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.

The cytotoxic, genotoxic and mitotoxic effects of Atractylis gummifera extract in vitro.

Background: The Mediterranean thistle Atractylis gummifera L. (Asteraceae; AG) has diterpenoid glucosides; atractyloside and carboxyatractyloside that interact with mitochondrial protein adenine nucleotide translocator (ANT) and resulted in ATP inhibition. Despite its well-known toxicity, acute poisonings still occur with this plant. Although most symptoms are attributed to ANT and diterpenoids interaction, in-depth investigation of the effects of AG extract on various cellular processes has not been performed. Objective/method: We tested in vitro induction of mitochondrial permeability transition pore (MPTP) opening in bovine liver mitochondria and evaluated its cytotoxicity and genotoxicity using Allium cepa test. Cell division, mitotic index (MI) and total chromosomal and mitotic aberrations (TAs), that all seem potentially affected by ATP shortage, were studied in root cells of Allium cepa exposed to Atractylis gummifera extract. Results: With the two different doses of two purified AG fractions, stronger induction of MPTP was observed compared to the induction with the standard pure atracyloside. Aqueous AG extract exerted inhibition root growth in A. cepa at 6 different doses. The TAs was increased in a dose-dependent manner too, while mitotic index was decreased at the same doses. Evaluation of mitotic phases revealed mitodepressive effect of AG on A. cepa roots. Conclusion: this work highlights cellular and mitochondrial adverse effects of Atractylis gummifera extracts. A purified fraction that likely corresponds to ATR derivatives induces MPTP opening leading to swelling of mitochondria and its dysfunction. Allium cepa test provides the evidence for A. gummifera genotoxicity and cytotoxicity.

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.