Glutathione-S-transferase activity in various organs of Crocodylus siamensis and its attenuation role in aflatoxin B1-induced cell apoptosis in human hepatocarcinoma cells

Abstract

Background and Aim: The crocodile is a model for studying relevant sources of environmental contamination. They were determined an appropriate biomonitoring species for various toxins. The cytosolic and microsomal fraction of crocodiles plays a role in detoxifying xenobiotics. Cytochrome P450 1A2 (CYP1A2) metabolizes aflatoxin B1 (AFB1) to aflatoxin M1, while glutathione-S-transferase (GST) catalyzes carcinogenic agents. This study aimed to investigate the GST activity in various organs of Crocodylus siamensis. Further, the fate of microsomal and cytosolic fractions from various crocodile organs against AFB1-induced apoptosis in human hepatocarcinoma (HepG2) cells was investigated. Materials and Methods: The liver, lungs, intestines, and kidneys tissues from a 3-year-old crocodile (C. siamensis) (n=5) were collected. The cytosolic and microsomal fraction of all tissues was extracted, and protein concentrations were measured with a spectrophotometer. Subsequently, a comparison of GST activity from various organs was carried out by spectrophotometry, and the protective effects of CYP450 and GST activity from various crocodile organs were studied. In vitro AFB1-induced apoptosis in HepG2 cells was detected by reverse transcription-quantitative polymerase chain reaction. Comparisons between the metabolisms of the detoxification enzyme in organs were tested using the Kruskal–Wallis one-way analysis of variance and Dunn's multiple comparison tests. All kinetic parameters were analyzed using GraphPad Prism software version 5.01 (GraphPad Software Inc., San Diego, USA). Results: Total GST activity in the liver was significantly higher than in the kidneys, intestines, and lungs (p<0.05, respectively). The highest GST pi (GSTP) activity was found in the liver, while the highest GST alpha-isoform activity was in the crocodile lung. The kinetics of total GST and GST mu activity in the liver had the highest velocity compared to other organs. In contrast, the kinetics of GSTP enzyme activity was the highest in the intestine. The in vitro study of microsome and cytosol extract against apoptosis induced by AFB1 revealed that the level of messenger RNA expression of the Bax and Bad genes of HepG2 cells decreased in the treatment group in a combination of cytosolic and microsomal fractions of the crocodile liver but not for Bcl-2. Interestingly, the downregulated expression of Bax and Bad genes was also found in the microsome and cytosol of crocodile kidneys. Conclusion: The crocodile liver revealed very effective GST activity and expression of the highest kinetic velocity compared to other organs. The combination of liver microsomal and cytosolic fractions could be used to prevent cell apoptosis induced by AFB1. However, further study of the molecular approaches to enzyme activity and apoptosis prevention mechanisms should be carried out.