Toxicity Evaluation of 6-Mercaptopurine Using Accelerated Cytotoxicity Mechanism Screening (ACMS) techniques

6- Mercaptopurine (6-MP) is widely used in clinic as an immunosuppressive for treatment of acute lymphocytic leukemia, Crohn's disease, and ulcerative colitis with documented unpredictable hepatotoxicity. The potential molecular cytotoxic mechanisms of 6-MP against isolated rat hepatocytes were searched in this study using ―Accelerated Cytotoxicity Mechanism Screening (ACMS)‖ techniques. The concentration of 6-MP required to cause 50% cytotoxicity in 2 hour at 37∘C was detected to be 400 μM. A significant increase in 6-MP induced cytotoxicity and reactive oxygen species (ROS) formation, % mitochondrial membrane potential (MMP), lysosomal damage were observed. The addition of chloroquine (lysosomotropic agent), L-carnitine (inhibitor of membrane permeability transition (MPT), Diphenyleneiodonium (DPI) as an inhibitor of production of superoxide, and H2O2 by mitochondria and Dimethyl sulfoxide (DMSO) as a radical scavenger decreased 6-MP-induced cytotoxicity, ROS formation, collapse of MMP, and lysosomal damage. Results from this study suggest that 6-MP -induced cytotoxicity in isolated rat hepatocytes due to ROS formation, mitochondrial and lysosomal damages that resulted in crosstalk toxicity between mitochondrial and lysosomal damage and finally cell death

The Use of Methanolic Extract of Persian Gulf Sea Cucumber, Holothuria, as Potential Anti-Cancer Agents

Hepatocellular carcinoma (HCC) is the fifth most malignant of liver cancer globally. Melanoma is also a highly aggressive and deadly cancer with a poor prognosis given its drug resistance.A defect in apoptosis pathway is one of the key mechanisms that contribute to drug resistance in cancer. An important sea marine animal is the Holothuria, also known as the sea cucumber, which has various pharmacological activities. Scientists have begun to further investigate on the natural bioactive compounds found in marine animals showing anti-inflammatory and anti-cancer properties. H. sabra and H. parva, sea cucumbers are known to show the mentioned properties. Cancerous induced animals were applied for mitochondrial investigations. The mitochondria isolated from Hepatocytes and melanoma cells via differential centrifuges was treated with various concentrations (250, 500 and 1000 µg/ml) of metanolic extract of H. parva and H. sabra. Reactive oxygen species (ROS) formation, mitochondrial swelling, mitochondrial membrane potential (MMP) and cytochrome c release resulting from the selective toxicity of various dilution of methanolic extract of H. sabra and H. parva on mitochondria isolated from cancerous induced animals were determined. Our results show that different concentrations (250, 500 and 1000µg/ml) of themethanolicextract significantly (p<0.05) induce reactive oxygen species (ROS) formation and mitochondrial swelling, decreased mitochondrial membrane potential (MMP) disruption and increased cytochrome c release only in HCC mitochondria and melanoma mitochondria, in a time-and concentration dependent manner (In comparison to the control group). Our study suggest that given bioactive compounds found in mentioned sea cucumbers can potentially introduced as anti-cancer molecules. Further studies such as molecular identification,  in vivo experiments and clinical trials can confirm these results. &nbsp

Selective Toxicity of Persian Gulf Stonefish (Pseudosynanceia Melanostigma) Venom on Human Acute Lymphocytic Leukemia B Lymphocytes

Persian Gulf Stonefish (Pseudosynanceia melanostigma) is one of the poisonous fish which is naturally found in Indian Ocean and Persian Gulf. The venom, which is isolated from this species, is suspected to use as an anticancer agent. In this study, we showed the cytotoxic effect of stonefish crude venom on lymphocytes, which obtained from acute lymphoblastic leukemia (ALL) patients and normal donors. Our results demonstrated that crude venom of Persian Gulf Stonefish could affect cancerous lymphocytes by reactive oxygen species (ROS) generation and mitochondrial membrane damage without any significant effect on normal cells.Highlights Persian Gulf Stonefish (Pseudosynanceia melanostigma) is one of the poisonous fishThis venom is composed of various proteins with cardiotoxic and the hemolytic activityCrude venom of Persian Gulf Stonefish can affect cancerous lymphocytes by ROS generation

Toxicity of industrial waste water containing Ag/Tio2 on muscle mitochondria isolated from Solen dactylus scallop

Industrial waste water is of global concern due to its severe effects on the environment. Recent studies have suggested that these compounds can cause toxicity via mitochondria-involved mechanisms. According to the fact, that these compounds are freely dispersed in the environment, it is crucial to determine the exact mechanism of toxicity. Introduction: Compared with municipal waste water, industrial waste water generally contains high concentration of toxic or non-biodegradable pollutants. Recently, it was shown that scallop could filter waste water, but heavy metal such as Ag and TiO2 could induce Reactive Oxygen Species (ROS) in mitochondria isolated from scallop. Recent studies have suggested that mitochondria have a key role in toxicity via mitochondrial membrane potential collapse and generation of ROS. Therefore, it was decided to determine the mechanistic toxicity of waste water contained heavy metals towards isolated scallop muscle mitochondria using new and reliable methods. Methods and Results: Isolated scallop mitochondria were obtained by differential ultracentrifugation at before and after exposure to waste water. Our results showed that two heavy metals induced mitochondrial dysfunction via an increase in mitochondrial ROS production and membrane potential collapse, which correlated to cytochrome C release. Conclusions: Our results suggest that waste water contained heavy metals-induced toxicity in scallop is result of a disruptive effect on the mitochondrial respiratory chain and increasing the chance of cell death signaling in scallop muscle cells

Hexavalent Chromium Induced Oxidative Stress and Toxicity on isolated human lymphocytes

Introduction: The most toxic form of chromium (Cr) in the environment is the oxyanion chromate (Cr (VI)). In this form it is soluble and is transported into the cells. Chromate structurally resemble phosphate and sulfate, and can be transported into cells by the anion carrier.  Methods and Results: In this study, toxicity effects of Cr (VI) on isolated human lymphocytes was studied using accelerated cytotoxicity mechanisms screening (ACMS) technique. Human lymphocytes were isolated from blood of healthy persons using Ficoll-paque PLUS standard method. The trypan blue dye was used to cytotoxicity assay. The mechanistic parameters including reactive oxygen species (ROS), lysosomal membrane destabilization, mitochondrial membrane potential (MMP) collapse, lipid peroxidation, GSH and GSSG levels were assessed after 1, 2 and 3 hrs in potassium dichromate treated lymphocytes. The results indicate that toxicity of Cr (VI) was concentration dependent in human lymphocytes. Cr (VI) significantly (p<0.05) induced ROS production, MMP reduction, lysosomal membrane destabilization and lipid peroxidation in human lymphocytes. There was also a decrease in intracellular GSH and raise in extracellular GSSG levels in Cr (VI) treated lymphocytes.  Conclusion: OOur findings revealed that ROS formation with subsequent cellular damages is the molecular mechanism for Cr (VI) induced human blood lymphocyte toxicity

Mitochondrial Transplantation Attenuates Toxicity in Human Lymphocytes Caused by Clozapine and Risperidone

Background: Clozapine (CLZ) and risperidone (RIS) are drugs that have the ability to disrupt mitochondrial function. Also, these drugs increase the level of free radicals. Mitochondrial dysfunction plays a role in the etiology of various diseases. Replacement and treatment of defective mitochondria with healthy mitochondria have been considered. Mitochondrial therapy (mitotherapy) or exogenous mitochondria transplantation is a method that can be used to replace dysfunctional mitochondria with healthy mitochondria. This method can help in the treatment of diseases related to mitochondria. Methods: In this study, we investigated the transplantation effect of isolated lymphocyte mitochondria on the toxicity induced by CLZ and RIS on human blood lymphocytes. Lymphocytes were isolated using the Ficoll standard method. Mitochondria of human lymphocytes were used for mitotherapy. This study was conducted in 6 groups. After treatment, the level of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), reduced glutathione (GSH) content, oxidized glutathione (GSSG) content, and adenosine triphosphate (ATP) content were evaluated. Results: Our data showed that CLZ (70 µm) and RIS (24 nM) caused cytotoxicity on human blood lymphocytes which are associated with ROS generation, collapse in MMP, decrease in GSH content, increase in GSSG content and change in ATP content. Mitochondria transplantation results showed that adding mitochondria of lymphocytes could protect the lymphocytes against the toxicity effects caused by CLZ and RIS. Furthermore, the results showed that pre-incubation with cytochalasin D considerably reserved the protective effects of mitotherapy in the human lymphocytes. Conclusion: We proposed that mitochondria transplantation or mitotherapy-affected blood lymphocytes with exogenous mitochondria could be used to treat CLZ and RIS-induced toxicity