Effects of Agkistrodon halys Metalloproteinase in vitro and in vivo

Snake venom contains a number of different, pharmacologically-active proteins and peptides. Most of the haemorrhagic proteins of snake venoms are metalloproteinases. Agkistrodon halys metalloproteinase (AHM) was isolated from the snake venom of Pallas (Mol wt. 23145). In vitro toxicological effects of AHM (0.1-2 mM) on human macrophages (THP-1 and U-937), lung fibroblasts (MRC-5) and murine lung epithelial (LA4) cells were evaluated by (2,3-bis- (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-hydroxide) assay and light microscopy. AHM strongly inhibited cell proliferation and adhesion to extracellular matrix, as well as induced morphological changes in a dose-dependent manner. Apoptosis was evaluated using propidium iodide (PI) staining and a terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay for DNA fragmentation. PI staining indicated an accumulation of cells at the sub-G1-phase following AHM treatment, and there was also DNA fragmentation as shown by TUNEL staining. Besides cell-based assays, an in vivo assessment of AHM (1.56-300 mg/kg, body weight) in mice was also done. Histopathology of muscle fibers revealed massive necrotic aggregations after AHM exposure. There were translucent vacuoles in the purkinje cells, which may cause substantial damage to kidney tubular epithelium. There were also clear areas in the cerebellum due to cell death, deposition of fibrinogen or fibrin on the intestinal epithelium, and skin necrosis following an AHM dose of 300 mg/kg. We also observed marked erythrocyte accumulation in lung alveolar walls that resulted in infarction, along with a consequent reduction of the alveolar space and necrosis linked to neutrophil infiltration. These results cumulatively suggest that AHM induces lethality at high doses, inhibits cell proliferation, and induces morphological changes in various cell types.