton S males were treated with the potent mutagen EMS in concentration of 0.75 ppm, alone and combined with methanol extracts obtained from underground or aerial part of G. asclepiadea and G. cruciata in concentration of 5%, separately. Although EMS in concentration of 0.75 ppm increased the mutation frequency in all three broods, post-treatments with methanol extracts obtained from the underground and aerial part of G. asclepiadea and G. cruciata in concentration of 5%, respectively, drastically reduced the frequency of sex-linked recessive lethal mutations induced by EMS. Compared to the sucrose, as a negative control, methanol extract obtained from underground part of G. cruciata showed the most potent antigenotoxic activity. Extracts from the underground and aerial part of the two species of Gentiana genus, G. asclepiadea L. and G. cruciata L. from Serbia used in our experiments showed a clear antimutagenic effect, reducing the frequency of mutations induced by a strong mutagen such as EMS.

In vivo antigenotoxic potential and possible mechanism of action of selected 4‐hydroxy‐2H‐chromen‐2‐one derivatives

The in vivo sex-linked recessive lethal test was carried out in Drosophila melanogaster to investigate whether or not five substituted 4-hydroxy-2H-chromen-2-ones can modulate the genotoxicity of the well-established mutagenic agent ethyl methanesulfonate (EMS). For this purpose, 3 days old Canton S males were treated with the potent mutagen EMS alone in concentration of 0.75 ppm, as well as in combination with one of the five 4-hydroxycoumarins, namely diethyl 2-(1-(4-hydroxy-2-oxo-2H-chromen-3-yl)ethylidene)malonate (2b), 3-(1-(4-hydroxy-2-oxo-2H-chromen-3-yl)ethylidene)pentane-2,4-dione (6b), 4-(4-(4-hydroxy-2-oxo-2H-chromen-3-yl)thiazol-2-ylamino) benzenesulfonic acid (4c), 4-hydroxy-3-(2-(2-nitropheny lamino)thiazol-4-yl)-2H-chromen-2-one (9c), and (E)-4-hydroxy-3-(1-(m-tolylimino)ethyl)-2H-chromen-2-one (5d), in concentration of 70 ppm. The frequency of germinative mutations increased significantly after the treatment with EMS and decreased after treatments with coumarins. The maximum reduction was observed after treatments with 2b, 6b, 4c, and 5d. By the formation of hydrogen bonds or electrostatic interactions with O(6) of DNA guanine, tested coumarins prevent EMS-induced alkylation. The results indicate a protective role of five 4-hydroxycoumarins under the action of a strong mutagen.

Study of genotoxicity and antigenotoxicity of the Cotinus coggygria Scop. methanol extract by Drosophila melanogaster sex-linked recessive lethal test

The genotoxic and antigenotoxic effects of Cotinus coggygria Scop. methanol extract was investigated using the Drosophila sex-linked recessive lethal (or SLRL) test. The results presented here show that the methanol extract of Cotinus coggygria in a concentration of 5% and artificial chemical agent ethyl methanesulfonate EMS (0.75 ppm) induce recessive lethal mutations on X-chromosome on Drosophila melanogaster in all broods (I, II and III). Post-treatment with lower concentration of the methanol extract of Cotinus coggygria (2%) was effective in reducing genotoxicity ofmutagen.

Genotoxic potential of Cotinus coggygria Scop. (Anacardiaceae) stem extract in vivo.

The intention was to evaluate the possible in vivo genotoxic potential in different cell-types, of a methanol extract obtained from the plant stem of Cotinus coggygria Scop., using the sex-linked recessive lethal (or SLRL) test and alkaline comet assay. The SLRL test, revealed the genotoxic effect of this extract in postmeiotic and premeiotic germ-cell lines. The comet assay was carried out on rat liver and bone marrow at 24 and 72 h after intraperitoneal administration. For genotoxic evaluation, three concentrations of the extract were tested, viz., 500, 1000 and 2000 mg/kg body weight (bw), based on the solubility limit of the extract in saline. Comet tail moment and total scores in the group treated with 500 mg/kg bw, 24 and 72 h after treatment, were not significantly different from the control group, whereas in the groups of animals, under the same conditions, but with 1000 and 2000 mg/kg bw of the extract, scores were statistically so. A slight decrease in the comet score and tail moment observed in all the doses in the 72 h treatment, gave to understand that DNA damage induced by Cotinus coggygria extract decreased with time. The results of both tests revealed the genotoxic effect of Cotinus coggygria under our experimental conditions.

The protective role of parsley extract against vincristine mutagenicity in Drosophila melanogaster

In this study, Drosophila melanogaster males were treated with parsley plant extract and the anticancer drug vincristine (VCR) singly and in combined treatments (pre, co and post-treatments) to detect the mutagenic effects by using sex-linked recessive lethal test (SLRL) and estimation of cholinesterase enzyme (ChE) activities in order to compare the sensitivity of the two test systems. A wild type strain Oregon-R (Or-R) male flies of D. melanogaster were reared on a medium containing one concentration of each of VCR and parsley (4 ml/100 ml medium) in each single and combined treatment. Also the activity of ChE was estimated in some insects of the two generations: F1 females, F2 bar eye females (heterozygous) and F2 wild types males. The results indicate that both of parsley and vincristine did not cause significant increases of SLRL test in either the single or combined treatments. In contrast, estimation of ChE activities showed significant increase in all the broods within single and combination treatments, except female of the second generation of spermatid brood which treated with parsley and VCR at the same time. It is concluded that enzyme estimation is more sensitive than SLRL test for detection the mutagenic effect for parsley’s extract and vincristine.

Mutagenic Effect of Three Nitrogenous Fertilizers on Drosophila melanogaster

The present study was carried out in order to evaluate the mutagenic effect of 3 nitrogenous fertilizers; urea, ammonium nitrate and calcium nitrate; on Drosophila melanogaster. Newly hatched larvae were treated with the LC25 and LC50 of the 3 tested nitrogenous fertilizers. The classic sex-linked recessive lethal (SLRL) test was used to detect the differential mutagenic effect of the fertilizers on the 3 germ stages. The results showed that all fertilizers exhibited variable mutation frequencies in the different broods of spermatogenesis after a single treatment. Urea exhibited a direct mutagenic effect on post-meiotic stages whereas ammonium nitrate and calcium nitrate revealed an indirect mutagenic effect on meiotic and pre-meiotic stages. The electrophoretic separation of proteins (SDS-PAGE) showed that treatment with these fertilizers resulted in polymorphetic changes in different low and high molecular weight protein bands, causing alteration in the electrophoretic patterns and densities of proteins. This study showed that the tested nitrogenous fertilizers possess mutagenic potentialities which are related to their nitrogen content.

Genotoxicity testing of the methanol extract of the plant Cotinus coggygria and gallic acid on Drosophila melanogaster

The genotoxic activity of methanol extract obtained from the stem of Cotinus coggygria Scop. and synthetic gallic acid were investigated using the Drosophila sex-linked recessive lethal test (or SLRL test). In the tested methanol extract of C. coggygria (1 g), 62.50 mg of pyrocatechol equivalent of phenols was detected. Also, 46.76 mg of flavonoids and 15.75 mg of nonflavonoids were observed in 1 g of dry weight of extract. Methanol extract of C. coggygria in a concentration of 5% and 5% synthetic gallic acid were shown to be clearly genotoxic, inducing sex-linked recessive lethal mutations on the X-chromosome of Drosophila melanogaster males in all three broods.

Application of the sex-linked recessive lethal test in Drosophila melanogaster for monitoring the work environment of a rubber factory.

The intention of the study was to try the applicability of a biologic test system for the detection of potentially hazardous ambient exposures in a rubber factory. Drosophila populations (strain “white”) were exposed for five generations in the calendering and the vulcanization areas. No significant increases in SLRL frequencies were observed. The difference was statistically slightly significant (P ≤ 0.05) between the pooled materials of the exposed cages. Drosophila males (strain “white”) were exposed for 14 days in four manufacturing departments, vulcanization, weighing, calendering and moulding (the area where methylene-o-chloro aniline (MOCA) is used). Only in the pooled material from the exposures in the vulcanization area and in the MOCAroom was the SLRL frequency statistically slightly significantly (P ≤ 0.05) increased as compared with the control. The results suggest that potentially mutagenic air-borne contaminants may be found in specific areas, such as the vulcanization area and in the MOCA-room, and that monitoring of ambient exposures can be performed with the Drosophila SLRL system.

In vitro analysis of clastogenic effects of adrenaline on human lymphocytes

and physiological effects of adrenaline and other catecholamines, little is known about possible genotoxic and mutagenic effects. There is an interesting experimental finding that dopamine induces DNA strand breaks in human skin fibroblasts and gene mutations in mouse lymphoma cells (M o l d e u s et al., 1983). However, dopamine did not exhibit genotoxic effects in the Salmonella/mammalian-microsome mutagenicity test, the sex-linked recessive lethal test in Drosophila melanogaster, the sister chromatid exchange (SCE) test in human lymphocytes , or the in vivo micronucleus assay in mouse and rat (M o l d e u s et al., 1983). Likewise, in cytogenetic analysis, adrenaline did not exhibit genotoxic effects on cultured human peripheral blood lymphocytes (D j e l i c , 1997). On the other hand, mutagenic effects of various catecholamines on mouse lymphoma cells resulted from the creation of superoxide anion (M c G r e g o r et al., 1988). In addition, catechol derivates, including adrenaline, induce DNA strand breakage by ferryl species, whereas the induction of 8-hydroxyguanine (8OHG) is due to a hydroxyl radical (·OH) (M i u r a et al., 2000). More recent studies revealed that noradrenaline induces primary DNA damage in the Comet assay on purified human lymphocytes (D j e l i c and A n d e r s o n , 2003) and sperm (D o b r z y n s k a et al., 2004), most likely due to formation of reactive oxygen species (ROS). This result is in agreement with findings that adrenaline and other catecholamines can be involved in redox cycling under the influence of superoxide anion (G e n o v a et al., 2006). Interestingly, superoxide anion may induce chromosome breakage and sister chromatid exchange (SCE) (M ’ b e m b a -M e k a et al., 2007).

Anti-genotoxic effect of aloe vera gelR on the mutagenic action of ethyl methanesulfonate

The antimutagenic effect of aloe vera gelR was investigated using the Drosophila sex-linked recessive lethal test (or SLRL test). In this assay, 3-day-old adults were treated with a direct-acting mutagen - ethyl methanesulfonate (EMS), which was the positive control. The other group of individuals of the same age was firstly treated with EMS, and then with aloe vera gelR (co-treatment). When co-treatment experiments with aloe were carried out, it was effective in reducing genotoxicity of the direct-acting mutagen. .

Final Amended Report on the Safety Assessment of Oxyquinoline and Oxyquinoline Sulfate as Used in Cosmetics1

Oxyquinoline is a heterocyclic phenol and Oxyquinoline Sulfate is its salt, both of which are described as cosmetic biocides for use in cosmetic formulations. In an earlier Cosmetic Ingredient Review (CIR) safety assessment, the available data were found insufficient to support safety. Currently, some uses are reported to the Food and Drug Administration (FDA) by industry, but industry reports to CIR indicate no use. In Europe, Oxyquinoline and Oxyquinoline Sulfate are accepted for use as stabilizers for hydrogen peroxide in rinse-off and leave-on hair care preparations, with concentration limitations. Oxyquinoline is metabolized and excreted in the urine as glucuronides. Oxyquinoline and Oxyquinoline Sulfate exhibit little acute or subchronic toxicity in animal studies. A 100-mg dose of Oxyquinoline was only slightly irritating to the eye. Oxyquinoline and Oxyquinoline Sulfate were genotoxic in certain Salmonella typhimirium strains with metabolic activation and in a mouse lymphoma assay. There was some evidence of increased chromosome aberrations in an in vitro study, and an increase in sister-chromatid exchanges (but not chromosome aberrations) in rats treated with Oxyquinoline, but no genotoxicity was found in a Drosophilia sex-linked recessive lethal test, mouse bone marrow micronucleus test, a rat bone marrow and hepatocyte micronucleus test, and unscheduled DNA synthesis in rat hepatocytes. Oxyquinoline did bind to DNA in the presence of liver enzymes. Although the International Agency for Research on Cancer concluded that the existing evidence is inadequate to determine carcinogenicity in animals, Oxyquinoline was noncarcinogenic in several rodent feeding studies, and newly available studies using genetically altered mice, in one case carrying the human c-Ha-ras gene, demonstrated that Oxyquinoline was not carcinogenic. In clinical tests, Oxyquinoline is neither an irritant nor a sensitizer when tested at 1% in petrolatum. The available data demonstrate that Oxyquinoline and Oxyquinoline Sulfate are safe as stabilizers for hydrogen peroxide in rinse-off hair care cosmetic products in the present practices of use. For leave-on cosmetic products, however, the absence of impurities and ultraviolet (UV) absorption data resulted in a finding that the available data are insufficient to support safety. The data needed in order to complete the safety assessment of Oxyquinoline and Oxyquinoline Sulfate in leave-on cosmetic products are (1) UV absorption data -- if significant absorption occurs, then photoirritation/photosensitization data will be needed; and (2) data on impurities.

Effect of mutagen-induced cell lethality on the dose response of germline mutations.

Molecular tests for mutations require a sample of tissue from which DNA is extracted, to determine the presence or absence of one or more mutations per sample. To ensure mutation fixation each sample must consist of an equal number of cells that have had one or more DNA replications. In an in vivo test, surviving stem cells compensate to give the same number of cells per sample, leaving as the only evidence for stem cell lethality the increase in mutants of clonal origin because the mutant clone developed from a population of fewer stem cells. A problem is that an increase in mutagen dose increases stem cell death, resulting in a decreased number of surviving target cells, thus giving a downward bias of samples with one or more mutations per sample. To compare in vivo tests with molecular tests we will use as a model system the sex-linked recessive lethal (SLRL) test for germ cell mutations in Drosophila melanogaster. Spermatogonia cells in male larvae were exposed to ENU and mutations detected in sperm cells from adults. The same SLRL data were analyzed by two methods: (1) The conventional analysis of SLRL data, in which each mutation of a cluster of mutations of common origin was counted. (2) An analysis was used to simulate a sample for molecular analysis by determining mutations per male with an equal size sample of progeny per male. With this second analysis a correction factor is required based on the change in cluster size of mutants of common origin.

Mutagenicity ofCombretum erythrophyllum in sex-linked recessive lethal test in Drosophila

The roots of the herb Combretum erythrophyllum are prescribed and consumed as a medicine for sexual diseases, as an aphrodisiac, as a purgative and for treating leprosy. This plant is also a source of gums used as food adulterants. In earlier studies, the aqueous extract of this plant induced oxidative and base-pair mutations in Salmonella. We employed the sex-linked recessive lethal test to evaluate its mutagenicity in Drosophila melanogaster. In the current paper we report that the aqueous extract causes mutations in the meiotic stage of Drosophila melanogaster. © 1997 John Wiley & Sons, Ltd.

Genotoxicity testing of two red dyes in the somatic and germ line cells of drosophila

Two red dyes, rhodamine B and amaranth, were tested for their genotoxic effects in the somatic (wing primordia) and germ line cells of Drosophila melanogaster following the wing spot and the sex-linked recessive lethal tests. Second- and third-instar larvae, carrying suitable genetic markers, were subjected to chronic exposure to different concentrations of the test dyes. The results indicate that rhodamine is genotoxic in both somatic and germ line cells and amaranth is non-genotoxic.

The hypermutability conferred by the mus308 mutation of Drosophila is not specific for cross-linking agents.

The hypersensitivity of the mus308 mutant of D. melanogaster to cross-linking agents has been suggested to be the consequence of a possible defect of this mutant in DNA cross-link repair. Moreover, the mus308 mutation has been proposed as an animal model for the study of Fanconi's anemia. In order to obtain more information about the function controlled by this locus, we have measured the mutability of the mus308 mutant to several mutagens with different modes of action using the sex-linked recessive lethal test. We show that this mutation confers hypermutability not only to the cross-linking agents tested, hexamethylphosphoramide and hexamethylmelamine, but to the point mutagen N-ethyl-N-nitrosourea as well, whereas the response to methyl methanesulfonate was normal. The results suggest that the mus308 locus is not defective in a repair pathway specific for cross-links but is rather involved in a step of a more general post-replication repair process responsible for the removal of non-excised adducts.