Introduction: Apoptosis, or programmed cell death, is a crucial process for maintaining tissue homeostasis and regulating cell growth. This process is controlled by the balance between pro-apoptotic genes like BAX and anti-apoptotic genes like BCL-2. Disruption in this balance can lead to various diseases, including cancer and developmental abnormalities. Benzalkonium chloride (BAC) is a widely used biocide in the poultry and pharmaceutical industries. Given the extensive use of this compound, it is essential to investigate its effects on key biological processes such as apoptosis and angiogenesis.
The chick embryo chorioallantoic membrane (CAM) is a well-established model for studying angiogenesis and apoptosis due to its simple structure and the ability to directly observe these processes. This study aims to investigate the effects of BAC on the expression of BAX and BCL-2 genes in the CAM vasculature, marking the first study on BAC's influence on apoptosis in this model.
Methods: This study utilized 14 fertilized eggs obtained from Mahan Kerman Poultry Hatchery. The eggs were randomly divided into two groups of 7 eggs each. The first group was treated with 0.2% BAC, while the second group received PBS (phosphate-buffered saline) as a control. The treatments were administered intra-egg at 24, 48, and 72 hours post-incubation. The CAMs were harvested 24 hours after the final treatment for gene expression analysis.
RNA extraction from the CAM tissues was performed using commercial RNA extraction kits, followed by cDNA synthesis. Gene expression of BAX and BCL-2 was analyzed using Real-Time PCR, with GAPDH serving as the reference gene for normalization. All PCR reactions were conducted in triplicates using specific primers and a Real-Time PCR system. Statistical analysis of the results was performed using SPSS version 25, and a T-test was applied to determine the significance of the data, with a significance threshold set at P<0.05.
Results: Real-Time PCR results demonstrated that BAC significantly increased the expression of the BAX gene in the treated group (P<0.05). Conversely, the expression of BCL-2 was significantly reduced in this group compared to the control (P<0.05). These findings indicate an increase in apoptosis within the CAM vasculature as a result of BAC treatment.
The analysis of the BAX/BCL-2 expression ratio revealed a marked increase in the BAC-treated group, strongly suggesting the activation of pro-apoptotic pathways and the suppression of anti-apoptotic activity.
Conclusion: The findings of this study clearly indicate that BAC has a direct impact on apoptosis in the chick embryo chorioallantoic membrane. The increase in BAX expression and decrease in BCL-2 expression provide strong evidence of BAC-induced apoptosis. These results offer valuable insights into the potential biological impacts of BAC in both biomedical and industrial contexts.
In mammals, angiogenesis plays a crucial role in the formation and growth of the placenta and the fetus. The use of BAC could potentially induce apoptosis, leading to adverse effects on fetal development. However, further research is needed to confirm these effects in mammalian models. Given the widespread use of BAC as a preservative in the pharmaceutical industry and its demonstrated negative effects on fetal development, caution is advised when administering medications containing BAC during pregnancy.
On the other hand, the pro-apoptotic and anti-angiogenic properties of BAC suggest its potential utility in the treatment of angiogenesis- and apoptosis-related diseases, such as controlling tumor growth. Inducing apoptosis in vascular cells could offer a promising strategy for inhibiting tumor angiogenesis and growth, positioning BAC as a potential agent in cancer treatment.