div dir="ltr">>Abstract:
This study was conducted for investigating the effect of purified toxin A produced from Pseudomonas aeruginosa isolate on killing of tumor cells in vivo and in vitro . A total of 161 clinical samples were collected from patients suffering from burns, wounds, otitis media, and urinary tract infection from three hospitals in Baghdad. Bacterial isolates were identified by microscopic examination, cultural characteristics, biochemical tests and API 20 E which revealed that only (123) isolates (76.3%) gave typical morphological characteristics and biochemical tests that related to Pseudomonas aeruginosa. Protease production test on skim milk agar was used for detecting the ability of Pseudomonas aeruginosa isolates to produce protease enzyme. Results revealed that only three isolates from the total of 123 isolates that related to Pseudomonas aeruginosa gave negative results for this enzyme. Depends on the results of protease test , protease negative isolates were selected for production of toxin A, the crude supernatant was precipitated by zinc acetate , then the toxicity was detected after injection, intraperitoneally to mice . Results revealed that Pseudomonas aeruginosa (26A) isolate gave high killing rate in mice after 24 hrs, therefore it was selected for production of toxin A. Partial purification of toxin A by ion - exchange chromatography column (DEAE) revealed presence of two peaks, both of them were toxic to mice when injected intraperitoneally. Further purification of toxin A was performed in to gel filteration column Sephacryl S-200, results showed that two peaks were appeared ,and when injected to mice, intraperitoneally, only peak 2 was toxic to mice while peak one was not. Molecular weight of the purified toxin A was detected by Sephacryl S-200 column after using four standards enzyme and protein (Aldolase,
Bovine serum albumin, Ovalbumin and Chymotrypsin) .The molecular weights of the purified toxin A was obtained from the standard curve by blotting the relationships between Ve/Vo and Log of molecular weights of the standard protein. Results showed that the molecular weight of the purified toxin A was estimated to be (65.12) kilodalton. Protein concentration of toxin A were detected at each purification step by the standard Bradford procedure. SDS- Poly acrylamide gel lectrophoresis was applied for detecting purity of toxin A using six standard protein and enzyme (Esterase, y- globulin, Transferrin, bovine serum albumin, Trypsin and lysozyme) .Results declared that toxin A was migrated as a single band in the gel. The pathogenicity of Pseudomonas aeruginosa isolates (26A) was detected in the burn mice model which were subjected to 30 second
burning with hot metal before (1-2) x 108 live cells in (0.5) ml of physiological saline were injected, subcutaneously, in the burned area. Lethality was appeared in untreated group after 24 hrs, of injection . LD50 of the purified toxin A was detected after injecting different concentrations of purified toxin A (0.05, 0.1, 0.2, 0.25, 1,2 and 4) μg/ ml in mice then lethality was recorded for five days . Results showed that LD50 of purified toxin was 1 μg /ml. On the other hand, histopathological examination of liver, spleen and kidney in mice injected with high doses revealed the presence of significant histopatholgical degenerations. Evaluating the mouse serum level of TNF-α after injection of purified toxin at different concentrations proved that TNF- α was reaching two peaks (250, 290) pg/ml after injection purified toxin A at concentrations (0.6,0.8) μg /ml for 36 hrs, respectively. In vitro evaluating the cytotoxic effects of purified toxin A at concentrations (10, 20, 40, 80, 100, 200) ng/ml first on normal cell line which included rat embryo fibroblast cell line (REF) and African green monkey cell line (Vero), and second on tumor cell line including Rhabdomyosarcoma cell line (RD) and mammary adenocarcinoma cell line (AMN3) cell line .Results revealed that the purified toxin A has
significant inhibitory effects on the normal cell line which was dose and time dependent. On the other hand , cytotoxic effects of purified toxin A on tumor cell line (AMN3) and (RD) was dose dependent but time independent only. Transplantation of tumor in vivo was done by injecting AM3 tumor cells, subcutaneously, to mice in the shoulder area ,and after appearance of tumor mass, histopathological examination of tumor mass were done in control and treated groups. For detection of the antitumor activities of purified toxin A in transplantable mice, purified toxin A (0.1) ml was injected at two concentrations (50, 80) ng/ml for three weeks, the concentration (80 ng/ml) cause significant inhibition of tumor mass in which the growth inhibition GI reached (65.5%) after 3 weeks, while the growth inhibition in mice treated with the purified toxin A at concentration (50) ng/ml reached (40.1 %) after 3 weeks. For detecting the ability of the purified toxin A to induce mitochondrial apoptosis in vitro, four cell line (Vero, RD, REF and AMN3) were used and exposed to the purified toxin A at concentrations (80, 250) ng /ml for 24 hrs. Toxin A at concentration (80) ng/ml caused apoptosis at percentages of (80.9%, 61.4%, 35.9% and 40.1%), respectively after 24 hrs, while concentration (250) ng/ml caused apoptosis at percentages of (90.3%, 91.2%, 70.4% and 77.3%)respectively after 24 hrs of exposure. Exposure of these lines to purified toxin A at concentration of (250) ng/ml for 48 hrs lead to complete destruction of all cell lines