## 4 Discussion  
Although the NPs formed agglomerates and aggregates, single particles still existed and acted as a major source of particles inside the cells. TEM analysis revealed that ZnO NPs were present inside the yeast cells as single or small aggregates of particles (Figure 3b) and accumulated deep inside the nucleus or protoplasm. The penetration of NPs into the cells or their adhesion to the cell membrane results in membrane destruction, and the cells eventually die. Despite no significant change in mortality, the E2 estrogenic activity in yeast exposed to ZnO NP was reduced as a function of the particle concentration (Figure 2a). Similar response patterns in mortalities and estrogenic activities were observed in the experiment using Al2O3 NP, suggesting a toxicity profile similar to that of ZnO NP (Figure 2b). [PERSON] et al. [16] reported that ZnO NP and bulk ZnO both dose-dependently inhibited the growth of yeast. However, the effects of non-cytotoxic concentrations of ZnO NP still remain unknown. The toxicity of metallic NPs in mammalian cells, as well as the adverse effects, such as mitochondrial dysfunction, membrane damage, intracellular oxidative stress, and abnormal cell morphologies, has been previously reported [20; 21; 22; 23; 24]. From the results obtained in this study, it can be inferred that exposure to non-cytotoxic concentrations of NPs (5-50 mg/L for ZnO NP and 10-50 mg/L for Al2O3 NP) may lead to cell membrane damage. The damaged membranes, in turn, may incidentally accelerate the cellular uptake of NPs. The toxicity of given NPs may lead to cell death, or merely alter a specific function of the cell. Yeast two-hybrid system applied in this study is worked by biological multi-scale processes, such as ligand-receptor binding, protein-protein and DNA-protein interactions, gene expression, and enzyme reaction. If not cell death, the signal reduction of the YES assay indicates the hindrance of binding between E2 and receptor, or the malfunction of other biological processes mentioned above. It can be hypothesized that NPs' inner or outer cells reduce E2 levels, by means of the sorption between NPs and E2. A previous study showed that the E2-sorption capacity of particles was negligible [25], even when the particles were coated with humic acids. Therefore, reduction of available E2 levels by the sorption between NPs and E2, in this case, is unlikely. It is worth noting that at lower concentrations (i.e., \(<\)0.5 mg/L), Ag NP stimulated the E2 estrogenic activities (Figure 2c). Although the E2 estrogenic activity was not significantly increased, a similar pattern was repeatedly observed in the tests using other metallic NPs. This phenomenon may be due to the inadequate destructive strength of Ag NPs to kill the yeast cells in a short time. This insufficient destructive strength may affect the cell membrane, resulting in the easier transport of exogenous chemicals, i.e., E2, into the cell. Finally, the activity of the indicator (i.e., \(\beta\)-galactosidase) increased within a short period. With increasing particle concentrations, more yeast cells were killed, and the estrogenic activity was consequently reduced. The high correlation coefficients between yeast viability and estrogenic activity confirm this proportional relationship, which also supports the above theory. Many studies about NPs have, up to now, been done based on a precautionary approach, because of the lack of tools to detect and quantify NPs in the environment [26]. [PERSON] et al. [27] have, however, succeeded in tracing the TiO2 NP emitted from exterior paints in a small stream. This means that the NPs in the environment become real concerns. Many in vitro assays, including YES assay, are being used to screen the adverse effects of environmental contaminants. According to the present study, metallic NPs could disturb the processes of in vitro assays, resulting in underestimating the adverse effects of contaminants.