Author: Endonita Hajzeraj
“Identification of biochemical and cytotoxic markers in cocaine treated PC12 cells”
Cocaine is a powerful drug that causes addiction and many deaths every year. It is a public health issue that researchers are constantly exploring to come up with new ideas to combat addiction. Cocaine has a lipophilic structure, which allows it to cross the blood brain barrier easily and once it reaches various domains of the central nervous system, it triggers different levels of cellular toxicity.1Badisa et. al aimed to reproduce cocaine toxicity in differentiated PC12 cells. PC12 is a cell line derived from pheochromocytoma of the rat adrenal medulla. With previous quantitative knowledge on biochemical and cytotoxicity markers, they were able to reproduce toxicity.
Injuries to neurons can severely affect neuronal structure. Neuronal development, which involves the generation, migration, and differentiation of neurons is important for the complete functioning of the nervous system. External injury can affect neuronal structures and the networking processes of neurons. This can cause severe and irreversible changes as well as cell death. Previous studies have shown that external injuries, like the use of drugs, can induce changes in cell structure and integrity.2 Cocaine, because of its lipophilic nature, causes psychostimulatory effects in the central nervous system. Along with physical symptoms such as depression, anger, aggressiveness and paranoia, cocaine also contributes to physiological changes such as increased blood pressure and increased cytotoxicity in all vital organs. These effects are caused by an imbalance of neurotransmitters induced by cocaine usage. Badisa et al. evaluated several biochemical changes including dopamine levels, general mitochondrial activity, membrane potential, lactate release, and glutathione levels. The cytotoxicity was measured with markers like the production of reactive oxygen species, lactate dehydrogenase release, GSH level, and nitric oxide generation.
PC12 cells were grown in a culture medium and exposed to NGF, a nerve growth factor, for five days. Badisa and colleagues observed differentiated cells and intercellular connections at several areas. Figure 1 shows the morphological features of NGF exposed PC12 cells.3 The authors observed the differences of neuronal characteristics. They stained both undifferentiated and differentiated cells for the presence of neurofilaments. They observed that differentiated PC12 cells behave as dopaminergic neurons as they released more dopamine in the culture medium. They tested the biochemical affects of cocaine at in vivo pharmacological doses on different cellular parameters. The authors treated the cells with various concentrations of cocaine. These concentrations ranged from 0.001 to 0.5mM for 48 hours to stimulate and evaluate cell viability.
Differentiated PC12 cells behaved like dopaminergic neurons, so to test the effects of cocaine on dopamine levels, the cells were treated with cocaine for 48 hours. Data indicated that the amount of dopamine levels was much higher in dopaminergic cells than in the control cells. The next observation was on mitochondrial activity. The cells were treated with various concentrations of cocaine and they observed that general mitochondrial activity was not bothered. However, when they measured the mitochondrial membrane potential using fluorescence probe Rh 123, the data indicated that cocaine treatment significantly decreased the mitochondrial membrane potential in cells compared to the control. When there is disruption in the mitochondrial potential, the cell switches to anaerobic glycolysis to survive. During this time, pyruvate is oxidized to lactate and is released in the medium. To determine if the loss of membrane potential was due to the release of lactate they treated the cells with three different doses of cocaine for 48 hours. They observed that cocaine treatment caused significant release of lactate. From this information, the authors wanted to further investigate cocaine effects of GSH levels.
In vitro studies were also conducted. Several parameters were evaluated when testing the cells with 2, 3, and 4mM cocaine for 48 hours. Increased concentrations decreased cell viability, decrease in neurite elongation. The authors speculated that the decrease in cell viability was due to reactive oxygen species’ interaction with the mitochondria in the cell. They tested their speculation by staining the cells with fluorescence and it was observed that there were dose-dependent increases in ROS levels compared to control cells.4 An increase in cocaine concentrations also caused a decrease in glutathione levels in the cell.
Overall, cocaine treatment has caused a variety of effects on neuronal cells. From increase in dopamine levels, to increase in reactive oxygen species which then results in a loss of cell viability. The results presented in this research suggest that the mitochondria are the primary targets of cocaine induced toxicity.5 ROS species that are generated produce a lot more problems in the body. ROS are radicals that are byproducts during the mitochondrial electron transport. ROS cause many problems in the body ranging from forming radicals and causing cell death, gene expression alteration, and cell signaling effects. 6 Understanding the effects of cocaine toxicity in terms of the biochemical results it has such as a decrease in glutathione and an increase in ROS causes many metabolic issues for our bodies. This study aimed to prove that cocaine does not only affect neurons in the brain, but essentially effects a lot of differentiated cells. Since dysfunction in the mitochondria can lead to psychiatric illnesses and cocaine usage causes dysfunction of the mitochondria, future work can include studying whether cocaine users are at a higher risk for these psychiatric illnesses.
.Haasen, C. et al. Relationship between cocaine use and mental health problems in a sample of European cocaine powder or crack users. World Psychiatry 4(3), 173–176 (2005).
.Cuntz, H. et al. One rule to grow them all: A general theory of neuronal branching and its practical application. PLoS Comput. Biol. 6(8), e1000877 (2010).
.Badisa, R. et al. Identification of biochemical and cytotoxic markers in cocaine treated PC12 cells. Scientific Reports 8, Article number:2710 (2018) doi: 10.1038/s41598-018-21182-7
 Badisa, R. et al. Identification of biochemical and cytotoxic markers in cocaine treated PC12 cells. Scientific Reports 8, Article number:2710 (2018) doi: 10.1038/s41598-018-21182-7
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