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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.

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., Highlights • VD3 was not toxic, mutagenic neither carcinogenic to Drosophila melanogaster. • VD3 has modulatory effects on the mutagenicity and carcinogenicity induced by DXR in Drosophila. • VD3 revealed a modulatory effect without altering the recombinogenic activity of DXR. • VD3 showed satisfactory interactions with the Ecdysone Receptor (EcR).

The aim of this study was to determine ecotoxicological parameters for biomonitoring of environmental risk of native soils from a ceramic industrial area that had been contaminated with cadmium (Cd) and chromium (Cr) by using the earthworm, Eisenia fetida. Initially, lab tests were conducted to compare earthworm (Eisenia fetida) growth, survival, morphology, behavior, and reproduction rates following exposure to six concentrations of contaminated soil at 0%, 6.25%, 12.5%, 25%, 50%, or 100% mixed in artificial soil and cow dung following a 28-d incubation period. The second experiment consisted of utilizing Eisenia fetida in a predetermined lowest observed effect concentration to measure heavy metals bioaccumulation from superficial soil collected from a ceramic industrial area following a 56-d exposure. Data demonstrated that in the lab earthworms maintained at 6.25% of contaminated soil, exhibited significant increase in mean weight, bioaccumulation of Cd and Cr associated with a significant decrease in the amount of Cd and Cr in the soil. At field testing, similar results that were observed as in the lab as evidenced by rise in mean weight, higher levels of Cd and Cr in the earthworm tissue accompanied by significant fall in soil levels of Cd and Cr. In conclusion, at tested relevant environmental concentrations, the use of Eisenia fetida for assessing ecotoxicological risk arising from contaminated soil due to ceramic industrial pollutant emissions was found to be an effective tool for biomonitoring program.

Melipona scutellaris Latreille, 1811 (Hymenoptera, Apidae) is a pollinator of various native and cultivated plants. Because of the expansion of agriculture and the need to ensure pest control, the use of insecticides such as fipronil (FP) has increased. This study aimed to evaluate the effects of sublethal doses of FP insecticide on M. scutellaris at different time intervals (6, 12, and 24 h) after exposure, via individually analyzed behavioral biomarkers (locomotor activity, behavioral change) as well as the effect of FP on different brain structures of bees (mushroom bodies, antennal cells, and optic cells), using sub-individual cell biomarkers (heterochromatin dispersion, total nuclear and heterochromatic volume). Forager bees were collected when they were returning to the nest and were exposed to three different concentrations of FP (0.40, 0.040, and 0.0040 ng a.i/bee) by topical application. The results revealed a reduction in the mean velocity, lethargy, motor difficulty, paralysis, and hyperexcitation in all groups of bees treated with FP. A modification of the heterochromatic dispersion pattern and changes in the total volume of the nucleus and heterochromatin were also observed in the mushroom bodies (6, 12, and 24 h of exposure) and antennal lobes (6 and 12 h) of bees exposed to 0.0040 ng a.i/bee (LD50/100). FP is toxic to M. scutellaris and impairs the essential functions required for the foraging activity.

Nanoparticles have been widely used in several sectors and their long-term effect on the body and environment remains unknown. To evaluate the mutagenic, recombinogenic and carcinogenic potential of 11 nm titanium dioxide nanocrystals (TiO2 NCs), the Somatic Mutation and Recombination Test (SMART) and the Test for Detection of Epithelial Tumors Clones (Warts-Wts) were used, both in Drosophila melanogaster. Third-instar larvae (72 + 4 h), obtained in both tests, were treated with different concentrations of TiO2 NCs ranging from 6.25 to 100 mM. Ultrapure water and urethane were used as negative and positive controls, respectively. At ST cross, all concentrations of TiO2 NCs showed a significant increase in the frequencies of mutant spots, demonstrating higher recombination rates. At the HB cross, only the 50 mM concentration showed a negative result. In the Wts Test, all used concentrations were carcinogenic, except for the 100 mM one, which was toxic. No relationship was demonstrated between the used concentrations and the obtained responses. There was no interference of the cytochrome P450 enzyme complex in the induction of mutant spots.

Thiamethoxam (TMX) belongs to a class of neuro-active insecticides referred as neonicotinoids, while actara® (AC) is one of the most popular TMX-based products in Brazil. The aim of this study was to evaluate the mutagenic, recombinogenic and carcinogenic potential of TMX and AC insecticides. The mutagenic and recombinogenic effect of TMX and AC were evaluated in vivo by the Somatic Mutation and Recombination Test (SMART) while carcinogenic effects were evaluated through the Test for Detection of Epithelial Tumor Clones (wts test), both in somatic cells of Drosophila melanogaster. In the SMART, third instar larvae from standard (ST) and high bioactivation (HB) crosses were treated with different concentrations of TMX and AC (2.4; 4.8; 9.7 × 10−4 mM and 1.9 × 10−3 mM). The results revealed mutagenic effects at the highest concentrations tested in the HB cross. In the test for the detection of epithelial tumor, third instar larvae resulting from the cross between wts/TM3, Sb1 virgin females and mwh/mwh males were treated with the same concentrations of TMX and AC used in the SMART. No carcinogenic effect was observed at any of the concentrations tested. In this work, the inhibition of the mechanism of repair by homologous recombination was observed in flies exposed to 9.7 × 10−4 and 1.9 × 10−3 mM of AC. In conclusion, TMX and AC demonstrated to be a promutagen in the highest concentrations tested.

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.

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Tiametoxam (TMX), Imidacloprido (IMI), e Acetamiprido (ACP) são inseticidas neurotóxicos neonicotinóides, agonistas aos receptores nicotínicos da acetilcolina. Actara® (AC), Premier® (PRM) e Mospilan® (MOP) são produtos formulados à base de TMX, IMI e ACP, respectivamente. O presente estudo teve como objetivo avaliar a toxicidade e os potenciais mutagênicos, recombinogênicos e carcinogênicos desses inseticidas. Os efeitos mutagênicos e recombinogênicos foram avaliados in vivo por meio do “Somatic mutation and recombination test” (SMART) em Drosophila melanogaster. Larvas de 72h, descendentes dos cruzamentos padrão (ST) e de alta bioativação metabólica (HB), foram tratadas com diferentes concentrações de TMX, IMI, ACP, AC, PRM ou MOP, por aproximadamente 48h. Todos os inseticidas foram tóxicos nas maiores concentrações. TMX e AC não foram mutagênicos no cruzamento ST, mas induziram aumentos estatisticamente significativos nas frequências de manchas mutantes nas concentrações de 9,7x10-4 e 1,9x10-3 mM do cruzamento HB. IMI induziu aumento significativo apenas nos tratados com 2,4 e 4,8x10-4 mM do cruzamento HB, enquanto o produto formulado PRM foi mutagênico em todas as concentrações (de 1,2 a 9,7x10-4 mM), em ambos os cruzamentos. ACP e MOP não foram mutagênicos no cruzamento ST. No cruzamento HB o ACP induziu aumento significativo de manchas mutantes em todas as concentrações utilizadas, enquanto o MOP foi mutagênico apenas nas concentrações de 1,9 e 3,9x10-3. Os efeitos carcinogênicos dos inseticidas foram avaliados por meio do Epithelial Tumor Test (ETT) em D. melanogaster. Larvas de 72h resultantes do cruzamento entre fêmeas virgens wts/TM3, Sb¹ e machos da linhagem mwh/mwh foram tratadas com as mesmas concentrações testadas no SMART. Foi observada atividade carcinogênica apenas nos tratados com o inseticida PRM. Conclusão: Os inseticidas e os respectivos produtos formulados foram tóxicos, mutagênicos após serem ativados por enzimas do citocromo P450 (CYP6A2) (com exceção do PRM, que foi mutagênico mesmo em níveis enzimáticos basais) e não carcinogênicos (com exceção do PRM, que apresentou efeitos carcinogênicos). Os ingredientes inertes interferem na toxicidade e mutagenicidade dos ingredientes ativos. Thiamethoxam (TMX), Imidacloprid (IMI) and Acetamiprid (ACP) are defined as neonicotinoid neurotoxic insecticides, acting as agonists to nicotinic acetylcholine receptors. Actara® (AC), Premier® (PRM) and Mospilan® (MOP) are formulated products composed of TMX, IMI and ACP, respectively. The present study aimed to assess the toxicity and mutagenic, recombinogenic and carcinogenic potential of these insecticides. The mutagenic and recombinogenic effects were evaluated in vivo through the somatic mutation and recombination test (SMART) in Drosophila melanogaster. Larvae of 72h resulting from descendants of standard crosses (ST) and high metabolic bioactivation (HB) crosses were treated with different concentrations of TMX, IMI, ACP, AC, PRM or MOP for approximately 48 h. All the insecticides were toxic at the highest concentrations. TMX and AC were non-mutagenic at the ST crossing, but induced statistically significant increases in mutant spot frequencies at the concentrations of 9.7x10-4 and 1.9x10-3 mM regarding the HB crossing. IMI induced a significant increase only in those treated with 2.4 and 4.8x10-4 mM of the HB crossing, whereas the formulated PRM was mutagenic at all concentrations (from 1.2 to 9.7x10-4 mM) in both the crosses. ACP and MOP were not mutagenic considering the ST crossing. At the HB crossing, ACP induced a significant increase of mutant spots at all concentrations tested, while MOP was mutagenic only at the concentrations 1.9 and 3.9x10-3. The carcinogenic effects of insecticides were evaluated using the Epithelial Tumor Test (wts) in D. melanogaster. Larvae of 72h descended from the crossing between virgin females wts/TM3, Sb1 and mwh/mwh males were treated with the same concentrations used in SMART. In this case, carcinogenic activity was observed only in those treated with the PRM insecticide. As conclusion, the findings revealed toxic and mutagenic effects of the insecticides and their formulated products after being activated by cytochrome P450 enzymes (CYP6A2) (except PRM, which was mutagenic even at basal enzyme levels) and non-carcinogenic (except PRM, which showed carcinogenic effects). Thus, the inert ingredients interfere on the toxicity and mutagenicity of the active ingredients. Tese (Doutorado)

Fipronil (FP) is an insecticide that belongs to the phenylpyrazole chemical family and is used to control pests by blocking GABA receptor at the entrance channel of the chlorine neurons. The aim of this study was to evaluate the mutagenic, recombinogenic and carcinogenic potential of FP. The mutagenic and recombinogenic effects were evaluated using the somatic mutation and recombination test (SMART) on wing cells of Drosophila melanogaster. Third instar larvae from standard (ST) and high bioactivation (HB) crosses were treated with different concentrations of FP (0.3, 0.7, 1.5 or 3.0 × 10-5 mM). The results showed mutagenic effects at all concentrations tested in the HB cross; and all concentrations tested in the ST cross, except at concentration of 0.7 × 10-5 mM. The carcinogenic effect of FP was assayed through the test for detection of epithelial tumor (warts) in D. melanogaster. Third instar larvae from wts/TM3 virgin females mated to mwh/mwh males were treated with different concentrations of FP (0.3, 0.7, 1.5 or 3.0 × 10-5 mM). All these concentrations induced a statistically significant increase in tumor frequency. In conclusion, FP proved to be mutagenic, recombinogenic and carcinogenic in somatic cells of D. melanogaster.

The aim of the present study was to appraise the mutagenic and recombinogenic potential of bupropion hydrochloride (BHc) and trazodone hydrochloride (THc). We used standard (ST) and the high bioactivation (HB) crossings from Drosophila melanogaster in the Somatic Mutation and Recombination Test. We treated third-instar larvae from both crossings with different concentrations of BHc and THc (0.9375 to 7.5 mg/mL). BHc significantly increased the frequency of mutant spots in both crossings, except for the lowest concentration in the ST crossing. ST had also the mostly recombinogenic result, and in the HB, BHc was highly mutagenic. On the other hand, THc significantly increased the frequency of mutant spots in both the ST and HB crossings at all concentrations. The three initial concentrations were recombinogenic and the highest concentration was mutagenic for the THc. BHc and THc at high concentrations were toxic, even though their mutagenicity was not dose-related. THc significantly increased the frequency of mutant spots when metabolized, probably as a result of the production of 1-(3′-chlorophenyl) piperazine. BHc was essentially recombinogenic and when metabolized, it became mutagenic. THc was recombinogenic in both crossings. Further studies are needed to clarify the action mechanisms from BHc and THc.