Mitochondrial Impact of Organophosphate Pesticide-Induced Cardiotoxicity: An In Silico and In Vitro Study.

Organophosphate pesticides are widely used; however, their use is limited due to neurotoxicity and, to a lesser extent, cardiotoxicity in humans. Given the high energy demands of cardiac muscle, which is characterized by a dense population of mitochondria, any damage to these organelles can exacerbate cardiotoxicity. This study aims to elucidate whether the cardiotoxic effects of organophosphate pesticides originate from mitochondrial dysfunction. To investigate this, in silico toxicogenomic analyses were performed using various tools, such as the Comparative Toxicogenomic Database, GeneMANIA, STRING, and Cytoscape. Results revealed that 11 out of the 13 WHO-recommended Class Ia organophosphate pesticides target genes associated with cardiotoxicity. Notably, three of these genes were mitochondrial, with catalase (CAT) being the common differentially expressed gene among parathion, methyl parathion, and phorate. Furthermore, protein-protein interaction analysis indicated a strong association between CAT and superoxide dismutase 2, mitochondrial (SOD2). Subsequently, isolated heart mitochondria were utilized to assess CAT and superoxide dismutase (SOD) activities in vitro. The findings demonstrated that at a concentration of 7.5 ng/µL, both methyl parathion and phorate significantly decreased CAT activity by approximately 35%. Moreover, phorate reduced total SOD and SOD2 activities by 17% and 19%, respectively, at the same concentration. In contrast, none of the three organophosphate pesticides induced the opening of the mitochondrial permeability transition pore. These results suggest that the reduction in CAT and SOD2 activities, critical antioxidant enzymes, leads to the accumulation of reactive oxygen species within mitochondria, ultimately resulting in mitochondrial damage. This mechanism likely underlies the observed cardiotoxicity induced by these organophosphate pesticides.

Exploring Natural Compounds Targeting PD-L1 and STAT3: Toxicogenomic Analysis, Virtual Screening, Molecular Docking, ADMET Evaluation, and Biological Activity Prediction.

BACKGROUND: One of the most important targets in cancer immunotherapy is programmed cell death ligand 1 (PD-L1). Monoclonal antibodies developed for this target have disadvantages due to their low bioavailability and some immune-related adverse effects. Additionally, small molecules targeting PD-L1 are still in the experimental stage. At this point, discovering non-toxic natural compounds that directly or indirectly target PD-L1 is essential. In this in silico study, a comprehensive literature search was conducted to identify publications reporting the master regulator of PD-L1, which was suggested as a Signal Transducer and Activator of Transcription 3 (STAT3). The relationship between STAT3 and PD-L1 was further investigated through bioinformatic analysis. METHOD: Subsequently, natural compounds targeting PD-L1 and STAT3 were screened, and compounds with suitable toxicity profiles were docked against both PD-L1 and STAT3. Following molecular docking, the selected molecules underwent DNA docking, ADMET profile analysis, and in silico assessment of biological activities. The relationship between PD-L1 and STAT3 was determined in 52 out of the 453 articles, and it was further demonstrated in genegene interactions. Following the virtual screening, 76 natural compounds were identified, and after pre-filtering based on physicochemical properties, drug-likeness, and ADMET profiles, 29 compounds remained. RESULT: Subsequent docking revealed that two compounds, 6-Prenylapigenin, and Gelomulide J, persisted. ADMET and biological activity prediction results suggested that 6-Prenylapigenin is non-toxic and has the potential to inhibit PD-L1 and STAT3 in silico. The present study highlights that STAT3 serves as the master regulator of PD-L1, and it further suggests that 6- Prenylapigenin exhibits the potential to modulate PD-L1 and/or STAT3. CONCLUSION: This finding could pave the way for the development of small molecules designed to block the PD-1/PD-L1 interaction by silencing the PD-L1 and/or STAT3 genes or reducing protein levels.

Role of Oxidative Stress and Reactive Metabolites in Cytotoxicity & Mitotoxicity of Clozapine, Diclofenac and Nifedipine in CHO-K1 Cells In Vitro.

AIM: To provide in vitro data on toxicity mechanisms of clozapine, diclofenac and nifedipine. BACKGROUND: CHO-K1 cells were used as in vitro model to explore mechanisms of cytotoxicity of the test drugs. OBJECTIVE: Cytotoxic mechanisms of clozapine (CLZ), diclofenac (DIC) and nifedipine (NIF) were studied in CHO-K1 cells in vitro. All three drugs induce adverse reactions in some patients with partially unknown mechanisms. METHOD: Following the determination of time- and dose-dependency of cytotoxicity by the MTT test, cytoplasmic membrane integrity was explored by the LDH leakage test. Both end-points were further examined in the presence of soft and hard nucleophilic agents, glutathione (GSH) and potassium cyanide (KCN), respectively, and either individual or general cytochrome P450 (CYP) inhibitors, whether CYP-catalysed formation of electrophilic metabolites play a role in the observed cytotoxicity and membrane damage. The generation of reactive metabolites during the incubations was also explored. Formation of malondialdehyde (MDA) and oxidation of dihydrofluorescein (DCFH) were monitored whether peroxidative membrane damage and oxidative stress take place in cytotoxicity. Incubations were also conducted in the presence of chelating agents of EDTA or DTPA to explore any possible role of metals in cytotoxicity by facilitating electron transfer in redox reactions. Finally, mitochondrial membrane oxidative degradation and permeability transition pore (mPTP) induction by the drugs were tested as markers of mitochondrial damage. RESULTS: The presence of an individual or combined nucleophilic agents significantly diminished CLZ- and NIF-induced cytotoxicities, while the presence of both agents paradoxically increased DIC-induced cytotoxicity by a factor of three with the reason remaining unknown. The presence of GSH significantly increased DIC-induced membrane damage too. Prevention of membrane damage by the hard nucleophile KCN suggests the generation of a hard electrophile upon DIC and GSH interaction. The presence of CYP2C9 inhibitor sulfaphenazol significantly diminished DIC-induced cytotoxicity, probably by preventing the formation of 4-hydroxylated metabolite of DIC, which further converts to an electrophilic reactive intermediate. Among the chelating agents, EDTA caused a marginal decrease in CLZ-induced cytotoxicity, while DIC-induced cytotoxicity was amplified by a factor of five. Both reactive and stable metabolites of CLZ could be detected in the incubation medium of CLZ with CHO-K1 cells, which are known to have low metabolic capacity. All three drugs caused a significant increase in cytoplasmic oxidative stress by means of DCFH oxidation, which was confirmed by increased MDA from cytoplasmic as well as mitochondrial membranes. The addition of GSH paradoxically and significantly increased DIC-induced MDA formation, in parallel with the increase in membrane damage when DIC and GSH combined. CONCLUSION: Our results suggested that the soft electrophilic nitrenium ion of CLZ is not responsible for the observed in vitro toxicities, and this may originate from a relatively low amount of the metabolite due to the low metabolic capacity of CHO-K1. A hard electrophilic intermediate may contribute to cellular membrane damage incubated with DIC, while a soft electrophilic intermediate seems to exacerbate cell death by a mechanism other than membrane damage. A significant decrease in cytotoxicity of NIF by GSH and KCN suggested that both soft and hard electrophiles contribute to NIF-induced cytotoxicity. All three drugs induced peroxidative cytoplasmic membrane damage, while only DIC and NIF induced peroxidative mitochondrial membrane damage, which suggested mitochondrial processes may contribute to adverse effects of these drugs in vivo.

Mitochondrial Biotransformation of Drugs and other Xenobiotics.

In vivo biotransformation of exposed chemicals is one of the major factors that determine the concentration and the duration of a substance at the systemic site of effect. Given that toxicity is expressed as a function of two factors, namely dose and time, the type and intensity of the toxicity are directly dependent on the chemical transformation of the exposed parent substance. This dependency involves two different situations. The amount of the chemical reaching the target will be decreased with the extent of metabolism if the parent chemical is toxic, and the opposite is true if the metabolite(s) is toxic instead. To date, the liver microsomal fraction in mammals has been justifiably considered as the center of biotransformation reactions because the liver and microsomes (i.e., endoplasmic reticulum component of the cell) possess the most abundant types and quantities of xenobiotic-metabolizing enzymes, especially the cytochrome P450 supergene enzyme family. These enzymes are common in all kingdoms of life, which strongly suggests that the origin of life is common. It is already known that various drugs enter mitochondria by different mechanisms, and this translocation is believed to be responsible for mitochondrial effects that are part of the therapeutic actions of various drugs such as lipid-lowering statins or antidiabetogenic thiazolidindiones. However, the discovery of mitochondrial forms of the xenobiotic-metabolizing enzymes provoked discussions about whether mitochondria metabolize drugs and other chemicals to some extent. This possibility may particularly be important as mitochondria have various critical cellular structures and functions. In the case of in situ generated metabolite(s), when there are adverse interactions with either these structures or functions, various toxic outcomes may appear. In this review, we compiled studies in the literature regarding biotransformation of drugs and other chemicals catalyzed by mitochondria where it is both an initiator and target of toxicity.

Bioactivation of clozapine by mitochondria of the murine heart: Possible cause of cardiotoxicity.

The mechanism of clozapine-associated cardiotoxicity has not been elucidated. The formation of a reactive nitrenium ion from the drug has been suggested as the cause, however, the reason why the heart is a target remains unknown. The heart is one of the most perfused organs; therefore, it contains a large number of mitochondria per cell; these organelles are responsible for both oxygen metabolism and energy production due to high energy expenditure. Given that mitochondria play critical roles in cellular homeostasis and maintenance, this study tested the hypothesis that cardiac mitochondria are both a target and initiator of clozapine-induced cardiotoxicity through activating the drug. We investigated whether murine heart receives a relatively high amount of systemically administered drug (20 mg/kg, i.p., Wistar albino rats) and whether cardiac mice (Swiss albino) and rat (Wistar albino) mitochondria locally activate clozapine (100 µM) to a reactive metabolite. We observed a relatively large distribution of clozapine to heart tissue as well as the formation of reactive metabolites by cardiac mitochondria in situ. Mitochondrial cytochrome P450 enzymes (CYP) in cardiac tissue responsible for biotransformation of clozapine were also characterized. CYP3A4 has been found to be the major enzyme catalyzes CLZ bioactivation, while CYP1A largely and CYP3A4 partially catalyzes the formation of stable metabolites of CLZ. At 100 µM concentration, clozapine caused a significant decline in mitochondrial oxygen consumption rate in vitro as much as positive control (antimycin A), while it did not induce mitochondrial permeability transition pore opening. These data provide an explanation as to why the heart is a target for clozapine adverse effects.

Inhibition of cell proliferation, migration and colony formation of LS174T Cells by carbonic anhydrase inhibitor.

BACKGROUND: Metastasis is the leading cause of cancer deaths. Migration of tumor cells is an important stage in metastasis. Therefore, recent studies have focused on clarifying migration and migration-dependent cell functions such as angiogenesis, wound healing, and invasion. OBJECTIVES: In the present study, we aimed to investigate the effect of acetazolamide, which is a classical carbonic anhydrase inhibitor, on the cell viability, migration, and colony forming capacity of human LS174T colorectal cancer cells. METHODS: Three different cell culture techniques (MTT test, wound healing and clonogenic assay) were performed in this in vitro study on colorectal cancer cells. RESULTS: Acetazolamide reduced the cell viability, migration and colony formation ability of cells depending on dose. There was no significant difference between the cells treated with acetazolamide with 1 µM dose and the control. However, it can be concluded that acetazolamide exerts its effect on human colorectal cancer cells at 10-1000 µM concentrations. CONCLUSION: Acetazolamide was observed to significantly inhibit the cell viability, colony forming capacity, and migration ability in the culture medium of LS174T cells. This inhibitor effect of acetazolamide was observed to be dependent on the concentration in medium.

Hemangioma mimicking dorsal nasal hump.

A 24-year-old female patient with a dorsal nasal hemangioma mimicking a prominent dorsal nasal hump, which was noticed during the rhinoplasty operation, is presented.

The effectiveness of steroid treatment in nasal polyposis.

OBJECTIVE: The objectives of the management of nasal polyposis are to eliminate or reduce the size of polyps, reestablish nasal breathing, reduce symptoms of rhinitis, restore the sense of smell, and prevent the recurrence of nasal polyps. Local or systemic steroids have been used in the treatment of nasal polyps, but efficacy of combined (local and systemic) steroids in nasal polyposis has been little investigated. The aim of this study was to evaluate the influence of combined steroid therapy on the symptoms and extent of the disease in patients with nasal polyposis. METHODS: Seventeen patients with nasal polyps were treated with combined steroids. Before and after the therapy, polyp size, nasal symptoms, sense of smell, and headache or facial pain were assessed by an established scoring system. RESULTS: After the therapy, symptom scores of all the patients improved. Of the patients, 12% showed a polyp-free nasal cavity, 76% a clear involution of polyps, and 12% no response to the therapy. There were statistically significant differences (P<0.001) for symptom scores and polyp size. Medical ablation of polyps using steroids was not achieved in 88% patients. CONCLUSION: Steroids can reduce polyp sizes and improve the symptoms, but are inadequate to eradicate the polyps. Surgery still plays a major part in the treatment of the nasal polyposis, but steroids can delay the necessity for surgical intervention.