## 4 Discussion
observed that exposure to CBZ led to a significant reduction of AChE activity in _Perna viridis_[21]. [PERSON] et al. observed that exposure to 10 ug/L CBZ led to slight reduction of AChE activity in zebrafish [47]. Interestingly, the present study showed that AChE activity in zebrafish was enhanced when exposed to combined stress of Cd and CBZ. The increase of AChE activity has been related to the production of free radicals [48] and to the apoptotic processes occurring in the organism [49; 50]. AChE activity increase leads to fast acetylcholine degradation and downregulation of acetylcholine receptors, influencing the cognitive functions of the organism [51]. Metabolic regulation plays a very important role in environmental stress tolerance, since supplying enough energy to meet the energy demand is crucial for survival [52]. To assess the alterations in the pathways of cellular energy production induced by toxicants, LDH was quite often used, since it is the terminal enzyme of anaerobic glycolysis located in the cytoplasm [53]. Some previous studies showed that heavy metals can influence the LDH activity. For example, [PERSON] et al. found that mercury exposure can reduce the LDH activity in gill of _L. marginailis_[54]. [PERSON] et al. found that LDH activity in rat can be reduced by mixed-metal exposure, including Pd, Cd, Co, and Cr [55]. In the present study, we found that exposure to Cd can deplete LDH activity, which potentially indicates the increase of energy costs associated with raised metabolic regulatory demands [56]. LDH activity was induced by CBZ exposure and the combined exposure of CBZ and Cd, suggesting that increased use of the anaerobic pathways for producing energy is likely to require additional energy to deal with this stress factor. The increase in use of this pathway under pollution stress has been observed in several organisms exposed to contaminants [57; 58; 59]. VTG is an egg yolk precursor that is normally produced in oviparous females, which provides energy for embryonic development in adult female ovipara [60]. VTG has been considered as a sensitive biomarker for monitoring estrogenic activity in the environment [61], since it responds to estrogens circulating in blood plasma [60; 61; 62]. In the present study, VTG content and E2 decreased sharply in Cd exposure groups. For the case of VTG, since Cd has a strong affinity with estrogen receptor [63], we hypothesize that the binding of Cd with this receptor might reduce the available binding sites for estradiol, resulting in the reduction of VTG content. This hypothesis needs further experimentation to verify. For the case of E2, Cd is likely to completely abolish the E2 induction of the receptor gene and the estrogen receptors [64]. CBZ inhibited the VTG content and E2 in female zebrafish gradually. Some previous studies showed that CBZ has the potential to disrupt the endocrine system. For example, [PERSON] et al. found that VTG expression was reduced after 21 days of exposure to CBZ [65]. [PERSON] et al. found that parent exposure to CBZ can disrupt the sexual differentiation in the F1 offspring [66]. The reduction of VTG content in female fish is presumably due to the decrease in E2 [67], which agrees with the results in the present study (Figure 4A). Interestingly, the VTG and E2 content in female fish were also reduced by the combined exposure of Cd + CBZ, but are lower than those in the CBZ single exposure group. The detailed mechanism needs further research.