The goal of this study was to describe the results of an untargeted lipidomic and metabolomic analysis of the effects of chlorpromazine at high therapeutic doses in brain endothelial cells using cell-based LC-MS metabolomics. This method has become increasingly popular and widely used in recent years [14–16]. The analyses presented in this study addresses BBB dysfunction and breakdown caused by high antipsychotic therapeutic dosage.
Chlorpromazine effects on brain endothelia cells
Our findings show that brain ECs treated with chlorpromazine at high therapeutic doses have significantly altered levels of non-polar and polar metabolites (as summarized in Supplementary Table 1 and 2) when compared to untreated cells. These findings are consistent with, and significantly add to, previous reports of lipid metabolism and cellular metabolism changes in the BBB [4,12]. Taken together, the findings suggest that high therapeutic doses of antipsychotics may be involved in BBB dysfunction.
Interestingly, examining the results of non-polar and polar metabolite changes reveals that chlorpromazine at high therapeutic doses affect the lipid profiles and intracellular metabolic profiles of brain ECs. In cellular processes, lipid metabolism is mainly involved in mitochondrial functions [17,18]. As a result, changes in lipid profiles and metabolic profiles may impair mitochondrial function. Antipsychotics have been shown to impair mitochondrial function [19–21] as well as cellular and mitochondrial membrane structure [12,19,22], leading to cell apoptosis [23–25].
Mitochondria are intricate organelles found in all human cells. They are in charge of producing the vast majority of cellular energy in the form of adenosine triphosphate (ATP). ATP is the primary source of energy for almost all cellular processes. Impaired mitochondrial function leads to impaired bioenergetics, which results in a lack of cellular energy [4,12,19]. Elmorsy et al. [4,12,22] recently demonstrated bioenergetic impairment in brain ECs treated with antipsychotics and subsequent mitochondrial dysfunction.
In brain ECs treated with chlorpromazine, the levels of acetoacetic acid, D-pantethine, (iso)butyrylcarnitine, 3-hydroxyactanoyl carnitine, L-carnitine, succinic acid semialdehyde, and D-arabitol significantly changed as summarized in Supplementary Table 1 and 2. As illustrated in Figure 6, changes in these metabolites may be involved in mitochondrial dysfunction. Mitochondrial dysfunction impairs bioenergetics in chlorpromazine-treated ECs. As a compensatory mechanism for energy deficiency, fatty acids are used for energy production. Several acid substances are produced during fatty acid metabolism, including acyl carnitine compounds and related compounds, as well as acetoacetic acid. The levels of acyl carnitine compounds ((iso)butyryl carnitine and 3-hydroxyactanoyl carnitine) in our results were significantly higher, while carnitine (an amino acid that plays an important role in fatty acid transport into mitochondria was significantly lower. These changes could confirm that brain ECs treated with high doses of chlorpromazine have a high rate of fatty acid metabolism.
Furthermore, the levels of acetoacetic acid and D-pantethine were found to be significantly higher. ECs and other cells do not normally contain high levels of acetoacetic acid and D-pantethine. These metabolites increase when cells lack oxaloacetic acid for the conversion of acetyl-CoA to citric acid or when fatty acid metabolism is excessive [26,27]. The levels of fatty acids and related compounds were significantly altered as a result of the findings. These changes could imply that the rise in acetoacetic acid and D-pantethine levels is due to mitochondrial dysfunction and an excess of fatty acid metabolism, which results in an excess of acetyl-CoA. Excess acetyl-CoA is then converted to acetoacetic acid. A state of ketoacidosis is reached when even more acetoacetic acid accumulates, lowering the cell's pH to dangerously acidic levels. Ketoacidosis is a metabolic disorder characterized by high levels of ketone bodies (acetoacetic acids), which causes EC dysfunction and apoptosis .
Furthermore, mitochondrial dysfunction and increased fatty acid metabolism can result in reactive oxygen species (ROS), which causes oxidative stress in cells. This stress can severely damage all cell components, including DNA, protein, and lipids . Elmorsy et al.  demonstrated that antipsychotc treatment disrupted the cellular structure and morphology of brain ECs. The antipsychotics caused this disruption, which was linked to oxidative stress. Our results revealed pronounced changes in some lipid compounds in brain ECs treated with chlorpromazine. These lipids are cellular membrane constituents that play roles in cell proliferation, differentiation, apoptosis, and oxidative stress .
Moreover, there are a variety of antioxidant defenses in cells to detoxify fluctuations in ROS and its reactive intermediates, but ROS generation frequently exceeds the antioxidant capacity of cells . The results show that the levels of hydroxymelatonin in brain ECs treated with chlorpromazine were significantly increased and decreased, respectively. Hydroxymelatonin is a byproduct of melatonin's interaction with reactive oxygen species (ROS) in cells . Melatonin has been shown to act as a direct scavenger of ROS (thus not only regulating sleep and circadian rhythms) and to produce hydroxymelatonin in human cells . The level of 3-hydroxymelatonin in cells is proportional to the level of oxidative stress caused by ROS . Antipsychotics have been shown to increase the production of ROS in cells [35,36]. The findings, which are consistent with and significantly add to previous reports of ROS-induced oxidative stress in brain ECs, could imply that chlorpromazine is involved in ROS generation, resulting in oxidative stress.
In conclusion, chlorpromazine at high therapeutic dosage could affect the bioenergetics pathway due to mitochondrial dysfunction resulting in keto-acidosis and inducing oxide stress by reactive oxygen species generation in brain ECs.