Toxicity assessment of emamectin benzoate and its commercially available formulations in Pakistan by in vivo and in vitro assays.

Emamectin benzoate (EMB) is generally considered a safe insecticide in agriculture and veterinary practices, yet, it can cause cytotoxic and genotoxic effects. Hence, the aim of this study was to evaluate toxic effects of 80% EMB and its commercially used formulations (Tycon 1.9% EC and Tycon plus 5% EW) in Pakistan and tested for acute toxicity in albino rats, rabbits and fish (Labeo rohita). Genotoxicity was investigated by in vivo comet assay and bone marrow micronucleues test in the rats. In vitro mutagenicity was tested in Salmonella typhimurium TA98 and TA100. The tested EMB formulations were found moderately toxic (oral LD50: 122-168 mg/kg), causing severe eye irritation in rabbits, highly toxic to fish (LC50: 9-43 µg/L) and found non mutagenic. Oral administrations of EMB (80% and 5%) at 100 mg/kg of body weight to male rats reduced red blood cells, hemoglobin, and slightly increased the blood glucose, urea and liver enzymes levels but had no significant damage to DNA. EMB induced bone marrow toxicity was observed as reduction of polychromatic erythrocytes. Overall, EMB exposure was highly toxic to fish, and caused hemo- and hepatotoxicity in rats. These findings warrant cautious use of EMB formulations in agrochemicals and veterinary medicine.

Development of a one-plasmid system to replace the endogenous protein with point mutation for post-translational modification studies.

BACKGROUND: Post-translational modification (PTM) is one of the major regulatory mechanism for protein activities. To understand the function of PTMs, mutants that prevent or mimic the modification are frequently utilized. The endogenous proteins are usually depleted while the point mutations are expressed. A common strategy to accomplish these tasks includes two-steps: First, a cell line stably expressing shRNA for protein depletion is generated, then an RNAi-resistance construct is introduced to express mutant. However, these steps are time- and labor-consuming. More importantly, shRNA and mutant protein are frequently expressed in different cells at different time, which significantly disturbs the conclusions. METHODS: To overcome these technical problems, we developed a lentiviral based one-plasmid system that allowed concurrent expression of shRNA and mutant protein. The puromycin-resistant gene was inserted for the selection of stable-expression cells. RESULTS: Using this plasmid, we efficiently replaced the endogenous proteins with comparable levels of exogenous proteins for LDHB and PKM2, two glycolytic enzymes regulated by PTM in cancer cells. The system was also successfully exploited in evaluating the role of phosphorylation of LDHB serine 162 in multiple in vitro and in vivo assays. CONCLUSION: Thus, we have developed an efficient one-plasmid system to replace endogenous protein with point mutations for the functional study of PTM.

Energy restriction causes metaphase delay and chromosome mis-segregation in cancer cells.

ATP metabolism during mitosis needs to be coordinated with numerous energy-demanding activities, especially in cancer cells whose metabolic pathways are reprogramed to sustain rapid proliferation in a nutrient-deficient environment. Although strategies targeting the energy metabolic pathways have shown therapeutic efficacy in preclinical cancer models, how normal cells and cancer cells differentially respond to energy shortage is unclear. In this study, using time-lapse microscopy, we found that cancer cells displayed unique mitotic phenotypes in a dose-dependent manner upon decreasing ATP (i.e. energy) supply. When reduction in ATP concentration was moderate, chromosome movements in mitosis were barely affected, while the metaphase-anaphase transition was significantly prolonged due to reduced tension between the sister-kinetochores, which delayed the satisfaction of the spindle assembly checkpoint. Further reduction in ATP concentration led to a decreased level of Aurora-B at the centromere, resulting in increased chromosome mis-segregation after metaphase delay. In contrast to cancer cells, ATP restriction in non-transformed cells induced cell cycle arrest in interphase, rather than causing mitotic defects. In addition, data mining of cancer patient database showed a correlation between signatures of energy production and chromosomal instability possibly resulted from mitotic defects. Together, these results reveal that energy restriction induces differential responses in normal and cancer cells, with chromosome mis-segregation only observed in cancer cells. This points to targeting energy metabolism as a potentially cancer-selective therapeutic strategy.