S9-mix for a treatment period of 24 hours. Selection of concentrations and preparation of test solutions The selection of concentration range kratom suboxone interaction tests was based on the cytotoxicity data using trypan blue exclusion assay performed as described in the previous chapter (Chapter 2). Indo Kratom – Red Vein Borneo A 10g the default vehicle solution for MSE and MIT was ethanol. Arochlor 1254 rat liver S9-mix was used as the exogenous metabolising system and was prepared freshly on the day of the assay. The S9-mix was prepared by mixing 1 part of S9 with 9 parts of co-factor (5. M NADP (Na2) and 27.
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MSE in the presence of S9 turned out to be positive. RTG and also low RSG (24%) prior plating. Some genotoxic carcinogens could not be detected in in vitro genotoxicity assays unless the concentration tested induced some degree of cytotoxicity (ICH 1995).
Based on the long term use of this plant by humans testing for its genotoxic potential using mammalian cells was thought to be more appropriate than conventional first tier testing for gene mutation in bacteria. In fact the primary first tier bacterial genetic toxicology assay the Ames Salmonella assay is incapable of detecting large scale deletion or recombination events of the mutations. Such events are more kratom tea pain common in mammalian cell mutagenesis (Clive et al 1990). Mitchell et al 1997). In general MSE with or without the presence of metabolic activation (Arochlor 1254 induced rat liver S9) was negative for genotoxic potential.
The Indo Kratom – Red Vein Borneo A 10g nature of cell death observed was unknown and to the best of my knowledge there are no reports or information available on Mitragyna speciosa Korth toxicity on mammalian cells. In this study Indo Kratom – Red Vein kratom addiction and withdrawal Borneo A 10g therefore an attempt was made to characterise the MSE and MIT toxicity by looking at cell cycle distribution. Firstly attempt was made to look at the cell cycle distribution in different cell lines using flow cytometry approach. Propidium Iodide is one of the most common and recommended dyes to use to quantitatively assess DNA content by flow cytometry (Darzynkiewicz et al 2001). The dose response and temporal effects of treatment were Indo Indo Kratom – Red Vein Borneo A 10g Kratom – Red Vein Borneo A 10g examined in this assay in order to maximally evaluate the effect on the cell cycle. Cell cycle analysis was initially performed using HEK 293 cells and the DNA profile was determined manually using the Cellquest Pro software (Fig.
Other pathways may be considered for MSE induced cell death with no involvement of caspase activation but yet following the
programmed fashion. Involvement of several enzymes from lysosomal pathways such cathepsins and calpains were shown to highly correlate to apoptotic-like or even necrotic cell death (Jiang et al 2006; Yamashita et al 2003). Mitochondria which play a key role in the intrinsic pathway for apoptosis may also again be involved as apoptotic inducing factor (AIF) which is usually released after activation of Bcl-2 family acted with the EndoG protein released from plasma membrane to trigger apoptotic-like cell death ( Jiang et al 2001). Many agents are currently known to induce cell death via caspase independent pathways as described above such as campothecin doxorubicin and paclitaxel. The necrotic type of cell death induced by MSE which is morphologically seen in cell lines such as kratom for narcotic withdrawal MCL-5 and HEK 293 cells could not be confirmed biochemically due to time limitations. Unlike MSE MIT treated SH-SY5Y cells have shown a different mechanism of cell death
in which there was an involvement of caspases 3 and 7.