## 4 Discussion
As for the last objective of this study, the results from the SMART and Comet assays showed that BPA exposure only causes genetic damage in Drosophila cells at environmentally unrealistic exposure doses, with no genotoxic effects at the concentration that corresponds, in flies, to the human LOAEL (1 mM). Nevertheless, the use of these higher concentrations allowed us to establish a concentration-effect relationship, since a significant increase in genetic damage with increasing concentration was observed. Besides BPA, the SMART assay has already been used in a study evaluating the genotoxicity of bisphenol A-glycidyl methacrylate (BisGMA), a monomeric resin used in dentistry [81]. In that study, however, the results were negative for all concentrations used, which may be because those concentrations were inferior to the ones used in this work. Another explanation provided by the authors to justify the obtained results was the fact that the dimension and inflexibility of BisGMA could render it difficult for it to access the nucleus via nuclear pores. No similar studies using the Comet assay in Drosophila are available to enable a direct comparison of the results; however, there are studies involving other model organisms, such as the invertebrate midge _Chironomus triparius_[82; 83; 84]. In that study, the authors observed an increase in the extent of DNA damage in fourth-instar larvae, as compared to control solvent-exposed individuals, in both concentrations tested (0.5 and 3 mg/L). Interestingly, they tested the effect of two different exposure times (24 h and 96 h), and found that the extent of DNA damage at 0.5 mg/L decreased with exposure time, possibly due to the activation of DNA-repairing mechanisms under prolonged exposure to BPA. That conclusion may thus explain the fact that, in our study, no significant increase in DNA damage was observed at the lowest concentration (1 mM), since the treatment we used was administered until the third stage of larval development. The present study involved relatively high (millimolar) concentration ranges of BPA, varying from 0.5 to 50 mmol/L. This was chosen to assess sublethal and acute toxic effects in _D. melanogaster_, according to the experimental toxicology approaches generally used in invertebrates [85; 86; 87]. It is also a standard approach in ecological and ecotoxicological essays to detect cellular and molecular effects that may remain undetectable at lower doses. This is particularly important for mechanisms that may exhibit non-linear or threshold-dependent responses. Although these doses exceed typical environmental or dietary exposure levels, they may be applied in mechanistic studies in order to elicit, and thereby elucidate, physiological, behavioural and developmental responses, which are quantitatively assessed. The use of a wide concentration gradient, including high doses, allows for the identification of toxicity thresholds, the characterization of dose-dependent effects and the investigation of potential compensatory stress-response mechanisms. High concentrations are particularly important when working with _Drosophila_ species, including _D. melanogaster_, which generally exhibit high tolerance to xenobiotics, due to their rapid metabolism and protective barriers. Moreover, similar concentrations of BPA have already been used in previous studies involving invertebrate models, including _Drosophila_, aiming to study endocrine-disrupting activity, reproductive toxicity, oxidative stress and gene-expression alterations. Therefore, the chosen concentration range may be justified within the context of ecotoxicology and exploratory toxicology, being consistent with the goals of assessing the mechanistic impacts of BPA at different intensity levels. [PERSON] et al. [85] exposed adult _D. melanogaster_ to 0.5 mM BPA solution and observed significant morphological alterations in the midgut, which reduced the survival rate and impaired climbing ability. Oxidative stress was considered to be a key mechanism, justifying BPA-induced gastrointestinal toxicity.