Improved high bioactivation cross for the wing somatic mutation and recombination test in Drosophila melanogaster

doi:10.1016/0165-1161(92)90032-H

Mutation Research/Environmental Mutagenesis and Related Subjects

Volume 271, Issue 1, February 1992, Pages 59-67

https://doi.org/10.1016/0165-1161(92)90032-H Get rights and content

Abstract

The two tester strains of the high bioactivation (HB) cross for the wing somatic mutation and recombination test (SMART) in Drosophila melanogaster developed by Frölich and Würgler possess high metabolic capacity to activate promutagens. These strains contain chromosomes 1 and 2 of the DDT-resistant stock Oregon R(R) which exhibit a high constitutive level of cytochrome P450. However, they show several disadvantages for routine application, such as disturbed wing hair patterns in certain areas of the wing, making spot classification difficult, and a delay in development of the larvae. We have established and evaluated an improved HB cross (ORR; flr³ females and mwh males) producing ORR heterozygous individuals. These develop normally and have a normal, undisturbed wing hair pattern while exhibiting high bioactivation. The hybrid larvae of the improved HB cross show P450-dependent bioactivation capacity equal to or even slightly higher than those of the original HB cross. This was demonstrated by measuring the genotoxic activity of the promutagens diethylnitrosamine, 7,12-dimethyl-benz[a]anthracene, N-nitrosopyrrolidine, and urethane. In addition, the improved HB cross has a sensitivity to the direct-acting alkylating agent ethyl nitrosourea equal to that of the standard cross. The main advantage of the improved HB cross is to combine the high bioactivation capacity with the ease of scoring the wings using the same criteria as for the standard cross.

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Complex interactions between nicotine and resveratrol in the Drosophila melanogaster wing spot test
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Nicotine (NIC) and resveratrol (RES) are chemicals in tobacco and wine, respectively, that are widely consumed concurrently worldwide. NIC is an alkaloid known to be toxic, addictive and to produce oxidative stress, while RES is thought of as an antioxidant with putative health benefits. Oxidative stress can induce genotoxic damage, yet few studies have examined whether NIC is genotoxic in vivo. In vitro studies have shown that RES can ameliorate deleterious effects of NIC. However, RES has been reported to have both antioxidant and pro-oxidant effects, and an in vivo study reported that 0.011 mM RES was genotoxic. We used the Drosophila melanogaster wing spot test to determine whether NIC and RES, first individually and then in combination, were genotoxic and/or altered the cell division. We hypothesized that RES would modulate NIC’s effects. NIC was genotoxic in the standard (ST) cross in a concentration-independent manner, but not genotoxic in the high bioactivation (HB) cross. RES was not genotoxic in either the ST or HB cross at the concentrations tested. We discovered a complex interaction between NIC and RES. Depending on concentration, RES was protective of NIC’s genotoxic damage, RES had no interaction with NIC, or RES had an additive or synergistic effect, increasing NIC’s genotoxic damage. Most NIC, RES, and NIC/RES combinations tested altered the cell division in the ST and HB crosses. Because we used the ST and HB crosses, we demonstrated that genotoxicity and cell division alterations were modulated by the xenobiotic metabolism. These results provide evidence of NIC’s genotoxicity in vivo at specific concentrations. Moreover, NIC’s genotoxicity can be modulated by its interaction with RES in a complex manner, in which their interaction can lead to either increasing NIC’s damage or protecting against it.
Assessment of the genotoxic potential of three novel composite nanomaterials using human lymphocytes and the fruit fly Drosophila melanogaster as model systems
2022, Chemical Engineering Journal Advances
Three novel composite nanomaterials, namely Clay/CNTs, GO/ADMA and Clay/ADMA were synthesized and tested for their genotoxic, mutagenic and cytotoxic potential. The cytokinesis block micronucleus (CBMN) assay, applied in human lymphocytes in vitro, demonstrated a lack of genotoxic activity of all three composite nanomaterials at all tested doses i.e. 10, 25 and 50 μg mL⁻¹. As far as their cytotoxicity is concerned, statistically significant cytotoxic activity was shown at the two highest doses (25 and 50 μg mL⁻¹) of Clay/CNTs and GO/ADMA whereas Clay/ADMA possessed slightly statistically significant cytotoxic potential only at the highest dose. Similarly, the in vivo SMART assay showed no increase in the frequency of the spontaneous mutation spots in Drosophila melanogaster indicating lack of genotoxic activity of all three composite nanomaterials tested at concentrations ranging from 100-4000 μg mL⁻¹. The present data assess the toxic profile of novel composite nanomaterials, demonstrating the plausible differences with their respective base counterparts and the ability to alleviate genotoxic effects leading potentially to further uses in medicinal applications.
Bioassay-guided study of the anti-inflammatory effect of Anoectochilus burmannicus ethanolic extract in RAW 264.7 cells
2021, Journal of Ethnopharmacology
Anoectochilus species is a small terrestrial orchid found in tropical and subtropical rain forest. These orchids are traditionally used extensively in China, Taiwan, and Vietnam due to their medicinal properties and therapeutic benefits. They are employed for treatment in different systems, such as stomach disorders, chest pain, arthritis, tumor, piles, boils, menstrual disorders, and inflammation. Aqueous extract of Anoectochilus burmannicus (AB) has been previously reported to exhibit anti-inflammatory activities, however there is a lack of evidence regarding its bioactive compounds and the mechanism of its actions.
The objectives of this study were to identify the anti-inflammatory compound(s) in an ethanolic extract of AB and to determine its anti-inflammatory mechanisms in LPS-stimulated macrophages and also its safety.
The ethanolic extract of AB (ABE) was prepared and subsequently subjected to polarity-dependent extraction using n-hexane and ethyl acetate, which would result in isolation of the n-hexane (ABH), ethyl acetate (ABEA), and residue or aqueous (ABA) fractions. The AB fractions were investigated to determine total phenolic and flavonoid content, antioxidant capacity, toxicity, and safety in RAW 264.7 macrophages, human PBMCs, and RBCs. After extraction anti-inflammation screening of each extract was performed by nitric oxide (NO) production assay. The active fractions were further examined for their effect on proinflammatory mediators. In addition, kinsenoside content in the active fractions was identified using LC-MS/MS. Cellular toxicity and genotoxicity of AB were also tested using the wing spot test in Drosophila melanogaster.
The data showed that ABEA had the highest phenolic content and level of antioxidant activities. ABE, ABEA, and ABA, but not ABH, significantly inhibited the LPS-stimulated NO production in the macrophages. Both ABEA and ABA reduced LPS-mediated expression of TNF-α, IL-6, iNOS, and COX-2 at both mRNA and protein levels. Besides, only ABEA notably diminished the LPS-stimulated p65 phosphorylation required for nuclear translocation and transcriptional activation of the nuclear factor-κB (NF-κB). Interestingly, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed ABA contained a high level of kinsenoside, a likely anti-inflammatory compound, while ABE and ABEA might require other compounds in combination with kinsenoside for the inhibition of inflammation. It was shown that all active fractions were neither cytotoxic nor genotoxic.
Our study demonstrated that the hydrophilic fractions of AB exhibit anti-inflammatory activity in LPS-stimulated macrophages. The mechanism used by the AB involves the scavenging of free radicals and the reduction of proinflammatory mediators, including IL-1β, IL-6, TNF-α, NO, iNOS and COX-2. The anti-inflammatory action of AB involves the suppression of the NF-κB signaling pathway by some unknown component(s) present in ABEA. This study found that kinsenoside is a major active compound in ABA which could be used as a biomarker for the quality control of the plant extraction. This study provides convincing significant information in vitro regarding the anti-inflammatory mechanism and preliminary evidence of the safety of Anoectochilus burmanicus. Therefore, the knowledge acquired from this study would provide supportive evidence for the development and standardization of the use of the extract of this plant as alternative medicine or functional food to prevent or treat non-communicable chronic diseases related to chronic inflammation.
Mutagenic and genotoxic activities of Phospholipase A<inf>2</inf> Bothropstoxin-I from Bothrops jararacussu in Drosophila melanogaster and human cell lines
2021, International Journal of Biological Macromolecules
Phospholipase A₂ Bothropstoxin-I (PLA₂ BthTX-I) is a myotoxic Lys49-PLA₂ from Bothrops jararacussu snake venom. In order to evaluate the DNA damage caused by BthTX-I, we used the Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster and Comet assay in HUVEC and DU-145 cells. For SMART, different concentrations of BthTX-I (6.72 to 430 μg/mL) were used and no significant changes in the survival rate were observed. Significant frequency of mutant spots was observed for the ST cross at the highest concentration of BthTX-I due to recombinogenic activity. In the HB cross, BthTX-I increased the number of mutant spots at intermediate concentrations, being 53.75 μg/mL highly mutagenic and 107.5 μg/mL predominantly recombinogenic. The highest concentrations were neither mutagenic nor recombinogenic, which could indicate cytotoxicity in the wing cells of D. melanogaster. In vitro, all BthTX-I concentrations (1 to 50 μg/mL) induced decrease in HUVEC cell viability, as well as in DU-145 cells at concentrations of 10, 25, and 50 μg/mL. The comet assay showed that in HUVEC and DU-145 cells, all BthTX-I concentrations promoted increase of DNA damage. Further studies should be performed to elucidate the mechanism of action of PLA₂ BthTX-I and its possible use in therapeutic strategies against cancer.
Modulating effect of vitamin D3 on the mutagenicity and carcinogenicity of doxorubicin in Drosophila melanogaster and in silico studies
2020, Food and Chemical Toxicology
Citation Excerpt :
These strains were kept in glass vials filled with a maintenance medium (i.e., agar-agar, banana, yeast, methylparaben, water and penicillin/streptomycin) in a Bio-Oxygen Demand-type (B.O.D.) chamber (Model: SL224, SOLAB – Equipamentos para Laboratórios Ltda., São Paulo, SP, Brazil) at 25 ± 1 °C, under 12 h light/12 h dark cycles of photoperiod. Two crosses were carried out to produce the experimental larval progeny: (1) Standard (ST) cross: mwh/mwh males crossed with flare-3 virgin females (Graf et al., 1984, 1989); (2) High bioactivation (HB) cross: mwh/mwh males crossed with ORR; flare-3 virgin females (Graf and van Schaik, 1992). These crosses yielded two types of offspring: marked trans-heterozygous (MH) (mwh +/+ flr3) flies with phenotypically wild-type wings and balanced heterozygous (BH) (mwh+/+TM3, BdS) flies with phenotypically serrated wings.
Vitamin D3 (VD3) deficiency increases DNA damage, while supplementation may exert a pro-oxidant activity, prevent viral infections and formation of tumors. The aim of this study was to investigate the mutagenicity and carcinogenicity of VD3 alone or in combination with doxorubicin (DXR) using the Somatic Mutation and Recombination Test and the Epithelial Tumor Test, both in Drosophila melanogaster. For better understanding of the molecular interactions of VD3 and receptors, in silico analysis were performed with molecular docking associated with molecular dynamics. Findings revealed that VD3 alone did not increase the frequency of mutant spots, but reduced the frequency of mutant spots when co-administered with DXR. In addition, VD3 did not alter the recombinogenic effect of DXR in both ST and HB crosses. VD3 alone did not increase the total frequency of tumor, but significantly reduced the total frequency of tumor when co-administered with DXR. Molecular modeling and molecular dynamics between calcitriol and Ecdysone Receptor (EcR) showed a stable interaction, indicating the possibility of signal transduction between VD3 and EcR. In conclusion, under these experimental conditions, VD3 has modulatory effects on the mutagenicity and carcinogenicity induced by DXR in somatic cells of D. melanogaster and exhibited satisfactory interactions with the EcR.
Betulinic acid modulates urethane-induced genotoxicity and mutagenicity in mice and Drosophila melanogaster
2020, Food and Chemical Toxicology
Betulinic acid (BA) is a pentacyclic triterpenoid found in several plant species. Urethane (URE) is a known promutagen. Here, we examine the genotoxicity and mutagenicity of BA alone or in combination with URE using the bone marrow micronucleus assay in mice bone marrow cells and the Somatic Mutation and Recombination Test in Drosophila melanogaster. Findings revealed that BA alone was not genotoxic, but reduced the frequency of micronucleus when compared to the positive control. No significant differences were observed in the cytotoxicity. Biochemical analyzes showed no significant differences for liver (AST and ALT) or renal (creatinine and urea) function parameters, indicating the absence of hepatotoxic and nephrotoxic effects. BA alone did not increase the frequency of mutant spots, but reduced the total frequency of mutant spots when co-administered with URE in both ST and HB crosses. In addition, BA reduced the recombinogenic effect of URE at the highest concentrations of both crosses. In conclusion, under experimental conditions, BA has modulatory effects on the genotoxicity induced by URE in mice, as well as in somatic cells of D. melanogaster. We suggest that the modulatory effects of BA may be mainly due to its antioxidant and apoptotic properties.

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Mutation Research/Environmental Mutagenesis and Related Subjects

Abstract

References (22)

Statistical methods to decide whether mutagenicity test data from Drosophila assays indicate a positive, negative or inconclusive result

Mutation Res.

New tester strains with improved bioactivation capacity for the Drosophila wing spot test

Mutation Res.

Drosophila wing spot test: improved detectability of genotoxicity of polycyclic aromatic hydrocarbons

Mutation Res.

Genotoxicity of ethyl carbamate in the Drosophila wing spot test: dependence on genotype-controlled metabolic capacity

Mutation Res.

Somatic mutation and recombination test in Drosophila melanogaster (wing spot test): Effects of extracts of airborne particulate matter from fire-exposed and non fire-exposed building ventilation filters

Chemosphere

Thirty compounds tested in the Drosophila wing spot test

Mutation Res.

Genetic regulation of the cytochrome P-450 system in Drosophila melanogaster, II. Localization of some genes regulating cytochrome P-450 activity

Chem.-Biol. Interact.

Genetic regulation of the cytochrome P-450 system in Drosophila melanogaster. I. Chromosomal determination of some cytochrome P-450-dependent reactions

Chem.-Biol. Interact.

Genetic variation in cytochrome P-450 and xenobiotic metabolism in Drosophila melanogaster

Biochem. Pharmacol.

Genotoxicity testing of antiparasitic nitrofurans in the Drosophila somatic mutation and recombination test

Mutagenesis

Biotransformation of xenobiotics in Drosophila melanogaster and its relevance for mutagenicity testing

Drug Metab. Rev.

Cited by (172)

Complex interactions between nicotine and resveratrol in the Drosophila melanogaster wing spot test

Assessment of the genotoxic potential of three novel composite nanomaterials using human lymphocytes and the fruit fly Drosophila melanogaster as model systems

Bioassay-guided study of the anti-inflammatory effect of Anoectochilus burmannicus ethanolic extract in RAW 264.7 cells

Mutagenic and genotoxic activities of Phospholipase A<inf>2</inf> Bothropstoxin-I from Bothrops jararacussu in Drosophila melanogaster and human cell lines

Modulating effect of vitamin D3 on the mutagenicity and carcinogenicity of doxorubicin in Drosophila melanogaster and in silico studies

Betulinic acid modulates urethane-induced genotoxicity and mutagenicity in mice and Drosophila melanogaster