## 3 Results and Discussion
ostreatus_-synthesized ZnO NPs. C1 (control with pure pET30 DNA), C2 (control with DNA+ H\({}_{2}\)O\({}_{2}\)), lanes 3.0–100 represent increasing concentrations of _P. ostreatus_-synthesized ZnO NPs in \(\upmu\)g/mL. mitochondrial dehydrogenases only occurs in viable cells; thus, the intensity of formazan absorbance is directly proportional to the percentage of viable cells in the sample [55]. The mushroom extract (0-2000 \(\upmu\)g/mL) caused a variable response in HepG2 cells, with cell viability above 100% for all treatments (Figure 5A). In contrast, the mushroom extract induced a dose-dependent decline in cell viability for the Hek293 cells, with the lowest cell viability of 22% recorded at 2000 \(\upmu\)g/mL (Figure 5B). The mushroom-synthesized ZnO NPs induced similar cytotoxic responses for the HepG2 (Figure 5C) and Hek293 (Figure 5D) cells. There was a sharp decline in cell viability from approximately 97% at 16 \(\upmu\)M to 12% at 100 \(\upmu\)M in HepG2 cells, and 94% and 22% cell viability was observed in Hek293 cells at the same concentrations. The observed results for mushroom-synthesized ZnO NPs are consistent with the findings of [PERSON] et al. [56], who stipulated that ZnO NPs have the potential to cause cytotoxicity in many cells, including HepG2 cells. Cell viabilities below 20% were recorded from the concentrations of 1000-2000 \(\upmu\)g/mL treatments in both HepG2 (Figure 5C) and Hek293 cells (Figure D). The decreased cell viability indicates a diminished capacity of mitochondrial dehydrogenases to convert the yellow tetrazolium salt into purple formazan crystals [55]. Thus, the lower IC50 values in Hek293 and HepG2 cells following ZnO treatment (Table 2) suggest increased sensitivity of the liver and kidney cells to this exposure (Figure 5C,D). In particular, these values denote mitochondrial toxicity since cell viability decreased. Recently, [PERSON] et al. [57] showed  
Figure 5: The dose response curves derived from the MTT assay. (**A**) The mushroom extract was not cytotoxic to HepG2, with increased cell viability occurring with increased concentrations. (**B**) Cell viability decreased gradually in Hek293 cells treated with increasing mushroom extract concentration. (**C**) ZnO mushroom nanoparticles decreased cell viability significantly between 10 and 100 \(\upmu\)g/mL in HepG2 cells, with more than 80% cell death from 1000 to 2000 \(\upmu\)g/mL. (**D**) The Hek293 cell response to ZnO mushroom nanoparticles indicates cytotoxicity at all concentrations. that ZnO NPs promote ROS generation, inducing mitochondrial dysfunction, and may impair mitochondrial biogenesis in cardiomyocytesocytes [55,57]. The ROS generation may be attributed to interference at complex III of the electron transport chain [58]. The acute cytotoxicity of the mushroom-synthesized ZnO NPs may be due to parameters such as particle size, surface area, and surface reactivity. Previous studies have identified these parameters for other nanoparticles [59]. For example, [PERSON] et al. [60] noted that smaller-sized nanoparticles are more toxic when compared to larger nanoparticles; however, it should also be considered that other factors can influence the toxicity of nanoparticles, as in the case of metallic particles [61]. In this study, the ISO norm that requires >75% cell viability for biomedical products was applied [61]. Thus, an IC25 where HepG2 and Hek293 cells retained 75% viability was applied for the mushroom ZnO NPs and mushroom extract to be deemed safe. The IC25 for the _P. ostreatus_ mushroom extract was 0.074 mg/mL in Hek293 cells (Table 2) and could not be calculated for HepG2 cells (Table 2), which retained cell viability (Figure 5A), indicating the concentrations retained 75% cell viability in both cell lines in accordance with the ISO norm. At the MIC for _P. aeruginosa_ (0.04 mg/mL), the Hek293 and HepG2 cell viability was around 83% and 112%, respectively. Thus, the _P. ostreatus_ mushroom extracts conformed to the ISO standard for the _P.