EC induced by chemical agents involves multi-step processes. Oxidative damage resulting from ROS generation could participate in all stages of the ESCC process. This study observed that NMBA exposure increases the mRNA levels of cyclin D1, cyclin E, and NF-κB p65, decreases the mRNA level of TGFß1 and increases the protein concentration of cyclin D1 in rat esophageal tissue, which was consistent with findings from other studies (6). In our study, 0.5 mg/kg body weight NMBA three times per week for 5 weeks produced only hyperplasia in the rat esophagus by 20 weeks, while another study showed that three doses/week at 0.5 mg/kg/NMBA over the course of 5 weeks induced esophageal tumors in 100% of the rats by week 20 (31), which may be due to rat species, or the purity of the NMBA.
We observed that absorption of selenium was not affected by NMBA, and there was no significant difference in serum selenium levels between the control and NMBA groups. Also, selenium in esophageal tissue was not meaningfully different in the NMBA group compared to the control group. However dietary selenium levels significantly increased the selenium concentrations in the serum and esophageal tissue. In the esophagus of rats treated with NMBA, increasing selenium levels increased the mRNA levels of cyclin D1 and NF-κB p65. Similar to the increased mRNA levels of cyclin D1, it was found in this study that the concentrations of cyclin D1 were also increased by the dietary selenium level. These changes in the mRNA levels and protein concentration were not dose-dependent.
Selenium, an essential element for human and animal health, shows a narrow range between deficiency and toxicity. Various studies show that the relationship between selenium status and disease onset status can be U-shaped. Regarding the mechanism of selenium toxicity, it is suggested that the high content of selenium in the body pool can lead to excessive production of ROS including superoxide and hydrogen peroxide during the reduction of active selenium compounds (32). Another mechanism for selenium toxicity is its ability to induce proteomic changes by substituting selenomethionine and selenocysteine in proteins in place of methionine and cysteine, respectively. Due to the role of cysteine in catalysis and stabilization of protein disulfide, the structure and function of proteins may be affected by this substitution and lead to toxic effects. Moreover, selenium has potential effects on the epigenetic regulation of DNA and histones (33).
Oxidative damage has been suggested as a major cause of EC, and selenium maybe promoted the development of EC by facilitating oxidative stress (34). Results from a multiple-dose, randomized controlled trial in a country of relatively low selenium status showed that a 300 µg/day dose of selenium taken for five years meaningfully increased all-cause mortality, especially cancer mortality (35). Assessment of elemental concentrations between tumor and non-tumor tissue from ESSC patients reported that the concentration of selenium in tumor tissue was significantly higher than in non-tumor tissue (36). Moreover, the study of the association between selenium concentration in cancerous and non-cancerous tissues with the risk of gastrointestinal cancer suggested that the concentration of selenium in esophageal and stomach cancer tissues was significantly higher than in non-cancerous tissue (37).
The incidence and development of ESSC is a complex process that is strongly associated with the imbalance of oncogenes and tumor suppressor genes (p53 signaling pathway, pathway in cancer), cell proliferation, and cell cycle (38, 39). The multi-step processes of esophageal carcinogenesis are related to overexpression of P53 and cyclin E, cyclin D1 dysregulation, and reduction of P21 (40). Cyclin D1, an essential regulator of the G1/S phase of the cell cycle involved in cell proliferation, is frequently up-regulated and amplified in 46.4% of ESCC cases (41). The NF-kB pathway controls the expression of genes involved in proliferation, migration, and apoptosis that play a key role in the development and progression of cancer. In patients with ESCC, the important proteins in the NF-κB pathway were overexpressed (42).
In this study, our results showed that parallel to the increased mRNA level of NF-κB also the mRNA level and protein concentration of cyclin D1 increased. In oxidative distress status, supra physiological concentration of ROS can irreversibly damage proteins, DNA, and lipids and alter their role and their physical properties (43). Therefore, ROS can be considered a toxic metabolite and one of the motivating factors of cancer (44). ROS activates NF-κB through rapid phosphorylation of NF-κB inhibitor (IκB) and following proteasomal degradation, which leads to the nuclear localization of NF-κB (45). The NF-κB signaling pathway plays an important role in the development of many human cancers. In most cases, cancer cells are characterized by NF-κB combination activation, uncontrolled proliferation, or insensitivity to cell death (46). In Specific types of cancer and at certain stages of cancer development, the role of NF-κB is frequently via increased cell proliferation rather than inhibition of apoptosis (47). A recent study reported that ROS induces the formation of pancreatic pre-neoplastic lesions by activating the NF-κB signaling pathway and promoting cell proliferation (48). Regarding the case of cell proliferation, NF-κB can regulate cyclin D1 expression and promote G1/S transition (49). Cyclin D1 protein is closely related to the incidence and development of ESSC (50).
Our results showed that excess dietary selenium did not affect the mRNA level of P53, KRAS, MGMT, TGFß1, and cyclin E in the esophagus of rats treated with NMBA. Alterations of KRAS, a regulator of cell proliferation, differentiation, and transformation, are considered to be a key biological factor in several cancer types. High KRAS mRNA expression and amplification have been reported in the primary gastroesophageal cancer tissue (51). The p53 tumour suppressor typically induces cell cycle arrest or apoptosis against genotoxic damage or oncogene activation (52). Dietary N- nitroso compounds result in the formation of O6-methylguanine in DNA. DNA repair genes such as MGMT through the removal of highly promutagenic and cytotoxic O6-alkyl adducts on guanine bases play an important role in the genomic stability maintenance and protection of cells from DNA damage (53). TGFß1plays the main role in stopping the proliferation of normal epithelial cells via inhibition of cyclin D and cyclin E mRNA expressions and promoting the invasion of tumor cells (54). A recent study showed that the expression of TGFß1 was significantly down-regulated after the intervention with selenium (55).
We found that excess dietary selenium did not significant effect serum MDA but significantly increased GPx activity. Increasing serum selenium levels, a main component of the antioxidant enzymes can enhance the activity of GPx and TR, thereby protect cell membranes from oxidative stress damage (56). Peroxidation of fatty acids in cell membranes leads to the production of small aldehydes including MDA, which can react with the free amino groups of nucleic acids and proteins. Increased MDA content is an important indicator of lipid peroxidation and reflection of cell membrane damage (57). A recent study reported that increasing plasma selenium concentrations greater than 110 µg/L were associated with increasing levels of 8-oxo-7, 8-dihydroguanine (8-oxo-dG), but with reducing MDA levels and oxidized to decreased glutathione (GSSG/GSH) ratio. These results suggest that high selenium concentrations may increase oxidative stress in some biological processes (58).