Antitumor, hepatotoxic and cytogenetic effect of the partially purified endotoxin extracted from locally isolated Salmonella typhimurium

This study was conducted for investigating the effect of Gram negative bacterial endotoxin extracted from enteric Salmonella typhimurium on the proliferation of tumor cells in vitro. A total of 95 stool samples were collected from pediatric patients suffering from diarrhea in order to isolate bacteria belonging to Salmonella typhimurium species. After performing microscopic examination, cultural characterization, biochemical and Api 20E identifications, only 9 isolates showed positive S.typhimurium. Following the separation of endotoxin from each isolate the biological activity of endotoxin was estimated by using E-TOXATE kit (Limulus amebocyte lysate test) and the isolate Salmonella typhimurium A3 showed the lowest concentration that gave positive result (0.5 μg/ml), therefore S.typhimurium endotoxin A3 was candidate for the next steps. Partial purification using sepharose Cl-6B gel chromatography for A3 endotoxin was applied and after purification two peaks were obtained. Chemical characterization involving the estimation of carbohydrate and
protein contents showed that the first peak contained the higher carbohydrate contents (38.6%) and lower protein contents (1.15%) as compared with the second peak which showed carbohydrate and protein contents 13.2 and 5.75% respectively. In addition, the first peak showed maximum activity of (≥0.1μg/ml), the second peak and the crude endotoxin showed 2.5 and 0.5μg/ml respectively. Three successive different experiments were undergone to the partially purified endotoxin as follows: Evaluating the cytotoxic effect of endotoxin (peak 1 and 2) treatment on three different tumor cell lines: Mammary Gland Adenocarcinoma (AMN3), Cervical Human Carcinoma (HeLa) and Laryngeal Epidermoid Carcinoma (Hep-2) and on normal rat embryo fibroblast (Ref) cell line in vitro, with the use of different concentrations (12.5, 25, 50, 100, 200 and 400 μg/ml) and in comparison with the negative control. Results showed that, partially purified endotoxin (peak 1) has significant inhibitory effects against AMN3 and Hep-2 cell lines, and the pattern of inhibition was dose dependent and to lesser extent time dependent. Thus 200 and 400 μg/ml showed the maximum inhibitory effect. On the other hand endotoxin recovered from peak 2 showed much less inhibitory action as compared with the endotoxin recovered from peak 1. However, neither endotoxin (peak 1) nor endotoxin (peak 2) showed significant cytotoxic inhibition effects toward HeLa and Ref cell lines. Estimating the cytogenetic effects of endotoxin (peak 1) on human blood lymphocyte culture through determining the frequency of micronucleus induction and using cytokinesis-blocked micronucleus assay (in vitro). Different concentrations were applied (12.5, 25, 50, 100, 200 and 400 μg/ml), in addition to the negative control (phosphate buffer) and positive control mitomycin C (5μg/ml). Results showed that the extracted endotoxin cause significant micronucleus and nucleoplasmic bridge induction, and such effect appeared to be dose dependent as compared with negative and positive treatments. Evaluating the mouse serum level of liver functional enzymes (AST, ALT and ALP) and determining mouse serum TNF-α after intraperitoneal administration of endotoxin (peak 1) for five successive days using these concentrations (12.5, 25, 50, 100, 200 and 400 μg/ml) as compared with the negative control (phosphate buffer). Results indicated that the endotoxin caused a significant dose dependent, increase in serum liver functional enzymes levels with maximum level following 400 μg/ml endotoxin injections. On the other hand liver histopathologicl sections were prepared to demonstrate any histopathological differences and results showed a simple liver damage with low concentrations with increasing hepatic necrosis by increasing the concentration. The amounts of released serum TNF-α as a result of endotoxin challenge were also detected by using TNF-α ELISA kit. The results as expected showed an increased in serum TNF-α by increasing the dose of endotoxin