At this stage it seems that despite having high MIT content in the MSE the
high dose MSE treatment in SH-SY5Y cells does not activate caspase enzymes. This probably could be due to other chemicals that present in MSE preventing the activation of caspase enzymes. Cell death of SH-SY5Y cells after MSE and MIT appeared to be predominantly via apoptosis based on its morphological appearance however biochemically the results discussed above fail to support a caspase mediating event. Kratom Is Not Dangerous as apoptosis could follow various pathways and often vary in different cells (Esposti and McLennan 1998 Hetts 1998) this prompted us to further investigate if other pathways could contribute.
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MIT has a lesser effect and cells arrest mainly at G1 phase in SH-SY5Y cells. The cell arrest occurring Kratom Is Not Dangerous at high doses of MIT was found to be correlated with p53 and p21 expression although the expression changes were marginal compared to control and lower dose groups. The mechanism for cell cycle arrest in the cells
treated with high doses of MSE remains unclear as there was no correlation with p53 and p21 as both proteins were lost after the treatment. The level of MSE toxicity for SH-SY5Y and HEK 293 cells was found o be increased 10-fold when metabolic activation system (post mitochondrial rat liver S9 induced with Arochlor 1254) was added to the treatment. This implies that MSE cytotoxicity requires metabolism for its activation and CYP2E1 was thought to be involved in this metabolic activation. However MIT in parallel experiments did not show any enhancement of toxicity in the presence of S9 and was inherently cytotoxic.
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Surprisingly this time a similar outcome was observed for both SH-SY5Y and MCL-5 cells and the shifting of the whole populations was evident at much lower concentrations of MSE than in the previous PI staining in chapter 2. This phenomenon is obviously due to the treatment effects as the control and lowest concentration of the MSE tested as seen in fig. The hypothesis of plasma membrane opening is supported with this finding. This kratom oil dose sanbornville phenomenon creates disadvantages for this assay as when the whole FACS profile shifts to the right side of the scale the determination of the stages of cell death is difficult to interpret as the cells are no longer located in specific quadrants. This observation is clearly in contrast with the previous cytological examinations which indicated that SH-SY5Y cells treated with high dose of MSE undergo apoptosis rather than necrosis. The right shifting phenomenon for MIT kraton berre treated cells observed in fig.