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Diclofenac Sodium and Mefenamic Acid: Potent Inducers of the Membrane Permeability Transition in Renal Cortex Mitochondria
Abstract
The ability of nonsteroidal anti-inflammatory drugs (NSAIDs) to induce Ca(2+)-mediated/cyclosporin A-sensitive mitochondrial membrane permeability transition (MMPT) was evaluated by monitoring swelling of isolated rat renal cortex mitochondria in the presence of 20 microM CaCl2. Dipyrone and paracetamol did not induce MMPT, while piroxicam and acetylsalicylic acid (and its metabolite salicylate) were poor inducers. In contrast, diclofenac sodium and mefenamic acid were potent triggering agents, inducing MMPT at 2 microM, a concentration below those previously shown to uncouple and/or inhibit oxidative phosphorylation. When compared to salicylate, a classical uncoupler and inducer of MMPT, the potency of diclofenac sodium and mefenamic acid was about 50-fold greater. Swelling was completely prevented by EGTA, cyclosporin A, or MgCl2, and only partially by ADP or dithiothreitol. Under the same experimental conditions as for the swelling assays, the drugs depressed the membrane potential of mitochondria, an effect prevented by cyclosporin A and restored by EGTA. Also, the drugs did not induce membrane lipid peroxidation or changes in GSSG levels, but led to a small decrease in protein thiol content, as well as to a substantial decrease in the NADPH levels of mitochondria. Hence, membrane depolarization and pyridine nucleotide oxidation seem to be involved in MMPT induction by these NSAIDs. The potency in eliciting the process, like the uncoupling activity, seems to be influenced by the lipophilic character of the molecules.
... Diclofenac sodium causes a rise in liver function, and has also been reported to cause hepatitis (14)(15)(16). The hepatotoxic and nephrotoxic effects of diclofenac sodium in both humans and experimental animals have been reported (16)(17)(18)(19). ...
... It is known to be an important target organ for the untoward effects of NSAID, which can produce acute, reversible or permanent effects (22,23,(30)(31)(32)(33)(34)(35)(36). The toxic effects of diclofenac can be acute or reversible (19). The NSAIDs adversely change the kidney functions (10) and may play a role in the induction of membranous nephropathy (37). ...
... Thus, an increased level of H 2 O 2 can also cause cell damage in kidney tissue (12). Diclofenac sodium causing nephrotoxicity could be associated with the potent inducer of the membrane permeability transition (MMPT) in renal cortex mitochondria (19). MMPT increases calcium uptake in mitochondria in the presence of reactive oxygen species. ...
In this study, we aimed to investigate the possible effects of high dose of diclofenac sodium on liver and renal tissues. Albino adult male rats (n:10) were injected intramuscularly a single high dose of 125 mg/kg diclofenac sodium daily. Changes in the liver and kidney tissues were determined and compared after 24 hours of the treatment. Mixed cells infiltration, proliferation of bile duct in portal areas and parenchymal cell degeneration of the liver were histologically observed. Peritubular lymphocyte infiltration and tubular epithelial cell degenerations were seen in the tissue samples of the kidney. The blood urine nitrogen (BUN), creatinine (CRE), activities of aspartate amino transferase (AST), alanin aminotransferase (ALT), alkaline phosphatase/(ALP), creatine phosphokinase (CPK), lactate dehidrogenase (LDH) in the serum, principal antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione reductase (GR), catalase (CAT), glucose 6-phosphate dehidrogenase (G6PD) in the liver and kidney tissues and malondialdehyde (MDA) levels, indicating lipid peroxidase in both the liver and kidney were determined biochemically. The significant increased in serum levels of LDH (p<0.05), BUN and CRE (p<0.001), indicated liver and renal functions, respectively. The levels of AST, ALT and CPK, on the other hand, remained unchanged. MDA levels which indicate the lipid peroxidation, in both tissues were found to be increased. The increased levels of antioxidant enzymes activities in liver might indicate a possible activation of antioxidant defense mechanism. The decrease in GSH-Px and GR activities and an increase in the SOD activity associated with the increased MDA levels might be related with an oxidative damage which occured in the kidney. The biochemical findings in the present study were in aggrement with the histological observations.
... Indomethacin or NS-398 cause not only COX inhibition, but also a three-to six-fold decrease in expression of dihydropyrimidine dehydrogenase mRNA in tumor cells and xenografts of colon cancer cells [126][127][128]. Several other studies have reported on the effects of the NSAIDs on mitochondrial function using either isolated tissues or tumor derived mitochondria, or intact cancer or other non-cancerous cells [129][130][131][132][133][134][135][136]. Thus, diclofenac, mefenamic acid and piroxicam in the low to middle micromolar range behaved as mitochondrial uncouplers and inhibited OxPhos in rat kidney mitochondria utilizing either glutamate + malate or succinate, whereas dipyrone, acetylsalicylic acid and paracetamol required in the low millimolar range [129]. ...
... Thus, diclofenac, mefenamic acid and piroxicam in the low to middle micromolar range behaved as mitochondrial uncouplers and inhibited OxPhos in rat kidney mitochondria utilizing either glutamate + malate or succinate, whereas dipyrone, acetylsalicylic acid and paracetamol required in the low millimolar range [129]. In extended studies, diclofenac and mefenamic acid at 2 µM, (concentrations well below the levels causing either uncoupling or inhibiting OxPhos) were 50-fold more potent than the classical uncoupler, salicylic acid at inducing Permeability Transition Pore Complex (PTPC) [136]. Similar results comparing a range of NSAIDs as uncouplers or inhibitors of OxPhos were obtained from studies using isolated rat liver or heart mitochondria or submitochondrial particles, intact hepatoma cells or isolated whole rat hearts [130]. ...
... Nabumetone inhibited respiratory Complex I but not Complex II activity and diclofenac inhibited ANT and ATP synthase activities, whereas naproxen did not affect mitochondrial function [130]. Thus, it can be summarized that the potency of the NSAID effects on mitochondrial function and killing of cancer cells does not correlate with their uncoupling activity or effects on OxPhos [130,136,137]. Neither did it relate to the severity of the NSAID effects on the gastrointestinal tract, suggesting that modifying mitochondrial function is not the direct cause of the high dose NSAID toxicity on normal cells [130]. ...
Tumor metastases that impede the function of vital organs are a major cause of cancer related mortality. Mitochondrial oxidative stress induced by hypoxia, low nutrient levels, or other stresses, such as genotoxic events, act as key drivers of the malignant changes in primary tumors to enhance their progression to metastasis. Emerging evidence now indicates that mitochondrial modifications and mutations resulting from oxidative stress, and leading to OxPhos stimulation and/or enhanced reactive oxygen species (ROS) production, are essential for promoting and sustaining the highly metastatic phenotype. Moreover, the modified mitochondria in emerging or existing metastatic cancer cells, by their irreversible differences, provide opportunities for selectively targeting their mitochondrial functions with a one-two punch. The first blow would block their anti-oxidative defense, followed by the knockout blow-promoting production of excess ROS, capitulating the terminal stage-activation of the mitochondrial permeability transition pore (mPTP), specifically killing metastatic cancer cells or their precursors. This review links a wide area of research relevant to cellular mechanisms that affect mitochondria activity as a major source of ROS production driving the pro-oxidative state in metastatic cancer cells. Each of the important aspects affecting mitochondrial function are discussed including: hypoxia, HIFs and PGC1 induced metabolic changes, increased ROS production to induce a more pro-oxidative state with reduced antioxidant defenses. It then focuses on how the mitochondria, as a major source of ROS in metastatic cancer cells driving the pro-oxidative state of malignancy enables targeting drugs affecting many of these altered processes and why the NSAIDs are an excellent example of mitochondria-targeted agents that provide a one-two knockout activating the mPTP and their efficacy as selective anticancer metastasis drugs.
... DCF exhibits anti-cancer effects [7][8][9][10] and is effective in the treatment of actinic keratosis 11 . DCF is a potent non-selective cyclooxygenase inhibitor 2,12 ; however, its full functional activity is thought to be related to a more complex mechanism of action, which has been investigated over the recent years 12,13 as well as toxic side-effects related to DCF therapies [14][15][16][17][18][19][20][21] . Since liver toxicity represents the most reported complication related to prolonged or high-dosage use of DCF, in vitro studies have mainly focused on hepatocytes. ...
... Moreover, the relevance of the previous studies has been questioned since they used very high concentrations which don't mimic a clinical therapeutic situation. While DCF hepato- [26][27][28][29][32][33][34][35] and nephro-toxicity 17,18,21,36 has been widely investigated, not that much is known about its activity as an anti-cancer drug [6][7][8][9][10]37 . For example, the mode of action of DCF in combination with hyaluronic acid in the local treatment of cutaneous actinic keratosis is largely elusive, but its chemotherapeutic activity could be associated with drug induced apoptosis 38,39 . ...
Electrospinning is an attractive method to generate drug releasing systems. In this work, we encapsulated the cell death-inducing drug Diclofenac (DCF) in an electrospun poly-L-lactide (PLA) scaffold. The scaffold offers a system for a sustained and controlled delivery of the cytotoxic DCF over time making it clinically favourable by achieving a prolonged therapeutic effect. We exposed human dermal fibroblasts (HDFs) to the drug-eluting scaffold and employed multiphoton microscopy and fluorescence lifetime imaging microscopy. These methods were suitable for non-invasive and marker-independent assessment of the cytotoxic effects. Released DCF induced changes in cell morphology and glycolytic activity. Furthermore, we showed that drug release can be influenced by adding dimethyl sulfoxide as a co-solvent for electrospinning. Interestingly, without affecting the drug diffusion mechanism, the resulting PLA scaffolds showed altered fibre morphology and enhanced initial DCF burst release. The here described model could represent an interesting way to control the diffusion of encapsulated bio-active molecules and test them using a marker-independent, non-invasive approach.
... Other agents inducing Fanconi syndrome include suramin, tyrosine kinase inhibitors and salicylates 91 . The latter have been reported to interfere with mitochondrial function and the induction of mitochondrial swelling [114][115][116] . ...
... While mitochondrial swelling upon NSAIDs was described, it was previously explained by the opening of the mPTP 115,116,238 . However, as discussed above, studies often attribute mitochondrial swelling to mPTP opening with insufficient hard evidence. ...
... Other NSAIDs also demonstrated increased cardiotoxic effects in patients who had prior heart conditions [53]. However, at least two studies with celecoxib suggest that the cardiovascular risk of this NSAID is similar to other non-selective NSAIDs or placebo [54,55]. When administered to patients with prior myocardial infarction, within weeks of treatment, diclofenac increased the risk of cardiac death by an average factor of 2.56, ibuprofen by 1.47, and celecoxib by 1.65 [56]. ...
... Hwang et al. Evidence also suggests that NSAIDs (such as diclofenac sodium and mefenamic acid) induce mitochondrial permeability transition (MPT) pores to open which leads to both reduction in oxidative phosphorylation (due to membrane depolarization) as well as initiation of apoptosis [55]. The consequences of an inadequate supply of ATP are numerous, but the most likely adverse effect related to increased risk of SCD is contractile dysfunction. ...
... In patients who developed hyperlactatemia during treatment with linezolid, decreased mitochondria mass and protein content were observed in their peripheral blood mononuclear cells, although mitochondrial membrane potential or intact cell oxidative capacity did not change significantly [44]. Mitochondrial permeability transition (MPT) has also been associated with cardiotoxicity of doxorubicin [45] and renal toxicity of non-steroid anti-inflammatory drugs [46]. Cerivastatin was withdrawn from the market due to its treatment-related rhabdomyolysis, kidney failure and deaths [47]. ...
... Cellular phenotypes can be useful pharmacodynamic endpoints for bridging cell/organ and clinical phenotypes. A mixed array of common and different molecular events can lead to mitochondrial toxicity, causing irreversible MPT, cell death and devastating injury to various organs [45,46,48]. A library of drugs that can cause mitochondrial toxicity, along with their individual molecular signatures in relevant cell lines/ tissues and clinical biomarkers, would be highly useful for modeling and establishing a predictive model for developing safer new drugs. ...
Advances in systems biology in conjunction with the expansion in knowledge of drug effects and diseases present an unprecedented opportunity to extend traditional pharmacokinetic and pharmacodynamic modeling/analysis to conduct systems pharmacology modeling. Many drugs that cause liver injury and myopathies have been extensively studied. Mitochondrion-centric systems pharmacology modeling is important since drug toxicity across a large number of pharmacological classes converges to mitochondrial injury and death. Approaches to systems pharmacology modeling of drug effects need to consider drug exposure, organelle and cellular phenotypes across all key cell types of human organs, organ-specific clinical biomarkers/phenotypes, gene-drug interaction, and immune responses. Systems modeling approaches, that leverage the knowledge base constructed from curating a selected list of drugs across a wide range of pharmacological classes, will provide a critically needed blueprint for making informed decisions to reduce the rate of attrition for drugs in development and increase the number of drugs with an acceptable benefit/risk ratio. This article is protected by copyright. All rights reserved.
... Oxidative stress is reported to play an important role in the pathogenesis of paracetamol-induced liver damage [10][11][12] . However, the mechanism of paracetamol-induced renal damage is not clear, though some studies suggest that lipid peroxidation plays a role, while others maintain that it may not [13][14][15] . Although lipid peroxidation is a commonly used measure of oxidative stress, indicators such as protein thiol, protein carbonyl content are also measured as the proteins regulate various metabolic pathways in the body and damage to proteins may result in cellular dysfunction and cell death. ...
... Glutathione level was further decreased and malondialdehyde level increased 24 hr after treatment, when there was no evidence of renal damage, suggesting that the increased malondialdehyde level may be a consequence of glutathione depletion. Similar results were reported by others workers 13,14 . Although, a recent study suggests that lipid peroxidation plays a role 15 , paracetamol-induced renal damage is reported to occur even in the absence of increase in lipid peroxidation and depletion of glutathione 9,18 . ...
The effect of administration of paracetamol (1 g/kg body wt) on oxidative damage to proteins and lipids in the kidney was studied at various time intervals in adult male Wistar rats. Iindicators of oxidative stress, such as protein thiol, protein carbonyl content and lipid peroxide levels were assayed along with thiol-dependent enzyme activities, glutamine synthase and glyceraldehyde-3-phosphate dehydrogenase. Paracetamol-induced renal damage after 4 hr of administration was evidenced by elevation in plasma creatinine levels and the presence of acute tubular necrosis on histological examination of the kidney. No significant change in any other parameters was observed, except for decreased glutathione level. An increase in lipid peroxide level was observed at 24 hr after treatment. The results suggest that oxidative stress may not play a causative role, but contribute to the pathogenesis of paracetamol-induced renal damage.
... Because L-carnitine inhibited Pal-CoA-induced MPT, its effect on MPT induced by other reagents was also investigated. In the presence of 2 M Ca 2ϩ , palmitic acid [7], T 3 [28], acetyl salicylate [29], and diclofenac [30] also induced swelling and depolarization of mitochondria by a mechanism that was suppressed by L-carnitine, BSA, chlorpromazine, and CsA (data not shown). ...
... It has been reported that the membranes of cells undergoing apoptosis are highly susceptible to the action of secretory PLA 2 (sPLA 2 ). For example, the accumulation of arachidonic acid followed by apoptosis occurred when PC12 cells were deprived of nerve growth factor and serum, when mast cells were deprived of hematopoietic cytokines, and when Fas-antigen treated monocytic U937 cells were stimulated via Fas antigen [30]. These facts indicate that the cell membranes of apoptotic cells are the potential targets for extracellular type II sPLA 2 and that LCFAs and carnitine play critical roles in certain types of apoptotic cell death. ...
Palmitoyl-CoA (Pal-CoA) lowered the respiratory control ratio (RCR), and induced mitochondrial membrane permeability transition (MPT) and cytochrome c (Cyt. c) release from isolated rat liver mitochondria. l-Carnitine suppressed the Pal-CoA-induced dysfunction, MPT, and Cyt. c release of isolated mitochondria. This suppression was inhibited by cephaloridine, an inhibitor of carnitine uptake into mitochondria. Cyclosporin A (CsA), an inhibitor of MPT, and BSA also suppressed the Pal-CoA-induced MPT. In the presence of inorganic phosphate (Pi), Ca2+-induced MPT was suppressed by BSA, l-carnitine, and chlorpromazine, an inhibitor of phospholipase A2. In the presence of a low concentration of Ca2+, 3,3′,5-triiodothyronine, long chain fatty acids, salicylic acid, and diclofenac induced MPT by a mechanism that was suppressed by BSA, l-carnitine, or chlorpromazine. During the incubation of mitochondria on ice, their respiratory competence decreased; l-carnitine and BSA also prevented this decrease. Mitochondrial depolarization in pheochromocytoma PC12 cells was induced by either serum deprivation or arachidonic acid by a mechanism that was suppressed by acetyl-l-carnitine. These results indicate that some MPTs may be regulated by fatty acid metabolism and that the Pal-CoA-induced MPT plays an important role in the induction of apoptosis.
... The final version may differ from this version. 1997;Ogata et al., 1999). Computational methods are then employed to model various experimental conditions, including gene functions in terms of gene networks and molecules, reconstruction of biochemical pathways and prediction of biological systems. ...
An increasing number of commonly prescribed drugs are known to interfere with mitochondrial function, which is associated with almost half of all FDA black box warnings, a variety of drug withdrawals and attrition of drug candidates. This can mainly be attributed to a historic lack of sensitive and specific assays to identify the mechanisms underlying mitochondrial toxicity during drug development. In the last decade, a better understanding of drug-induced mitochondrial dysfunction has been achieved by network-based and structure-based systems pharmacological approaches. Here, we propose the implementation of a tiered systems pharmacology approach to detect adverse mitochondrial drug effects during preclinical drug development, which is based on a toolset developed to study inherited mitochondrial disease. This includes phenotypic characterization, profiling of key metabolic alterations, mechanistic studies, and functional in vitro and in vivo studies. Combined with binding pocket similarity comparisons and bottom-up as well as top-down metabolic network modeling this tiered approach enables identification of mechanisms underlying drug-induced mitochondrial dysfunction. After validation of these off-target mechanisms, drug candidates can be adjusted to minimize mitochondrial activity. Implementing such a tiered systems pharmacology approach could lead to a more efficient drug development trajectory due to lower drug attrition rates and ultimately contribute to the development of safer drugs. Significance Statement Many commonly prescribed drugs adversely affect mitochondrial function, which can be detected using phenotypic assays. However, these methods provide only limited insight into the underlying mechanisms. In recent years, a better understanding of drug-induced mitochondrial dysfunction has been achieved by network-based and structure-based system pharmacological approaches. Their implementation in preclinical drug development could reduce the number of drug failures, contributing to safer drug design.
... Other explanations of the nephrotoxicity of diclofenac in a high dose were reported by many investigators. Diclofenac nephrotoxicity could be associated with the potent inducer of the membrane permeability transition in renal cortex mitochondria (Sergio and Antonio, 1997). ...
Non-steroidal anti-inflammatory drugs are in common use worldwide. These drugs may be used for long period and sometimes in high doses by mistake. In this study we investigate the effects of therapeutic and high doses of meloxicam and diclofenac sodium on liver and renal tissues. Forty adult (12-13 weeks) male albino rats weighing 180-200 g were divided equally into 5 groups. The rats in group A (control group, n=8) were each intramuscularly injected with normal saline. Rats in group B (n=8) and group D (n=8) were intramuscularly injected with meloxicam at a therapeutic and high doses of 0.22 and 1.1 mg/kg/day respectively, while the rats in group C (n=8) and group E (n=8) were intramuscularly injected with diclofenac sodium at a therapeutic and high doses of 2.2 and 11 mg/kg/day respectively. The injections were given in a single daily dose for sixty days. At the end of the experimental period (60 days), the animals were sacrificed. Livers and kidneys were obtained for histological, histochemical and morphometrical examinations.
The meloxicam and diclofenac treatments affected the histological, histochemical and morphometrical features of the two organs. Histological changes in the liver after therapeutic doses of meloxicam and diclofenac included vacuolar degeneration of hepatocytes, proliferation of bile duct in portal area, enlargement of portal area with mononuclear cell infiltration and focal sinusoidal and central venule dilatation. In addition, small areas of necrosis and fatty infiltration of some hepatocytes were seen in diclofenac treated group. Histochemicaly, glycogen content in the hepatocytes decreased as a result of these two drugs. Morphometrical parameters of liver were also affected by meloxicam and diclofenac administration (but they were less with meloxicam than diclofenac). The number of hepatocytes decreased significantly (P≤0.05), while the longest diameters of hepatocytes as well as the number of bile ducts and diameters of portal areas increased significantly (P≤0.05). The histological and morphometrical changes, besides, glycogen depletion in the liver tissues were increased and became widespread and intensive with increasing the doses of meloxicam and diclofenac.
Therapeutic doses of meloxicam and diclofenac affected the kidney as well resulting in vacuolar degeneration of the epithelial cells lining the proximal convoluted tubules, little peritubular mononuclear cell infiltration and congestion of glomeruli and interstitial blood vessels, in addition to pyknosis of some nuclei of the cells lining the proximal convoluted tubules and some atrophic glomeruli were observed in diclofenac treated group. Morphometrical parameters in the kidney were also affected by these drugs but were more with diclofenac than meloxicam group. The number of renal tubules as well as their lining cells and the longest diameters of glomeruli was decreased significantly (P≤0.05), the longest diameters of renal tubules and width of urinary space were significantly increased (P≤0.05). Glycogen content of tubular cells was normal. As the doses of both drugs increased, the histological and morphometrical changes increased in severity resulting in involvement of both proximal and distal convoluted tubules; in addition necrosis and sloughing of their epithelium occurred. Moreover glycogen content of tubular cells decreased.
These results suggest that, meloxicam and diclofenac in prolonged use are capable of producing hepatotoxicity and nehprotoxicity which were a dose dependant; in addition, the hepatic and renal toxicity of meloxicam was slightly less than that of diclofenac. Thus care should be taken when prescribing or using these drugs.
... Our hypothesis on the observed K-dependent mitochondrial membrane depolarization is that the drug has the ability to affect proton influx in the mitochondrial respiratory chain, similarly to salicylate derivatives. Also, it was previously shown that the uncoupling effect of Dic is about 50-fold greater than salicylate 74 . In agreement, our results show an alteration of mPTP in cells exposed to Dic; conversely cells exposed to K show the same behaviour of untreated cells. ...
The progressive consumption growth of non-steroidal anti-inflammatory drugs (NSAIDs) has progressively raised the attention toward the gastrointestinal, renal, and cardiovascular toxicity. Increased risk of cardiovascular diseases was strictly associated with the usage of COX-2 selective NSAIDs. Other studies allowed to clarify that the cardiovascular risk is not limited to COX-2 selective but also extended to non-selective NSAIDs, such as Diclofenac and Ketoprofen. To date, although a less favorable cardiovascular risk profile for Diclofenac as compared to Ketoprofen is reported, the mechanisms through which NSAIDs cause adverse cardiovascular events are not entirely understood. The present study aimed to evaluate the effects of Ketoprofen in comparison with Diclofenac in immortalized human cardiomyocytes. The results obtained highlight the dose-dependent cardiotoxicity of Diclofenac compared to Ketoprofen. Despite both drugs induce the increase in ROS production, decrease of mitochondrial membrane potential, and proteasome activity modulation, only Diclofenac exposure shows a marked alteration of these intracellular parameters, leading to cell death. Noteworthy, Diclofenac decreases the proteasome 26S DC and this scenario may be dependent on the intracellular overload of oxidized proteins. The data support the hypothesis that immortalized human cardiomyocytes exposed to Ketoprofen are subjected to tolerable stress events, conversely Diclofenac exposition triggers cell death.
... 25 It was reported that MFA is capable to inhibit oxidative phosphorylation, induce mitochondrial membrane permeability transition, cause mitochondrial swelling and increase the production rate of pro-oxidant radicals. 26 The MFA cytotoxicity may be also attributed to the formation of proteinreactive acylating metabolites by phase II metabolism that proceeds by oxidative biotransformation. 5 Repeated administration of MFA in mice can cause, at dose-dependent manner, glomerular necrosis, massive degeneration, inflammation, and tubular atrophy together with plasma elevation of blood urea nitrogen and creatinine. ...
Mefenamic acid (MFA) treatment is associated with a number of cellular effects that potentiate the incidence of renal toxicity. The aim of this study is to investigate the potential ultrastructural alterations induced by various preparations of MFA (free MFA, MFA-Tween 80 liposomes, and MFA-DDC liposomes) on the renal tissues. Sprague-Dawley rats were subjected to a daily dose of MFA preparations for 28 days. Renal biopsies from all groups of rats under study were processed for transmission electron microscopic examination. The findings revealed that MFA preparations induced various ultrastructural alterations including mitochondrial injury, nuclear and lysosomal alterations, tubular cells steatosis, apoptotic activity, autophagy, and nucleophagy. These alterations were more clear in rats received free MFA, and MFA-Tween 80 liposomes than those received MFA-DDC liposomes. It is concluded that MFA-DDC liposomes are less potential to induce renal damage than free MFA and MFA-Tween 80 liposomes. Thus, MFA-DDC liposomes may offer an advantage of safe drug delivery.
... This method is rapid and simple, and yields intact, reasonably pure, and functional mitochondria for analysis of different mitochondria-related processes (Fernández-Vizarra et al., 2010;Frezza et al., 2007;Pallotti and Lenaz, 2001) (Supplementary file). Different parameters including mitochondrial depolarization (Rhodamine 123 as the probe), ATP levels (HPLC analysis or standard kits), mitochondrial swelling and permeabilization (Light scattering method), mitochondrial dehydrogenases activity (Colorimetric method of methyl tetrazolium reduction), lipid peroxidation (Thiobarbituric acid reactive substances, TBARS, assay), and glutathione content (HPLC analysis or standard kits) could be assessed as biomarkers of mitochondrial function Heidari et al., 2018c, d;Heidari et al., 2018f;Lampl et al., 2015;Lash et al., 2001;Uyemura et al., 1997) (Supplementary file). Other aspects of mitochondrial function including biogenetical analysis, the interaction of mtDNA with xenobiotics, mitochondrial tRNA function, mitochondrial gene expression, protein transport, mitochondria respiratory complexes activity, and the analysis of mitochondrial oxygen consumption rate (OCR) could also be assessed based on the availability of appropriate laboratory facilities (Aparicio-Trejo et al., 2018;Iuso et al., 2017). ...
Fanconi's Syndrome (FS)is a disorder characterized by impaired renal proximal tubule function. FS is associated with a vast defect in the renal reabsorption of several chemicals. Inherited and/or acquired conditions seem to be connected with FS. Several xenobiotics including many pharmaceuticals are capable of inducing FS and nephrotoxicity. Although the pathological state of FS is well described, the exact underlying etiology and cellular mechanism(s)of xenobiotics-induced nephrotoxicity, serum electrolytes imbalance, and FS are not elucidated. Constant and high dependence of the renal reabsorption process to energy (ATP)makes mitochondrial dysfunction as a pivotal mechanism which could be involved in the pathogenesis of FS. The current review focuses on the footprints of mitochondrial impairment in the etiology of xenobiotics-induced FS. Moreover, the importance of mitochondria protecting agents and their preventive/therapeutic capability against FS is highlighted. The information collected in this review may provide significant clues to new therapeutic interventions aimed at minimizing xenobiotics-induced renal injury, serum electrolytes imbalance, and FS.
... Studies in rats have suggested that cell mitochondria of rat renal cortex are a prime target of prostaglandins may play a role in renal development [50]. diclofenac [35]. Furthermore, diclofenac can induce the The nephrotoxic effects of NSAIDs are attributed to the mitochondria permeability transition in isolated block of prostaglandin synthesis [51]. ...
Abstract: The present study was carried out to evaluate the effect of the non-steroidal anti-inflammatory drug diclofenac sodium (DS) on the renal cortex of fetuses of albino mice from the histological and ultrastructural points of view. Twenty pregnant female mice were allocated into 2 groups (10 mice each). The first group served as control and each animal was injected intraperitoneally (i.p.) with the solvent of the drug, daily for 8 days during pregnancy from day 7 till day 14 of gestation (GDs 7-14). The second group is the treated group; each animal was injected (i.p.) daily with 1.5 mg/kg body weight of DS for 8 days (GDs 7-14). Histological examination of the renal cortex of maternally treated fetuses showed atrophic glomeruli with widened capsular spaces of the renal corpuscles. Renal convoluted tubular cells had a vacuolated cytoplasm and pyknotic nuclei. Some proximal tubules showed disruption of their apical brush borders. Also, the lumina of some proximal and distal tubules were occluded with hyaline casts. Electron microscopic examination of the renal cortex of fetuses maternally treated with DS revealed conspicuous alterations, represented by thickening of the capillary basement membrane in some glomeruli. The foot processes of podocytes were frequently fused thus obliterating the infiltration slits. The cells of the proximal convoluted tubules displayed partial destruction of the microvilli of the apical brush borders and degeneration of mitochondria. Besides, many vesicles and large vacuoles were observed near the basal part of the microvilli. The cells of the distal convoluted tubules showed marked thickening of their basement membranes and their mitochondria lost their cristae and appeared vacuolated
(14) (PDF) Histological and Ultrastructural Studies on the Effect of Diclofenac Sodium on the Renal Cortex of Fetuses of Albino Mice. Available from: https://www.researchgate.net/publication/331533510_Histological_and_Ultrastructural_Studies_on_the_Effect_of_Diclofenac_Sodium_on_the_Renal_Cortex_of_Fetuses_of_Albino_Mice [accessed May 31 2019].
... In addition, studies using isolated mitochondria and renal cortex mitochondria have suggested the mechanism of membrane permeability for naproxen, salicylates, and diclofenac are also dependent on calcium. This leads to uncoupling of mitochondrial oxidative phosphorylation and mitochondrial swelling in the presence of high intra-mitochondrial calcium levels (Salgueiro-Pagadigorria et al., 2004;Uyemura et al., 1997). Studies using isolated rat kidney mitochondria have revealed diclofenac further contributes to mitochondrial stress by inhibiting the malate-aspartate shuttle, which transports malate and glutamate into the mitochondria. ...
Leber's hereditary optic neuropathy (LHON) is a maternally inherited mitochondrial disorder characterized by acute bilateral vision loss. The pathophysiology involves reactive oxygen species (ROS), which can be affected by medications. This article reviews the evidence for medications with demonstrated and theoretical effects on mitochondrial function, specifically in relation to increased ROS production. The data reviewed provides guidance when selecting medications for individuals with LHON mutations (carriers) and are susceptible to conversion to affected. However, as with all medications, the proven benefits of these therapies must be weighed against, in some cases, purely theoretical risks for this unique patient population.
... Specifically, in isolated liver mitochondria, diclofenac readily inhibits ATP synthesis and induces the MPT [94,97], leading to a collapse of the mitochondrial transmembrane potential (∆Ψ m ). In this context, diclofenac, which is a lipophilic and weak acidic compound, works as an uncoupler of oxidative phosphorylation [101], translocating protons across the inner mitochondrial membrane to cause uncoupling. In addition, some studies suggest that the uncoupling effect of diclofenac stimulates mitochondrial respiration and enhances superoxide generation, with the increased ROS oxidizing membrane thiols, which play important roles in opening of the MPT [94]. ...
Mitochondria are critical cellular organelles for energy generation and are now also recognized as playing important roles in cellular signaling. Their central role in energy metabolism, as well as their high abundance in hepatocytes, make them important targets for drug-induced hepatotoxicity. This review summarizes the current mechanistic understanding of the role of mitochondria in drug-induced hepatotoxicity caused by acetaminophen, diclofenac, anti-tuberculosis drugs such as rifampin and isoniazid, anti-epileptic drugs such as valproic acid and constituents of herbal supplements such as pyrrolizidine alkaloids. The utilization of circulating mitochondrial-specific biomarkers in understanding mechanisms of toxicity in humans will also be examined. In summary, it is well-established that mitochondria are central to acetaminophen-induced cell death. However, the most promising areas for clinically useful therapeutic interventions after acetaminophen toxicity may involve the promotion of adaptive responses and repair processes including mitophagy and mitochondrial biogenesis, In contrast, the limited understanding of the role of mitochondria in various aspects of hepatotoxicity by most other drugs and herbs requires more detailed mechanistic investigations in both animals and humans. Development of clinically relevant animal models and more translational studies using mechanistic biomarkers are critical for progress in this area.
... There are different molecular pathways involved in apoptotic effect of ASA recognized, these include; p38 MAP Kinase (Mitogen-activated protein kinases) activation in human fibroblast [37], effect on human telomerase reverse transcriptase in colorectal cancer cells [38], caspase activation [39,40], down regulation of the anti-apoptotic Bcl-2 protein [41,42], beside that it triggers alteration of the signaling pathway of the transcription factor NF-κB in human gastric cells [43]. ASA induce tumor apoptotic cell death through promoting the onset of the mitochondrial permeability transition [44,45]. ...
... Interestingly, enhanced mitochondrial ROS generation has also been implicated in the enhancement of MPTP sensitivity and/or opening[102], which leads to a decrease in mitochondrial trans-membrane potential, resulting in mitochondrial dysfunction[103]. These observations are highly relevant in context to the role of TNF in NSAID enteropathy as both NSAIDs and TNF have been frequently reported as MPTP inducers[104][105][106]and their cytotoxicity also depends in part on induction of MPTP M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 10 intestinal epithelial cells[112]. Importantly, these secondary bile acids have also been identified as potent MPTP inducers[113,114]. ...
The pathogenesis of Nonsteroidal anti-inflammatory drug (NSAID) enteropathy is still unclear and consequently there is no approved therapeutic strategy for ameliorating such damage. On the other hand, molecular treatment strategies targeting tumour necrosis factor (TNF) exerts beneficial effects on NSAID-induced intestinal lesions in rodents and rheumatoid arthritis patients. Thus, TNF appears to be a potential therapeutic target for both the prevention as well as treatment of NSAID enteropathy. However, the causative relationship between TNF and NSAID enteropathy is largely unknown. Currently approved anti-TNF agents are highly expensive and exhibit numerous side effects. Hence, in this review, the pivotal role of TNF in NSAID enteropathy has been summarized and plant-derived polyphenols have been suggested as useful alternative anti-TNF agents because of their ability to suppress TNF activated inflammatory pathways both in vitro and in vivo.
... Arachidonic acid is an important component of cellular structural integrity; the lack of its availability and the potentially increased permeability of cellular membranes may lead to corneal epithelial cell death and keratolysis. 24,25 According to the mean conjunctival and corneal fluorescein staining score in this study, the diclofenac group had significantly higher mean scores at 4 weeks postoperatively than the nepafenac group, and significantly higher mean scores were noted at 4 weeks postoperatively than preoperatively and at 1 week postoperatively within the diclofenac group. Twenty-six patients (87%) in the diclofenac group had an increase in only the corneal scores from 1 to 4 weeks postoperatively. ...
Background:
The aim of this study was to compare the effects of nepafenac ophthalmic suspension 0.1% (Nevanac) and diclofenac sodium ophthalmic solution 0.1% (Diclod) on the cornea, tear film, and ocular surface after cataract surgery.
Methods:
A total of 60 eyes (60 patients) were selected for this study, with no ocular diseases other than cataract (scheduled for cataract surgery by one surgeon). Patients were randomly enrolled to receive nepafenac or diclofenac in the perioperative period, and cataract surgery was performed using torsional microcoaxial phacoemulsification and aspiration with intraocular lens implantation via a transconjunctival single-plane sclerocorneal incision at the 12 o'clock position. We compared intra- and intergroup differences preoperatively and postoperatively in conjunctival and corneal fluorescein staining scores, tear film breakup times, Schirmer's tests, the Dry Eye Related Quality of Life Scores, and tear meniscus areas using anterior segment optical coherence tomography.
Results:
The diclofenac group had significantly higher conjunctival and corneal fluorescein staining scores at 4 weeks postoperatively compared with the nepafenac group (P<0.001). Within the diclofenac group, significantly higher conjunctival and corneal fluorescein staining scores were noted at 4 weeks postoperatively than those seen preoperatively (P<0.001) and at 1 week postoperatively (P<0.001). No statistically significant differences were found in any other items.
Conclusions:
Nepafenac ophthalmic suspension 0.1% is considered safe for the corneal epithelium after cataract surgery.
... Diclofenac sodium like other non steroid anti-inflammatory drugs (NSAIDs) expresses its anti-inflammatory, analgesic and antipyretic effects as well as adverse effect through decrease of the prostaglandin synthesis from arachidonic acid by inhibition of the cyclooxygenase enzyme activity [7,20,33]. Diclofenac sodium has undesirable side effects such as gastrointestinal ulceration, hepatotoxicity and nephrotoxicity [7,11,15,27,28,31,32]. Recent studies have shown that NSAIDs may play a role in the formation of radical cations co-oxidising GSH or NADH to generate reactive oxygen species (ROS), by becoming oxidised via peroxidase [25,26]. ...
This study investigated the eventual protective effects of L-carnitine against renal and liver damage caused by high doses of diclofenac sodium in mice. A pilot study, designed to determine the highest toxic dose of diclofenac, was conducted on 32 Swiss Albino adult mice randomly divided into 4 equal groups according to the drug dose: 0 (control), 2.5 (low), 5 (moderate) and 10 mg/kg/day (high dose) for 5 days by the subcutaneous route. Serum biochemical parameters (BUN and creatinine concentrations and AST, ALT and ALP activities) were measured as well as GSH and MDA contents in liver and in kidney at the end of the treatment. The 2 highest dosages of diclofenac have induced significant increases of the serum markers and MDA accumulation in tissues whereas the kidney and liver GSH contents were depressed in parallel. Besides, a strong dose-effect relationship was evidenced. In the second experimental step, 4 groups of 8 mice received subcutaneous injections for 5 days of saline solution (NaCl, 20 mL/g body weight/day) (group I), of L carnitine (500 mg/kg/day) (group II), of diclofenac sodium (10 mg/kg/day) (group III) and of diclofenac (10 mg/kg/day) plus L carnitine (500 mg/kg/day 3 days before and 2 days during the diclofenac treatment) (group IV) respectively. The diclofenac treatment alone or in combination with L carnitine induced liver and kidney damage as attested by significant increases of the serum markers and by tissue MDA accumulation. Nevertheless, these variations were significantly reduced in co-treated mice. Whereas the GSH pools in liver and kidney were markedly depressed in the group III, they were significantly enhanced in mice treated with L carnitine alone, and remained unaffected in co-treated mice (group IV) compared to the controls. These results demonstrated that the diclofenac toxicity is due to lipid peroxidation and impairment of the antioxidant systems in liver and kidney and that a co-treatment with L carnitine can partially alleviate it by restoring antioxidant capacity.
... The results of this study agree with previous reports on the toxicity of diclofenac sodium (7,8,(24)(25)(26)(27)(28). Overall, the toxicity of the diclofenac sodium eye solution might be resulted from the following 3 underlying mechanisms: 1) the lower Na + concentration, 2) the higher amount of preservatives, and 3) the blockage of the lipoxygenase pathway at high concentrations (22,23). ...
This study investigated the toxicity of commercial non-steroid anti-inflammatory drug (NSAID) eye solutions against corneal epithelial cells in vitro. The biologic effects of 1/100-, 1/50-, and 1/10-diluted bromfenac sodium, pranoprofen, diclofenac sodium, and the fluorometholone on corneal epithelial cells were evaluated after 1-, 4-, 12-, and 24-hr of exposure compared to corneal epithelial cell treated with balanced salt solution as control. Cellular metabolic activity, cellular damage, and morphology were assessed. Corneal epithelial cell migration was quantified by the scratch-wound assay. Compared to bromfenac and pranoprofen, the cellular metabolic activity of diclofenac and fluorometholone significantly decreased after 12-hr exposure, which was maintained for 24-hr compared to control. Especially, at 1/10-diluted eye solution for 24-hr exposure, the LDH titers of fluorometholone and diclofenac sodium markedly increased more than those of bromfenac and pranoprofen. In diclofenac sodium, the Na+ concentration was lower and amount of preservatives was higher than other NSAIDs eye solutions tested. However, the K+ and Cl- concentration, pH, and osmolarity were similar for all NSAIDs eye solutions. Bromfenac and pranoprofen significantly promoted cell migration, and restored wound gap after 48-hr exposure, compared with that of diclofenac or fluorometholone. At 1/50-diluted eye solution for 48-hr exposure, the corneal epithelial cellular morphology of diclofenac and fluorometholone induced more damage than that of bromfenac or pranoprofen. Overall, the corneal epithelial cells in bromfenac and pranoprofen NSAID eye solutions are less damaged compared to those in diclofenac, included fluorometholone as steroid eye solution.
... Kidney of rats of control group showed normal in total protein and albumin values. Tolman, Kappus and histologic architecture with normal proximal convoluted Sergio and antonio [30,32,33] reported that diclofenac tubules lined by cuboidal epithelium (Fig. 1E). However, sodium induced hepatotoxic and nephrotoxic effects in kidney of rats treated with diclofenac sodium at dose of both humans and experimental animals. ...
The present study was carried out to evaluate the adverse effects of a daily single injection of diclofenac sodium at different doses on some hematological, biochemical parameters and oxidative stress. Histological changes in liver and kidney induced by diclofenac sodium were also investigated in male albino rats. Forty five rats were randomly divided into 3 equal groups, the first group was given saline intramuscullary and kept as a control.The second group recieved diclofenac sodium at a dose of 6.75 mg / kg b.wt. i. m. once/day. The third group was given diclofenac sodium at a dose of 13.5 mg / kg b.wt. i. m. once/day. Diclofenac sodium and saline were administered for two consecutive weeks. The obtained results showed that, diclofenac sodium at dose of 13.5 mg / kg b.wt. induced a significant decrease in Hb, PCV, RBCs and WBCs values. Moreover there were a significant decrease in serum total protein and albumin levels and significant increase in aminotranseferases, alkaline phosphatase, urea and creatinine levels. In addition there were a significant reduction in reduced glutathione(GSH) levels and significant increase in malondialdehyde (MDA) content in liver and kidney homogenates. Histopathological alterations were found in livers and kidneys. However, diclofenac sodium at dose of 6.75 mg / kg b.wt. induced non signaficant changes in the previous parameters. It could be concluded that administration of diclofenac sodium at high dose induced some adverse effects on hematological, biochemical, oxidative parameters as well as histology of liver and kidney. That could be attributed to oxidative stress induced by the drug. However, these effects were reversible.
... In order to determine whether the IMM were co-immobilized with the OMM, the presence of SUR and mPTP was investigated. Glibenclamide, an antidiabetic sulfonyl urea, was used as the marker ligand for SUR [36], while diclofenac was used as the marker ligand for mPTP [1,37]. The binding affinities (K d values) obtained for glibenclamide and diclofenac, based on the frontal displacement experiments carried out on MMAC-(U87MG) using ammonium acetate [10 mM, pH 7.4] was 0.79 ± 0.39 μM and 1.20 ± 0.19 μM respectively (Table 3), which is consistent with previuosly reported values. ...
Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized on to Immobilized Artificial Membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC-(U87MG) column and the binding affinities (Kd) determined were 1.08 ± 0.49 and 0.0086 ± 0.0006 μM respectively, which was consistent with previously reported values. Further, binding affinities (Ki) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX and rotenone. Additionally, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC-(U87MG) was consistent with the obtained Ki values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy.
Copyright © 2015. Published by Elsevier Inc.
... Most notably, however, the finding that low dose treatment with Dcl causes up-regulation of several subunits of ATP synthase, an enzyme critical for mitochondrial energy production, may provide a mechanistic clue as to how sensitization of cells by therapeutic doses of Dcl in our model may be brought about. Indeed, mitochondria have been recognized as critical targets of Dcl based on the finding thatpreceding overt cell injury -Dcl causes ATP depletion, uncoupling of respiration and opening of the mitochondrial membrane permeability transition pore in perfused rat liver and heart (Moreno-Sanchez et al., 1999;Petrescu and Tarba, 1997), primary rat hepatocytes (Masubuchi et al., 2002;Masubuchi et al., 1998), isolated liver and kidney mitochondria (Masubuchi et al., 2002;Ng et al., 2006;Uyemura et al., 1997), and thymocytes (Krause et al., 2003) Although data on Dcl are lacking, it is interesting to note that ATP synthase subunits A and B have been identified as common target proteins of a range of compounds known to mediate their toxic effects through formation of reactive metabolites, including acetaminophen, naphtalene, benzene, Teucrin A, mycophenolic acid, monocrotaline pyrrole, and furan (summarized in the target protein database: http://tpdb.medchem.ku.edu:8080/protein_database/) (Moro et al., 2011). It is thus tempting to speculate that up-regulation of ATP synthase expression may occur in response to inactivation of ATP synthase through covalent binding of reactive metabolites. ...
Increasing experimental and clinical evidence suggest a contribution of non-drug related risk factors (e.g. underlying disease, bacterial/viral infection) to idiosyncratic drug reactions (IDR). Our previous work showed that co-treatment with bacterial endotoxin (LPS) and therapeutic doses of diclofenac (Dcl), an analgesic associated with drug idiosyncrasy in patients, induced severe hepatotoxicity in rats. Here, we used an integrated discovery to targeted LC-MS proteomics approach to identify mechanistically relevant liver and plasma proteins modulated by LPS/Dcl treatment, potentially applicable as early markers for IDRs. Based on pre-screening results and their role in liver toxicity, 47 liver and 15 plasma proteins were selected for targeted LC-MS analysis. LPS alone significantly changed the levels of 19 and 3 of these proteins, respectively. T-kininogen-1, previously suggested as a marker of drug-induced liver injury, was markedly elevated in plasma after repeated Dcl treatment in the absence of hepatotoxicity, possibly indicating clinically silent stress. Dcl both alone and in combination with LPS, caused up-regulation of the ATP synthase subunits (ATP5J, ATPA, and ATPB), suggesting that Dcl may sensitize cells against additional stress factors such as LPS through generation of mitochondrial stress. Additionally, depletion of plasma fibrinogen was observed in the co-treatment group, consistent with an increased hepatic fibrin deposition and suspected contribution of the hemostatic system to IDRs. In contrast, several proteins previously suggested as liver biomarkers, such as clusterin, did not correlate with liver injury in this model. Taken together, these analyses revealed proteomic changes in a rat model of LPS/Dcl co-administration that could offer mechanistic insight and may serve as biomarkers or safety alert for a drug's potential to cause IDRs.
Copyright © 2015. Published by Elsevier Ireland Ltd.
... Increased collagen fibers around central veins and portal tracts was also seen. These changes are in agreement with the results of other researchers who reported that diclofenac in high doses induced hepatotoxic and hepatocellular necrosis in the liver (23)(24)(25)(26) . Moreover, our study goes hand in hand with biochemical study of previous work who reported that, high doses of diclofenac produced impairment in the liver and kidney function testes which be taken as a markers of their injuries (22) . ...
Introduction: Diclofenac is one of the most frequently prescribed non-steroidal anti-inflammatory drugs (NSAIDs) and have been reported to cause multiple organs damage. Ginger extract has been used as an antioxidant and preventive agent against a number of diseases. Aim of the Work: This work aimed to study the possible histological and ultrastructural changes of liver induced by diclofenac treatment and to evaluate the possible protective effect of ginger extract. Materials and Methods: Forty adult male albino rats were divided into 4 main groups. Group I: served as control. Group II: received ginger extract orally in a dose of 250 mg/kg /day. Group III: animals received diclofenac intramuscularly at a dose 150 mg/kg /day. Groups IV: animals received ginger extract then diclofenac after two hours in the previous doses. The treatments were given for rats for 7 days, then, rats were sacrificed by ether anesthesia and specimens from liver were taken for light and electron microscopic examination. Results: Light microscopic examination of the liver treated with diclofenac revealed disorganized hepatic architecture. The affected lobules appeared with dilated congested central veins and blood sinusoids, vacuolated hepatocytes with dark nuclei, cellular infilteration and fibrosis. Ultrastructurally, hepatocytes showed disintegration of cellular organelles, destructed mitochondria and pyknotic nuclei. Coadministration of diclofenac with ginger extract showed a slight improvement in some hepatocytes that looked normal in both LM and EM examination but still others were markedly affected and showing signs of degeneration. Conclusion: Results obtained in this study demonstrated that high doses of diclofenac induced histological and ultrastructural changes in the liver due to oxidative stress and the use of ginger extract had partially improved the toxic effect of diclofenac.
... Here, these pathways induce events such as the release of cytochrome c and other proapoptotic molecules, including SMAC/Diablo, from the mitochondria [43], which irreversibly commit cells to the full apoptotic phenotype. Thus mitochondria clearly constitute a very important target of NSAID-induced apoptosis, as indicated by the many studies associating NSAID-induced apoptosis of mammalian cells with mitochondrial dysfunction , such as uncoupling of oxidative phosphorylation [99], induced mitochondrial permeability transition [100, 101], and inactivation of mitochondrial enzymes such as aconitase and respiratory chain proteins [45]. Likewise, it has been shown that the mitochondria of S. cerevisiae cells constitute a critical target of NSAIDs such as aspirin [32], the proapoptotic effects of which were shown to be associated with inhibition of the electron transport chain [93]. ...
Nonsteroidal anti-inflammatory drugs (NSAIDs) have long been used to treat pain, fever, and inflammation. However, mounting evidence shows that NSAIDs, such as aspirin, have very promising antineoplastic properties. The chemopreventive, antiproliferative behaviour of NSAIDs has been associated with both their inactivation of cyclooxygenases (COX) and their ability to induce apoptosis via pathways that are largely COX-independent. In this review, the various proapoptotic pathways induced by traditional and novel NSAIDs such as phospho-NSAIDs, hydrogen sulfide-releasing NSAIDs and nitric oxide-releasing NSAIDs in mammalian cell lines are discussed, as well as the proapoptotic effects of NSAIDs on budding yeast which retains the hallmarks of mammalian apoptosis. The significance of these mechanisms in terms of the role of NSAIDs in effective cancer prevention is considered.
... As a consequence of some pathological conditions prevalent in aged patients, non-steroid anti-inflammatory drugs (NSAID), including diclofenac and salicylate, are widely consumed. Both drugs inhibit in vitro KM and LM ATP production, also markedly increasing calciuminduced MPTP and activating apoptotic signaling, leading also to an over-production of ROS in the process (Al-Nasser, 1999;Battaglia et al., 2005;Masubuchi et al., 2002;Mingatto et al., 1996;Ng et al., 2006;Pigoso et al., 1998;Trost and Lemasters, 1997;Uyemura et al., 1997). ...
... One mechanism is the opening of the mPT pore, involving both the inner and outer membrane, which can be triggered by increased [Ca 2+ ], oxidant stress, and/or a collapse of the mitochondrial membrane potential (∆Ψ m ) (for recent reviews, see Baines [2009]; Halestrap [2009]). A number of NSAIDs indeed cause increases in cellular [Ca 2+ ] and oxidant stress (Tanaka et al., 2005), and many of them are uncouplers of OXPHOS, resulting in a dissipation of ∆Ψ m (Al-Nasser, 2000; Masubuchi et al., 2000Masubuchi et al., , 2002Tay et al., 2005;Uyemura et al., 1997). However, direct evidence that NSAIDs can cause mPT pore opening in the small intestinal mucosa has been limited to in vitro studies with cultured enterocytes, where high concentrations of diclofenac were able to cause cyclosporin A-sensitive changes in calcein/Co 2+ fluorescence, an accepted indicator of the mPT (LoGuidice et al., 2010). ...
Nonsteroidal anti-inflammatory drugs (NSAIDs) can cause serious gastrointestinal injury including jejunal/ileal mucosal ulceration, bleeding, and even perforation, in susceptible patients. The underlying mechanisms are largely unknown, but they are distinct from those related to gastric injury. Based on recent insights from experimental models, including genetics and pharmacology in rodents typically exposed to diclofenac, indomethacin, or naproxen, we propose a multiple-hit pathogenesis of NSAID enteropathy. The multiple-hits start with an initial pharmacokinetic determinant caused by vectorial hepatobiliary excretion and delivery of glucuronide NSAID or oxidative metabolite conjugates to the distal small intestinal lumen, where bacterial β-glucuronidase produces critical aglycones. The released aglycones are then taken up by enterocytes and further metabolized by intestinal cytochrome P450s to potentially reactive intermediates. The "first hit" is caused by the NSAID and/or oxidative metabolites that induce severe endoplasmic reticulum stress or mitochondrial stress and lead to cell death. The "second hit" is created by the significant subsequent inflammatory response that would follow such a first-hit injury. Based on these putative mechanisms, strategies have been developed to protect the enterocytes from being exposed to the parent NSAID and/or oxidative metabolites. Among these, a novel strategy already demonstrated in a murine model is the selective disruption of bacteria-specific β-glucuronidases with a novel small molecule inhibitor that does not harm the bacteria and that alleviates NSAID-induced enteropathy. Such mechanism-based strategies require further investigation, but provide potential avenues for the alleviation of the GI toxicity caused by multiple NSAID hits.
Significance:
Ample evidence has demonstrated an important role for autophagy as either a protective mechanism or a cause for hepatotoxicity, denoting the likely potentials for targeting autophagy in the prevention or treatment of hepatotoxicity.
Recent advances:
The functional role of autophagy is in the pathogenesis of hepatotoxicity has been gradually recognized. Mechanistically, autophagy-mediated either protective or promoting effect on hepatotoxicity is attributed to its functions to regulate oxidative stress, endoplasmic reticulum (ER) stress, lipid metabolisms, iron homeostasis, inflammatory response and programmed cell death. Targeting autophagy as a novel strategy for fighting against hepatotoxicity has demonstrated encouraging efficacy in a number of models.
Critical issues:
Clarifying the precise functional role of autophagy in different types of hepatotoxicity is essential for developing the type-specific autophagy-based intervention. Unraveling the molecular targets and identifying novel agents for effectively and accurately manipulating autophagy are needed for a better utilization of autophagy-based approach to exert beneficial effect on hepatotoxicity.
Future directions:
Well-designed clinical trials are needed to validate the efficacy of autophagy-targeting strategy for hepatotoxicity intervention. Further studies should be also focused on developing novel autophagy-targeting agents that can accurately regulate autophagy based on the characteristics of each type of hepatotoxicity.
The present study discusses comparative structural features of fourteen multicomponent solids of two non-steroidal anti-inflammatory drugs, Niflumic and Mefenamic acids, with amine and pyridine-based coformers. All the solids were structurally characterized through PXRD, SCXRD, DSC, and the monophasic nature of some of the solids was established through Rietveld refinement. The solid forms include salt, cocrystal, hydrate, and solvate. Except for two, all the solids reported here showed relatively higher solubility compared to the acids. The difference in pKa and similarity in structural features of both the molecules enabled us to study the effect of ΔpKa on crystallization outcome systematically. The structures of all the solids are described through acid-pyridine synthon perspective.
Natural food flavour (E)-2-hexenal, a green leaf volatile, exhibits potent antifungal activity on Aspergillus flavus, but its antifungal mechanism has not been fully elucidated. In this study, we evaluated (E)-2-hexenal-induced apoptosis in A. flavus conidia and explored the underlying mechanisms of action. Evidence of apoptosis in A. flavus conidia were investigated by methods including fluorescent staining, flow cytometry, confocal laser scanning microscope, and spectral analysis. Results indicated that 4.0 μL/mL (minimum fungicidal concentration, MFC) of (E)-2-hexenal application induced early markers of apoptotic cell death in A. flavus conidia with a rate of 38.4% after 6 h exposure. Meanwhile, typical hallmarks of apoptosis, such as decreased mitochondrial membrane potential (MMP), activated metacaspase activity, fragmented DNA, mitochondrial permeability transition pore (MPTP) opening and cytochrome c (Cyt C) release from mitochondria to the cytosol were also confirmed. Furthermore, intracellular ATP levels were reduced by 63.3 ± 3.6% and reactive oxygen species (ROS) positive cells increased by 31.1 ± 3.1% during A. flavus apoptosis induced by (E)-2-hexenal. l-Cysteine (Cys), an antioxidant, could strongly block the excess ROS generation caused by (E)-2-hexenal, which correspondingly resulted in a significant inhibition of MPTP opening and decrease of apoptosis in A. flavus, indicating that ROS palys a pivotal role in (E)-2-hexenal-induced apoptosis. These results suggest that (E)-2-hexenal exerts its antifungal effect on A. flavus conidia via a ROS-dependent mitochondrial apoptotic pathway.
Cervical cancer is one the commonest type of cancers affecting women worldwide with high mortality rate. Human Papilloma Virus (HPV) is the common risk factor for about 99% of cervical cancer infection worldwide. HPV causes cervical cancer through the up-regulation of COX. COX-1 and COX-2 which is present in small amount in every cell. It plays a key role in regulation of cervical cancer neoplasia. Cell viability was determined using cell titre blue by doubling dilution from 1,000,000 to 31,250 cells per ml on HT3. Dose response for Acetylsalicylic Acid ASA and SA was carried out at different concentrations from 0-20mM concentrations and incubated at different time intervals 24, 48 and 72hrs incubation. A 10mM concentration of ASA and Salisalic Acid (SA) was used to determine the caspase activity using caspaseglo on the cell line for the period of 0-24hrs incubations. Western blot was carried out using active anti-caspase3 antibody for caspase3 proteins. The number of cells was found to increase as the absorbance increases, but the relationship breaks up above 600000 cells per ml. Effects of drugs on the viability of HT3 indicated that SA has more effect by inhibiting the viability of HT3 from 10-20mM concentrations at 48 and 72hrs incubations. SA shows more differential effect on caspase with more increase in caspase activity from 8-16hrs incubations. Western blot shows no expression of protein for caspase3, using β actin as a housekeeping gene. This study indicates that SA has more effect on HT3 cervical cancer cell line. This finding might be as a result of differences in the chemical properties of the drugs.
The iron chelator Deferasirox (DFX) causes severe toxicity in patients for reasons that were previously unexplained. Here, using the kidney as a clinically relevant in vivo model for toxicity together with a broad range of experimental techniques, including live cell imaging and in vitro biophysical models, we show that DFX causes partial uncoupling and dramatic swelling of mitochondria, but without depolarization or opening of the mitochondrial permeability transition pore. This effect is explained by an increase in inner mitochondrial membrane (IMM) permeability to protons, but not small molecules. The movement of water into mitochondria is prevented by altering intracellular osmotic gradients. Other clinically used iron chelators do not produce mitochondrial swelling. Thus, DFX causes organ toxicity due to an off-target effect on the IMM, which has major adverse consequences for mitochondrial volume regulation.
Acanthamoeba keratitis is caused by a protozoal infection of the cornea, with 80% of cases involving the improper use of contact lenses. The infection causes intense pain and is potentially blinding. However, early diagnosis improves treatment efficacy and the chances of healing. Despite the apparent accessibility of the cornea, patients do not always respond well to current eye drop treatments largely due to rapid dose loss due to blinking and nasolacrimal drainage. Here, the topical drug delivery of voriconazole alone and in combination with diclofenac via drug-loaded contact lenses, were investigated in vitro. The contact lenses were applied onto excised porcine eyeballs and maintained at 32°C under constant irrigation, with simulated tear fluid applied to mimic in vivo conditions. The drug delivered to the corneas was quantified by HPLC analysis. The system was further tested in terms of cytotoxicity and a scratch wound repopulation model, using corneal epithelial cells. Sustained drug delivery to the cornea was achieved and for voriconazole, the MIC against Acanthamoeba castellanii was attained alone and in combination with diclofenac. MTT and scratch wound data showed reasonable cell proliferation and wound repopulation at the drug doses used, supporting further development of the system to treat Acanthamoeba keratitis.
Background & aims:
Non-steroidal anti-inflammatory drugs (NSAIDs) such as diclofenac (DFN) and indomethacin (INDO) are extensively used worldwide. Their main side effects are injury of gastrointestinal tract - erosions, ulcers and bleeding. Since gastric epithelial cells (GEPCs) are crucial for mucosal defense and are the major target of injury, we examined the extent to which DFN- and INDO- induced GEPC injury can be reversed by nerve growth factor (NGF), 16,16-dimethyl prostaglandin E2 (dmPGE2), and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) - the pharmacological activator of the metabolic sensor, AMP kinase (AMPK).
Methods:
Cultured rat gastric mucosal epithelial cells (RGM1) were treated with PBS (control), NGF, dmPGE2, AICAR and/or NSAID - DFN or INDO for 1-4 hours. We examined: 1) cell injury; 2) cell death/survival; 3) mitochondrial membrane potential; and 4) phosphorylation of AMPK.
Results:
DFN and INDO treatment of RGM1 cells for 2 hours decreased mitochondrial membrane potential and cell viability. NGF post-treatment (initiated 1 or 2 hours after DFN or INDO) reversed the dissipation of mitochondrial membrane potential and cell injury caused by DFN and INDO, and increased cell viability vs. cells treated for 4 hours with NSAID alone. Pre-treatment with dmPGE2 and AICAR protected these cells from DFN- and INDO- induced injury while dmPGE2 and AICAR post-treatment (initiated 1 hour after NSAID treatment) reversed cell injury, and significantly increased cell viability and rescued the cells from NSAID-induced mitochondrial membrane potential reduction.
Conclusions:
DFN and INDO induce extensive mitochondrial injury and GEPC death, which can be significantly reversed by NGF, dmPGE2 and AICAR.
Our previous studies showed colistin-induced neurotoxicity involves apoptosis and oxidative damage. The present study demonstrates a neuroprotective effect of rapamycin against colistin-induced neurotoxicity in vitro and in vivo. In a mouse model, colistin treatment (18 mg/kg/d; 14 days) produced marked neuronal mitochondria damage in the cerebral cortex and increased activation of caspase-9 and -3. Rapamycin co-treatment (2.5 mg/kg/d) effectively reduced this neurotoxic effect. In an in vitro mouse neuroblastoma-2a (N2a) cell culture model, rapamycin pre-treatment (500 nM) significantly decreased colistin (200 μM) induced cell death from ~50% to 72%. Moreover, rapamycin showed a marked neuroprotective effect in the N2a cells by decreasing intracellular reactive oxygen species (ROS) production and by up-regulating the activities of the anti-ROS enzymes superoxide dismutase and catalase, and recovering GSH levels to normal. Moreover, rapamycin pre-treatment protected against colistin-induced mitochondrial dysfunction, caspase activation and subsequent apoptosis by up-regulating autophagy and activating the Akt/CREB, NGF and Nrf2 pathways, while inhibiting p53 signaling. Taken together, this is the first study to demonstrate that rapamycin protects against colistin-induced neurotoxicity by activating autophagy, inhibiting oxidative stress, mitochondria dysfunction and apoptosis. Our data highlight that regulating autophagy to rescue neurons from apoptosis may become a new targeted therapy to relieve the adverse neurotoxic effects associated with colistin therapy.
Mefenamic acid (MFA) is used as an anti-inflammatory, antinociceptive, and antipyretic agent for treatment of a wide range of pathological disorders. While the uncertainty of its safety and the poor oral bioavailability constitute the major limiting factors of its medical use, considerable efforts including liposomal encapsulation are needed to achieve maximum therapeutic advantages. The current work was conducted to investigate the ultrastructural alterations in the liver induced by free MFA and its liposomal preparation. Female Sprague-Dawley rats were treated with daily oral doses of either free MFA or MFA entrapped in Tween 80 inoculated liposomes at the concentration of 80 mg/kg for 28 days. Ultrathin sections were prepared from biopsies taken from the liver of each member of all animals under study and subjected to examination by transmission electron microscopy. The liver of rats that were exposed to liposomal MFA showed more ultrastructural alterations than the rats treated with the free drug. While both groups of rats demonstrated sinusoidal dilatation, Kupffer cell hyperplasia, mitochondrial damage, and nuclear alterations, rats treated with liposome-encapsulated MFA induced an increase in the multiple lysosomes formation, hepatocytic steatosis, and apoptotic activity than free MFA-treated rats. The ultrastructural findings of the present study indicate that the use of liposomal MFA induces more hepatic damage than the use of free MFA.
Introduction
Pain after surgical extraction of third molars has been a nemesis for oral surgeons with clinicians, thus striving for an analgesic modality. NSAIDs are among the most widely used therapeutic classes of analgesics. Transbuccal diclofenac sodium patches have been developed as an innovative drug delivery system using buccal mucosa as a gifted choice, hence overcoming first pass metabolism and offering the advantage of sustained drug delivery with reduced incidence of systemic adverse effects.
Aim
A comparative study was conducted to evaluate the efficacy of diclofenac sodium for pain control, administered via the far-fetched and gifted novel route through the transbuccal patch and by ever popular per oral route and also to assess the adverse effects vis-à-vis for transbuccal diclofenac patch and oral diclofenac following extraction of bilaterally symmetrical impacted mandibular third molars under local anaesthesia.
Methodology
Thirty healthy subjects of both the sexes in the age of 12 to 50 years with asymptomatic bilaterally symmetrical mandibular third molars underwent extraction under LA. It is a split-mouth study, i.e. after the extraction of tooth on one side, diclofenac sodium (50 mg) via oral route was given and then in another visit, when the same patient is comfortable and asymptomatic, extraction on contralateral side was executed and transbuccal patched diclofenac sodium (20 mg) was applied. Pain was measured on visual analog scale and verbal rating scale by the patient for 3 days and adverse effects if any were noted.
Result
Statistical analysis showed that transbuccal diclofenac sodium was significantly efficacious when compared to the drug administered orally. Also, statistically significant results were obtained in percentage reduction in pain from 1st to 3rd postoperative day in transbuccal group. No significant difference is seen for adverse reactions.
Conclusion
Transbuccal diclofenac sodium patch is more efficacious and can be used for pain control.
The liver is an important organ of the body, which has a vital role in metabolic functions. The non-steroidal anti-inflammatory drug (NSAID), diclofenac causes hepato-renal toxicity and gastric ulcers. NSAIDs are noted to be an agent for the toxicity of body organs. This review has elaborated various scientific perspectives of the toxicity caused by diclofenac and its mechanistic action in affecting the vital organ. This review suggests natural products are better remedies than current clinical drugs against the toxicity caused by NSAIDs. Natural products are known for their minimal side effects, low cost and availability. On the other hand, synthetic drugs pose the danger of adverse effects if used frequently or over a long period. Copyright
RP-HPLC method was developed in mobile phase containing Acetonitrile:Monobasic potassium dihydrogen phosphate (60:40,v/v) using C 8 Luna (150 mm × 4.6 mm id, 5+μm) at wavelength 215nm. The method was linear in the concentration range of 2-6μg/ml for DICY and 50-150μg/ml for MEF. The method was validated for linearity, accuracy and precision as per ICH guidelines. The developed and validated method was successfully used for the quantitative analysis of commercially available dosage form.
Drug-induced liver injury (DILI) induced by nonsteroidal antiinflammatory drugs (NSAIDs) is clearly idiosyncratic (i.e., host-dependent). Therefore, a discussion on mechanisms underlying the hepatic toxicity of NSAIDs includes both those that determine the compound-specific hazard and those that determine an individual's risk to develop DILI. Significant liver injury is confined to a relatively small number of NSAIDs rather than being based on a general pharmacological class effect or chemical family effect. Insights into the compound-specific mechanisms have mostly been obtained from cell or rodent studies with the model NSAID, diclofenac, and, to a lesser extent, sulindac or nimesulide. Toxicity is multifactorial and includes toxicokinetic factors (metabolic bioactivation and disposition), as well as toxicodynamic factors (disruption of mitochondrial function, endoplasmic reticulum stress, oxidant stress, apoptosis, activation of proinflammatory pathways, and innate or adaptive immune reactions). The patient-specific mechanisms are largely unknown, but a causal association between certain HLA haplotypes and NSAID-induced DILI has been emerging. Owing to the unique sensitivity of rodents to NSAID-induced gastrointestinal injury, there are no validated animal models available. Instead, efforts should be concentrated on analyzing DILI patient-specific determinants of susceptibility.
Background: Diclofenac sodium eye drops, pranoprofen eye drops and bromfenac sodium hydrate eye drops are three clinical commonly used nonsteroidal anti-inflammatory drugs (NSAIDs). The variation of cytoxicity among these drugs and whether the cytoxicity is related to the supplements are also unknown. Objective: This study was to compare the cytotoxicity of three non-steroidal anti-inflammatory eye drops and their active components with cultured human corneal epithelial cells (HCECs) in vitro, and to discuss toxic origins of these drugs. Methods: HCECs were cultured in different drugs with the final concentration of 0.10%, 0.05%, 0.02% and 0.01%. Cell proliferation was evaluated by MTT assay. Then, 0.002% eye drops (1:50) was added, and the migration and damage of the cells were deceted by transwell migration assay and lactate dehydrogenase (LDH) assay. Results: The cytotoxicity of three nonsteroidal anti-inflammatory eye drops on HCECs was concentration-dependent (all at P=0.00). Diclofenac sodium eye drops showed the most dominant effects on the proliferation, migration and damage of HCECs among the three eye drops, while bromfenac sodium eye drops showed the least effect on the cell damage (proliferation; Fdrug=20.25, P=0.00; migration: F=103.43, P=0.00; damage; Fdrug=164.16, P=0.00). Compared with the eye drops, their active components showed less cytoxicity. Pranoprofen appeared the least effects on the proliferation, migration and damage of HCECs (proliferation; Fdrug=332.27, P=0.00; migration; F=332.27, P=0.00; damage; Fdrug=154.83, P=0.00). Conclusions: The cytotoxicity of diclofenac sodium eye drops is more obvious than that of pranoprofen eye drops or bromfenac sodium hydrate eye drops. The cytotoxicity of the three eye drops originates from their supplements or the interaction between the supplements and active components.
The nonsteroidal antiinflammatory drug diclofenac causes rare but significant cases of serious hepatotoxicity, typically with a delayed onset (>1-3 months). Because there is no simple dose relationship and because liver injury cannot be reproduced in current animal models, individual patient-specific susceptibility factors have been evoked to account for the increased risk. While these patient factors have remained undefined, a number of molecular hazards have been characterized. Among these are metabolic factors (bioactivation by hCYP2C9 or hCYP3A4 to thiol-reactive quinone imines, activation by hUGT2B7 to protein-reactive acyl glucuronides and iso-glucuronides, and 4'-hydroxylation secondary to diclofenac glucuronidation), as well as kinetic factors (Mrp2-mediated concentrative transport of diclofenac metabolites into bile). From the toxicodynamic view, both oxidative stress (caused by putative diclofenac cation radicals or nitroxide and quinone imine-related redox cycling) and mitochondrial injury (protonophoretic activity and opening of the permeability transition pore) alone or in combination have been implicated in diclofenac toxicity. In some cases, immune-mediated liver injury is involved, inferred from inadvertent rechallenge data and from a number of experiments demonstrating T cell sensitization. Why certain underlying diseases (e.g., osteoarthritis) also increase the susceptibility to diclofenac hepatotoxicity is not clear. To date, cumulative damage to mitochondrial targets seems a plausible putative mechanism to explain the delayed onset of liver failure, perhaps even superimposed on an underlying silent mitochondrial abnormality. Increased efforts to identify both patient-specific risk factors and disease-related factors will help to define patient subsets at risk as well as increase the predictability of unexpected hepatotoxicity in drug development.
This study outlines the analysis of mitochondrial toxicity for a variety of pharmaceutical drugs extracted from Zhang et al. These chemicals were grouped into categories based upon structural similarity. Subsequently, mechanistic analysis was undertaken for each category to identify the Molecular Initiating Event driving mitochondrial toxicity. The mechanistic information elucidated during the analysis enabled mechanism-based structural alerts to be developed and combined together to form an in silico profiler. This profiler is envisaged to be used to develop chemical categories based upon similar mechanisms as part of the Adverse Outcome Pathway paradigm. Additionally, the profiler could be utilised in screening large dataset in order to identify chemicals with the potential to induce mitochondrial toxicity.
In this research, we investigated the cytotoxic mechanisms of one of the widely used pharmaceuticals that are regularly associated with the adverse effects on the liver, sometimes leading to acute liver failure, diclofenac. Diclofenac liver cytotoxicity was associated with reactive oxygen species (ROS) formation and lipid peroxidation which were inhibited by antioxidants and ROS scavengers, ferric chelator, inhibitors of reduced CYP2E1 and CYP2C9, mitochondrial permeability transition (MPT) pore sealing agents and endocytosis inhibitors. Incubation of hepatocytes with diclofenac caused rapid hepatocyte glutathione (GSH) depletion which is another marker of cellular oxidative stress. Most of the diclofenac-induced GSH depletion could be attributed to the expulsion of GSSG. Diclofenac cytotoxicity was also associated with mitochondrial injury, lysosomal membrane rupture and release of digestive proteases which were prevented by antioxidants, MPT pore sealing agents, lysosomotropic agents and inhibitors of cytochrome P450 isoenzymes. These events could cause cytochrome C release from the mitochondrial intramembrane space to cytosol. The cytochrome C release could trigger activation of caspase-3 and apoptosis. We finally concluded that diclofenac hepatotoxicity is a result of metabolic activation by CYP2E1 and CYP2C9 and ROS formation, leading to a mitochondrial/lysosomal toxic cross-talk in the liver hepatocytes.
Rhein, a compound found as a glucoside in the root of rhubarb, is currently a subject of interest for its antitumor properties. The apoptosis of tumor cell lines induced by rhein was observed, and the involvement of mitochondria was established, however the role of mitochondrial permeability transition (MPT) remains unknown. Here we report that MPT plays an important role in the apoptosis of human hepatocellular carcinoma Hep-G2 cells induced by rhein. After adding rhein to the isolated hepatic mitochondria, swelling effects and the leakage of Ca(2+) were observed. These alterations were suppressed by cyclosporin A (CsA), an MPT inhibitor. Furthermore, in Hep-G2 cells, the decrease of ATP production, the loss of mitochondrial transmembrane potential (MTP), the release of cytochrome c (Cyto c), and the activation of caspase-3 were also observed. These toxic effects of rhein can also be attenuated by CsA as well. Moreover, TUNEL assay confirmed that in the presence of CsA, rhein-induced apoptosis was largely inhibited. These results suggest that MPT plays a critical role in the pathogenesis of Hep-G2 cell injury induced by rhein, and imply that MPT may contribute to the anti-cancer activity of rhein.
In the present study, the mechanism of colistin-induced neurotoxicity was investigated with a focus on behavioral characters, mitochondrial ultrastructures and functions of the central nerve tissues in mice followed by administrating intravenously 15 (divided into two dose and 12h apart), 7.5 and 5mg/kgbw colistin sulfate for 1, 3 or 7 days successively. To assess the recoverability of colistin-induced neurotoxicity, the neurotoxicity was also examined on day 15 (8 post colistin sulfate administration for 7 days). The results showed that, the spontaneous activities of mice were significantly decreased on days 3 and 7 in the 15mg/kg group compared with the correspondingly control group. The abnormal ultrastructure changes of mitochondria were presented in their nervous tissues and changed in a dose- and time-dependent manner, e.g. severe vacuolation and fission on days 3 and 7 in the 15mg/kg group and more slight on day 7 in the 7.5mg/kg group. In addition, mitochondrial permeability transition (MPT), membrane potential (Δψ(m)) and activities of mitochondrial succinate dehydrogenase changed, showing that colistin affected the mitochondrial functions. The recoverability of colistin-induced neurotoxicity was showed and only slight injury occurred in the nerve tissues of mice on day 15 in the 15mg/kg group and it had no abnormal changes in the behavioral and neuropathology characters in mice on day 15 in the 7.5 and 5mg/kg groups. The results suggested that mitochondrial dysfunction might partly account for the mechanism of neurotoxicity induced by colistin sulfate.
Addition of high concentrations (>1 mM) of inorganic phosphate (Pi) or arsenate to Ca-loaded mitochondria was followed by increased rates of H2O2 production, membrane lipid peroxidation, and swelling. Mitochondrial swelling was only partially prevented either by butylhydroxytoluene,
an inhibitor of lipid peroxidation, or cyclosporin A, an inhibitor of the mitochondrial permeability transition pore. This
swelling was totally prevented by the simultaneous presence of these compounds. At lower Pi concentrations (1 mM), mitochondrial swelling is reversible and prevented by cyclosporin A, but not by butylhydroxytoluene.
In any case (low or high phosphate concentration) exogenous catalase prevented mitochondrial swelling, suggesting that reactive
oxygen species (ROS) participate in these mechanisms. Altogether, the data suggest that, at low Pi concentrations, membrane permeabilization is reversible and mediated by opening of the mitochondrial permeability transition
pore, whereas at high Pi concentrations, membrane permeabilization is irreversible because lipid peroxidation also takes place. Under these conditions,
lipid peroxidation is strongly inhibited by sorbate, a putative quencher of triplet carbonyl species. This suggests that high
Pi or arsenate concentrations stimulate propagation of the peroxidative reactions initiated by mitochondrial-generated ROS because
these anions are able to catalyze C-aldehyde tautomerization producing enols, which can be oxidized by hemeproteins to yield the lower C-aldehyde in the triplet state. This proposition was also supported by experiments using a model system consisting of phosphatidylcholine/dicethylphosphate
liposomes and the triplet acetone-generating system isobutanal/horseradish peroxidase, where phosphate and Ca cooperate to increase the yield of thiobarbituric acid-reactive substances.
Thein vitrointerference of some of most important nonsteroidal anti-inflammatory drugs (NSAIDs) with the respiration of rat kidney (renal cortex) mitochondria and ATP synthesis was evaluated. Acetylsalicylic acid, diclofenac sodium, mefenamic acid, and piroxicam both uncoupled and inhibited oxidative phosphorylation in mitochondria energized with glutamate plus malate or with succinate, while dipyrone only uncoupled and paracetamol only inhibited it. The drug concentrations affecting mitochondrial respiration were in the low to middle micromolar range for diclofenac, mefenamic acid, and piroxicam, and in the low millimolar range for acetylsalicylic acid, dipyrone, and paracetamol. The pattern of inhibition, except for the paracetamol, was similar to that expressed by the respiratory chain inhibitors. NSAIDs also inhibited the rate of ATP synthesis in mitochondria energized with glutamate plus malate, as well as the phosphorylation potential of mitochondria. The IC50values for rate of ATP synthesis, using 2 mMADP, were about 0.1 mMfor diclofenac sodium and mefenamic acid, 0.7 mMfor piroxicam, and in the range of 5–8 mMfor acetylsalicylic acid, dipyrone, and paracetamol. The potential for renal energetic cytotoxicity of NSAIDs is discussed considering their ability to interact with the oxidative phosphorylation in rat renal cortex mitochondria. A comparison is made with the interference of salicylate, the main metabolite of acetylsalicylic acid, and a classical uncoupler of oxidative phosphorylation.
The efflux of Ca2+ from mitochondria respiring at steady state, and much of uncoupler-induced Ca2+ efflux, is shown to be a consequence of the Ca2+-induced membrane transition (the Ca2+-induced transition is the Ca2+-dependent sudden increase in the nonspecific permeability of the mitochondrial inner membrane which occurs spontaneously when mitochondria are incubated under a variety of conditions (D. R. Hunter, R. A. Haworth, and J. H. Southard, 1976, J. Biol. Chem.251, 5069–5077)). Ca2+ release from mitochondria respiring at steady state is shown to be transitional by four criteria: (1) Ca2+ release is inhibited by Mg2+, ADP, and bovine serum albumin (BSA), all inhibitors of the transition; (2) release is selective for Ca2+ over Sr2+, a selectivity also found for the transition; (3) the time course of Ca2+ release is identical to the time course of the change in the mitochondrial population from the aggregated to the orthodox configuration; and (4) from kinetics, Ca2+ release from individual mitochondria is shown to occur suddenly, following a lag period during which no release occurs. Ca2+ release induced by uncoupler is shown to be mostly by a transitional mechanism, as judged by four criteria: (1) release of Ca2+ is ruthenium red-insensitive and is an order of magnitude faster than Sr2+ release which is ruthenium red-sensitive; (2) release of Ca2+ is strongly inhibited by keeping the mitochondrial NAD+ reduced; (3) the kinetics of Ca2+ release indicates a transitional release mechanism; and (4) uncoupler addition triggers the aggregated to orthodox configurational transition which, at higher levels of Ca2+ uptake, occurs in the whole mitochondrial population at a rate equal to the rate of Ca2+ release. Na2+-induced Ca2+ release was not accompanied by a configurational change; we therefore conclude that it is not mediated by the Ca2+-induced transition.
Linolenic, linoleic, oleic, palmitic and stearic acids (FFA) collapse the electrical potential of pea stem mitochondria in the absence or in the presence of 0.5 mM Mg2+. Higher concentrations of this cation (5 mM) lower the rate of dissipation caused by linoleic, oleic and palmitic acids, while abolishing that induced by stearic acid. Carboxyatractyloside and ADP do not reverse the FFA-induced collapse both in the presence or absence of Mg2+. EDTA, EGTA or BHT do not influence the dissipation caused by FFA that, in addition, is not linked to lipid peroxidation evaluated as malondialdehyde or conjugated diene formation. Only linolenic acid sustains a peroxidation which, however, appears to be caused by its own oxidation catalysed by lipoxygenases rather than by membrane lipoperoxidation induced by this free fatty acid. These results suggest that neither the ATP/ADP exchanger nor lipid peroxidation appear to be involved in FFA-induced uncoupling in pea stem mitochondria.
John A. Oates, M.D., Editor Alastair J.J. Wood, M.D., Associate Editor THE nonsteroidal antiinflammatory drugs (NSAIDs) are very commonly prescribed. This fact reflects the high prevalence of rheumatic diseases; approximately 8 percent of people have a rheumatic symptom at any one time. In 1984, it was estimated that nearly one in seven Americans was treated with an NSAID,1 and in 1986 100 million prescriptions were written for these drugs.2 During the past 30 years, there has been a substantial increase in the number of NSAIDs, but their availability varies markedly between countries.3 In the United States, at least until recently, . . .
It has been firmly established that the rapid uptake of Ca2+ by mitochondria from a wide range of sources is mediated by a uniporter which permits transport of the ion down its electrochemical gradient. Several mechanisms of Ca2+ efflux from mitochondria have also been extensively discussed in the literature. Energized mitochondria must expend a significant amount of energy to transport Ca2+ against its electrochemical gradient from the matrix space to the external space. Two separate mechanisms have been found to mediate this outward transport: a Ca2+/nNa+ exchanger and a Na(+)-independent efflux mechanism. These efflux mechanisms are considered from the perspective of available energy. In addition, a reversible Ca2(+)-induced increase in inner membrane permeability can also occur. The induction of this permeability transition is characterized by swelling of the mitochondria, leakiness to small ions such as K+, Mg2+, and Ca2+, and loss of the mitochondrial membrane potential. It has been suggested that the permeability transition and its reversal may also function as a mitochondrial Ca2+ efflux mechanism under some conditions. The characteristics of each of these mechanisms are discussed, as well as their possible physiological functions.
This chapter discusses the use of isolated perfused organs in hypoxia and ischemia/reperfusion oxidant stress. Several methods are available to detect reactive oxygen species in isolated organs. A sensitive method without the necessity of additional chemicals interfering with the biological system or the use of expensive equipment is to monitor the formation and cellular release of glutathione disulfide (GSSG) as an index for the activity of the endogenous defense system against reactive oxygen species. The method is based on the very rapid enzymatic dismutation of intracellularly generated superoxide to molecular oxygen and hydrogen peroxide, which is then reduced to water through glutathione peroxidase. Glutathione (GSH) provides the reducing equivalents for this reaction and is oxidized to its disulfide (GSSG). Most of the GSSG formed is immediately reduced back to GSH through glutathione reductase with the cofactor nicotinamide adenine dinucleotide phosphate (NADPH). In the liver, GSSG is secreted mainly into bile against a steep concentration gradient. The biliary portion of the total GSSG export is a relatively constant value of about 80 to 85% as long as no major interference occurs with bile formation. GSSG is released mainly into the perfusate in other organs such as heart and lung in contrast to the 15 to 20% released into the perfusate in the liver. Although, only a small percentage of the GSSG generated is released from the cells, any change in the overall GSSG formation is reflected by similar changes in the cellular efflux. Thus, a significant increase in the efflux of GSSG into perfusate or bile (liver) indicates an enhanced activity of the defense system against reactive oxygen species (oxidant stress).
Recent work has shown that a sustained increase in cytosolic Ca2+ concentration is often linked to the onset of cytotoxicity. Sten Orrenius and colleagues describe several biochemical mechanisms that are stimulated by such a Ca2+ increase and can directly mediate cell death by causing disruption of the cytoskeleton, DNA fragmentation and extensive damage to other cell components.
Dysfunction of mitochondria after oxidation of endogenous NAD(P)H, especially after calcium accumulation, has been abundantly reported, but the causes of membrane perturbations did not receive a full explanation. In light of several additional observations reported in this study, we propose a general scheme which shows the sequential processes that are likely involved in the appearance of calcium-induced membrane leakiness. Addition of acetoacetate, oxaloacetate, or ketomalonate to rotenone-treated mitochondria led to a massive oxidation of both NADH and NADPH. Under these conditions, stimulation of fatty acid oxidation could be observed. This process was shown to be accompanied by a reduction of intramitochondrial NADP+. The reduction of NADP+ was inhibited by uncouplers, electron transfer inhibitors and N,N'-dicyclohexylcarbodiimide. It was thus probably catalyzed by the mitochondrial transhydrogenase. Oxidation of pyridine nucleotides in the presence of acetoacetate induced (i) a slight decrease in the number of sulfhydryl groups reactive with N-ethylmaleimide (but no change in the amount of intramitochondrial reduced glutathione) and (ii) modifications of the kinetics and the orientation of the ADP/ATP carrier. In the presence of calcium ions, acetoacetate-stimulated fatty acid oxidation promoted an extensive swelling of mitochondria. Uptake of calcium ions into the matrix was a critical factor for triggering the swelling. Thiols, if they were added at a sufficiently high concentration, suppressed the swelling. Also ligands of the ADP/ATP carrier which stabilized the m-state conformation of the protein, exerted an efficient protective action. Three essential interacting factors emerge from this study: (i) The crucial role of the ADP/ATP carrier orientation in promoting the calcium-induced membrane destabilization. More precisely, it has been shown that the ADP/ATP carrier adopts the c-state conformation (i.e., nucleotide binding site facing the cytoplasm) during fatty acid oxidation. (ii) The modification of a very small number of sulfhydryl groups of mitochondrial protein. These groups are probably in an oxidized state when the level of reduced pyridine nucleotides is low. (iii) The prevailing role of the transhydrogenase, the function of which is also intimately associated with fatty acid oxidation. After energization, transhydrogenase can hinder thiol oxidation and therefore partially protect the membrane structure.
The effects of Reye's plasma, allantoin, and salicylates on mitochondrial structure and Ca2+ transport have been investigated. Measurements of Ca2+ transport showed that when 20-30 microM Ca2+ was added to isolated rat liver mitochondria preincubated with one of these agents, Ca2+ uptake was followed by its spontaneous release into the medium. This was accompanied by large-amplitude swelling; the onset preceded the Ca2+ release. No further Ca2+ release was induced by uncoupler or the Ca2+ ionophore, A23187. The mitochondria continued to swell even after all of the Ca2+ had been released. The time between the addition of Ca2+ and the onset of swelling (or Ca2+ release) depended on the concentration of the agent added and the preincubation time; the extent of swelling did not. These effects were prevented, but not reversed, by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid, ruthenium red, rotenone, or adenine nucleotides. The massive swelling and membrane disruption were confirmed by electron microscopy of the treated vs untreated mitochondria. Similar results concerning swelling and Ca2+ release were also seen with Ca2+ alone, but the time scale was much longer (i.e., greater than 3-4 min), indicating that these agents act by potentiating Ca2+-induced alterations in mitochondrial structure, as suggested by our earlier work (T.Y. Segalman and C.P. Lee (1982) Arch. Biochem. Biophys. 214, 522-530; M.E. Martens and C.P. Lee (1984) Biochem. Pharmacol. 33, 2869-2876). Our data show, therefore, that allantoin, salicylates, and the "toxic" agent in Reye's plasma severely limit the ability of isolated rat liver mitochondria to maintain their structural integrity under conditions of limited Ca2+ loading.
This chapter summarizes the chief methods in use for thiols in general. These methods exploit either of the two principal properties of the SH group—namely, its capacity for oxidation or substitution. Oxidizing agents may be of analytical value if they do not oxidize thiols further than disulfides (that is, to sulfinic or sulfonic acids) ⁷⁵Se-labeled and also give absorbance changes of sufficient magnitude. The chapter describes aromatic disulfides that fulfill these requirements best and, they are the reagents of first choice. Aromatic disulfides dominate the field because they are easily reduced by thiol-disulfide exchange, the equilibrium over-whelmingly favoring the formation of aromatic thiol. Mixed disulfides are stable intermediate products with protein thiols, but with nonprotein thiols they are themselves rapidly reduced. In either case, the overall stoichiometry is one aromatic thiol generated for each biological thiol originally present. Various organic oxidants methods described are lodosobenzoic acid, diphenylpicrylphenylhydrazine, benzofuroxan, quinines, 4, 4’-bisdimethylaminodiphenylcarbinol, and trinitrobenzenesulfonic acid. Various inorganic oxidants methods are also described. The chapter explores substitution products exploited for spectrophotometric assay that includes mercurials, nitrous acids, maleimides, halides, and others.
The nonsteroidal antiinflammatory drugs represent a valuable tool in the medical therapy of rheumatologic diseases. For this reason, their widespread use is understandable and generally justifiable. It is clear, however, that a wide array of clinical renal syndromes may be associated with their use. These include acute renal failure, for which patients with preexisting renal disease or compromised renal perfusion are at greatest risk; sodium retention and edema; hyponatremia; and hyperkalemia. These syndromes are probably attributable to the suppression by NSAIDs of renal prostaglandin biosynthesis. It is possible that an etiologic role of cyclooxygenase inhibitors in the development of papillary necrosis will also be established. In addition, nephrotic syndrome and acute interstitial nephritis have been reported with increased frequency in patients treated with NSAIDs. A role for altered prostaglandin metabolism in the pathogenesis of this disorder seems likely but remains to be established. At present we would urge physicians to exercise extreme caution in prescribing these drugs to patients who are at risk for renal complications. This high-risk group comprises patients with chronic renal insufficiency, congestive heart failure, hepatic cirrhosis, and volume contraction secondary to nephrotic syndrome or diuretic use. NSAID therapy should also be withheld from patients who are about to undergo surgery because of the risk of acute renal failures, as well as of impaired hemostasis due to the effects of these agents on platelet function. Patients receiving NSAIDs should have periodic monitoring of blood chemistry and renal function.
The effects of aspirin (acetylsalicylate, ASA) and related compounds in the presence of Ca2+ on the oxidative metabolism of isolated rat liver mitochondria were studied. Intact mitochondrial preparations preincubated with ASA + Ca2+ exhibited a transient stimulation of the state 4 respiratory rate with NAD+-linked substrates, followed by an inhibition which could not be released by the addition of ADP or uncoupler. Maximum respiratory rates were achieved by subsequent addition of NAD+ or succinate. The Ca2+-transport inhibitors ruthenium red and ethylene glycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) prevented these effects. Five brands of commercial aspirin were tested and were as effective as purified ASA. Tylenol (acetaminophen) could reproduce these effects only at much higher (greater than or equal to 10-fold) concentrations. Other salicyl derivatives showed results qualitatively similar to ASA, with potencies in the order: acid much much greater than ASA much greater than alcohol greater than or equal to catechol greater than amide, salicylate being approximately 10-fold more potent than ASA. The magnitude of the effect seen depended on the Ca2+ (endogenous + exogenous) and salicylate concentrations/mg mitochondrial protein, and on the length of the preincubation. Added inorganic phosphate was also required. That salicylate + Ca2+ induces an increase in the permeability of the mitochondrial inner membrane was demonstrated by the observation that 90% of the intramitochondrial NAD(P)+ was released into the surrounding medium upon preincubation of intact mitochondria with these agents. Salicylate + Ca2+ had virtually no effect on respiration with succinate (+ rotenone) as substrate at salicylate concentrations which markedly affected NAD+-linked substrate oxidation. The presence of rotenone in the preincubation mixture prevented the damaging effects of salicylate + Ca2+ on the mitochondrial membrane, suggesting that the redox state of intramitochondrial pyridine nucleotides can modulate these effects. The results reported here are similar to those reported previously by our laboratory for the effects of Reye's plasma and allantoin + Ca2+, and indicate that, like these agents, salicylate and salicyl compounds can potentiate the Ca2+-induced damage to the mitochondrial inner membrane and may be another factor responsible for Reye's syndrome.
In this paper we introduce monobromobimane, a thiol reagent, as a selective blocker of the recently identified dithiol whose oxidation-reduction status modifies voltage sensing by the mitochondrial permeability transition pore, a cyclosporin A-sensitive channel. Monobromobimane does not inhibit the phosphate carrier, nor does it interfere with Ca2+ transport, energy coupling or ATP production and transport. We show that monobromobimane selectively prevents the shift in pore gating potential caused by some dithiol oxidants or crosslinkers but not by increasing [Ca2+], allowing a clear distinction of the pore agonists which act at this site.
Treatment of isolated mitochondria with Ca2+ and inorganic phosphate (Pi) induces an inner membrane permeability that appears to be mediated through a cyclosporin A (CsA)-inhibitable Ca(2+)-dependent pore. Isolated mitochondria during inner membrane permeability undergo rapid efflux of matrix solutes such as glutathione as GSH and Ca2+, loss of coupled functions, and large amplitude swelling. Permeability transition without large amplitude swelling, a parameter often used to assess inner membrane permeability, has been observed. The addition of either oligomycin, antimycin, or sulfide to incubation buffer containing Ca2+ and Pi abolished large amplitude swelling of mitochondria. The GSH status during a Ca(2+)- and Pi-dependent mechanism of mitochondrial GSH release in isolated mitochondria was influenced significantly by metabolic inhibitors of the respiratory chain but did not prevent inner membrane permeability as demonstrated by the release of mitochondrial GSH and Ca2+. The release of GSH was inhibited by the addition of CsA, a potent inhibitor of permeability transition. Under these conditions we did not find GSSG; however, rapid oxidation of pyridine nucleotides and depletion of ATP and ADP with conversion to AMP occurred. The addition of CsA, prevented the oxidation of pyridine nucleotides and depletion of ATP and ADP. Since NADH and NADPH were extensively oxidized, protection against oxidative stress is reflected in maintenance of GSH and not observable lipid peroxidation. Evidence from transmission electron microscopy analysis, combined with the GSH release data, indicate that permeability transition can be observed in the absence of large amplitude swelling.
Nephrotoxicity can be grouped by the xenobiotics place of action, by the clinical presentation or by the generic toxic effect. The latter can be dose related, indirect, idiosyncratic or allergic. Nephrotoxicity of lithium, demeclocycline, aminoglycosides, cyclosporine, mercuric ion, nonsteroidal anti-inflammatory drugs, methoxyflurane, ethylene glycol, D-penicillamine and methicillin is reviewed in light of all these three viewpoints, but emphasis is on toxic mechanisms.
The mitochondrial inner membrane can undergo a permeability increase known as "permeability transition" elicited by Ca2+ and several other inducing agents. In general, the condition of oxidative stress acts as an inducer, at variance with antioxidants and reducing agents that inhibit the permeability transition. The action of mitochondrial respiratory substrates in preventing the permeability transition induced by Ca2+ and phosphate was examined; pyruvate, isocitrate, and glutamate proved to be particularly effective. The effect of substrates was evident also in the presence of an uncoupler, and, in addition, they were able to counteract the swelling stimulated by acetoacetate and tert-butylhydroperoxide. In the presence of various pyridine nucleotide-dependent substrates, mitochondria are able to reduce the disulfide 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) to an extent far larger than that calculated from the theoretical amount of total mitochondrial thiol groups, indicating the occurrence of a catalytic system. Similarly, the enzymes of the mitochondrial matrix in the presence of either NADH or NADPH are able to reduce DTNB. The results are discussed considering the existence of a close redox communication between pyridine nucleotides and membrane thiol groups, possibly mediated by dithiols such as thioredoxin and lipoic acid.
The mitochondrial permeability transition pore allows solutes with a m.w. approximately less than 1500 to equilibrate across the inner membrane. A closed pore is favored by cyclosporin A acting at a high-affinity site, which may be the matrix space cylophilin isozyme. Early results obtained with cyclosporin A analogs and metabolites support this hypothesis. Inhibition by cyclosporin does not appear to require inhibition of calcineurin activity; however, it may relate to inhibition of cyclophilin peptide bond isomerase activity. The permeability transition pore is strongly regulated by both the membrane potential (delta psi) and delta pH components of the mitochondrial protonmotive force. A voltage sensor which is influenced by the disulfide/sulhydryl state of vicinal sulfhydryls is proposed to render pore opening sensitive to delta psi. Early results indicate that this sensor is also responsive to membrane surface potential and/or to surface potential gradients. Histidine residues located on the matrix side of the inner membrane render the pore responsive to delta pH. The pore is also regulated by several ions and metabolites which act at sites that are interactive. There are many analogies between the systems which regulate the permeability transition pore and the NMDA receptor channel. These suggest structural similarities and that the permeability transition pore belongs to the family of ligand gated ion channels.
NSAIDs pose little threat of renal insult in normal, healthy persons at therapeutic dosages. However, NSAID administration to susceptible persons may cause decrements in renal plasma flow and glomerular filtration rate within hours. Such acute noxious renal effects are mediated by products of arachidonic acid metabolism. Precipitous decrements in glomerular filtration and renal ischemia, manifested by increased serum creatinine and urea nitrogen, are possible. However, these effects are usually fully reversible with prompt discontinuation of the offending NSAID. Risk factors for the development of these acute renal effects are known. Acute interstitial nephritis with or without nephrotic syndrome is a rare form of renal toxicity that typically occurs between 2-18 months of use. Renal impairment may be so severe as to require temporary hemodialysis; however, renal function usually returns to normal upon discontinuation of the NSAID. The mechanism of acute interstitial nephritis is presumed to be of allergic origin but could also be caused by a reactive metabolite. Fenoprofen use appears to be associated with a much higher risk for its development. In contrast to the acute effects of NSAIDs, irreversible, analgesic-associated nephropathy manifested by papillary necrosis and chronic interstitial nephritis may occur following months to years of high doses of analgesic mixtures. The mechanism by which combination analgesics produce this form of renal injury is unknown and could be either a result of medullary ischemia or a direct effect of a reactive metabolite. An important issue to be resolved is the relationship between the acute, reversible, prostaglandin-mediated renal effects of the NSAIDs and chronic, irreversible destruction, if such a relationship exists. Theoretically, continual or repeated decrements in renal function in patients with predisposing risk factors could cause or contribute to progressive deterioration in renal function. Elevations in blood pressure or interference with the effects of antihypertensive medications could theoretically also contribute to long-term renal deterioration. In addition to renal syndromes caused by NSAIDs that result in renal impairment, other transient effects on electrolyte and water metabolism may also occur. Reduced secretion of sodium may result in formation of edema, exacerbation of heart failure, or increased blood pressure. Hyporeninemic-hypoaldosteronism may produce hyperkalemia. Finally, reduced excretion of water has rarely caused hyponatremia. It has been suggested that NSAIDs may be renoprotective in patients with nephrotic syndrome. Others have suggested that sulindac is "renal-sparing" because of a unique metabolic pathway that supposedly limits the exposure of the kidney to the active sulfide metabolite.(ABSTRACT TRUNCATED AT 400 WORDS)
Studies were undertaken to investigate the principal actions underlying mercury-induced oxidative stress in the kidney. Mitochondria from kidneys of rats treated with HgCl2 (1.5 mg/kg i.p.) demonstrated a 2-fold increase in hydrogen peroxide (H2O2) formation for up to 6 hr following Hg(II) treatment using succinate as the electron transport chain substrate. No increase in H2O2 formation was observed when NAD-linked substrates (malate/glutamate) were used, suggesting that Hg(II) affects H2O2 formation principally at the ubiquinone-cytochrome b region of the mitochondrial respiratory chain in vivo. Together with increased H2O2 formation, mitochondrial glutathione (GSH) content was depleted by more than 50% following Hg(II) treatment, whereas formation of thiobarbiturate reactive substances (TBARS), indicative of mitochondrial lipid peroxidation, was increased by 68%. Studies in vivo revealed a significant concentration-related depolarization of the inner mitochondrial membrane following the addition of Hg(II) to mitochondria isolated from kidneys of untreated rats. This effect was accompanied by significantly increased H2O2 formation, GSH depletion and TBARS formation linked to both NADH dehydrogenase (rotenone-inhibited) and ubiquinone-cytochrome b (antimycin-inhibited) regions of the electron transport chain. Oxidation of pyridine nucleotides (NAD[P]H) was also observed in mitochondria incubated with Hg(II) in vitro. In further studies in vitro, the potential role of Ca2+ in Hg(II)-induced mitochondrial oxidative stress was investigated. Ca2+ alone (30-400 nmol/mg protein) produced no increase in H2O2 and only a slight increase in TBARS formation when incubated with kidney mitochondria isolated from untreated rats. However, Ca2+ significantly increased H2O2 and TBARS formation elicited by Hg(II) at the ubiquinone-cytochrome b region of the mitochondrial electron transport chain, whereas TBARS formation was decreased significantly when the Ca2+ uptake inhibitors, ruthenium red or [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), were included with Hg(II) in the reaction mixtures. These findings support the view that Hg(II) causes depolarization of the mitochondrial inner membrane with consequent increased H2O2 formation. These events, coupled with Hg(II)-mediated GSH depletion and pyridine nucleotide oxidation, create an oxidant stress condition characterized by increased susceptibility of mitochondrial membranes to iron-dependent lipid peroxidation (TBARS formation). Since increased H2O2 formation, GSH depletion and lipid peroxidation were also observed in vivo following Hg(II) treatment, these events may underlie oxidative tissue damage caused by mercury compounds. Moreover, Hg(II)-induced alterations in mitochondrial Ca2+ homeostasis may exacerbate Hg(II)-induced oxidative stress in kidney cells.
In this study, we show that mitochondrial membrane permeability transition in Ca(2+)-loaded mitochondria treated with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) or inorganic phosphate (P(i)) is preceded by enhanced production of H2O2. This production is inhibited either by ethylene glycobis(b-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) or Mg2+, but not by cyclosporin A. Permeability transition is prevented either by EGTA, catalase or dithiothreitol, suggesting the involvement of Ca2+, H2O2 and oxidation of membrane protein thiols in this mechanism. When mitochondria are incubated under anaerobiosis, no permeabilization or H2O2 production occurs. Based on these results we conclude that mitochondrial permeability transition induced by P(i) or FCCP-uncoupling is dependent on mitochondrial-generated reactive oxygen species.
The renal effects of nonsteroidal anti-inflammatory drugs are reviewed with special emphasis on the clinical, pathophysiologic, and risk factors for acute renal failure. Renal papillary necrosis and chronic renal insufficiency can occur with the prolonged use of these drugs, although the prevalence of this manifestation of nonsteroidal anti-inflammatory drug nephrotoxicity is unknown. Current recommendations based on a critical literature survey are provided, along with a list of suggested areas in which more research is needed.
The permeability transition pore (MTP) is a high conductance channel of the mitochondrial inner membrane inhibited by cyclosporin A. While the physiological role of the MTP has not been clarified yet, it is becoming clear that this channel plays an important role in the pathways leading to cell death. The recent demonstrations that the MTP is controlled by the membrane potential, that a variety of physiological and pathological effectors can modulate the threshold voltage at which pore opening occurs, and that surface potential may contribute to pore modulation provide a useful framework to describe the mechanistic aspects of pore function in isolated mitochondria. Here we (i) briefly review the key features of pore regulation, and report our recent progress on the role of oxidants and mitochondrial cyclophilin; and (ii) elaborate on how MTP regulation by cellular pathophysiological effectors (such as cytosolic [Ca2+] transients, oxidative stress, and changes in the concentration of polyamines, nitric oxide, and metabolites of both the sphingomyelin and phospholipase A2 pathways) might take place in vivo. Further definition of the MTP checkpoints should help in the design of specific modulators, and offers great promise for the development of new conceptual and pharmacological tools aimed at therapeutic intervention in pathological conditions where pore opening is a critical event.
Programmed cell death, or apoptosis, has in the past few years undoubtedly become one of the most intensively investigated biological processes. However, fundamental questions concerning the molecular and biochemical mechanisms remain to be elucidated. The central question concerns the biochemical steps shared by the numerous death induction pathways elicited by different stimuli. Heterogeneous death signals precede a common effector phase during which cells pass a threshold of 'no return' and are engaged in a degradation phase where they acquire the typical onset of late apoptosis. Alterations in mitochondrial permeability transition linked to membrane potential disruption precede nuclear and plasma membrane changes. In vitro induction of permeability transition in isolated mitochondria provokes the release of a protein factor capable of inducing nuclear chromatin condensation and fragmentation. This permeability transition is regulated by multiple endogenous effectors, including members of the bcl-2 gene family. Inhibition of these effects prevents apoptosis.
The extent of swelling undergone by deenergized mitochondria incubated in KCl/sucrose medium in the presence of Ca2+ alone or Ca2+ and t-butyl hydroperoxide decreases by decreasing molecular oxygen concentration in the reaction medium; under anaerobiosis no swelling occurs. This swelling is also inhibited by the presence of exogenous catalase or by the Fe2+ chelator o-phenanthroline in a time-dependent manner. The production of protein thiol cross-linking that leads to the formation of protein aggregates induced by Ca2+ and t-butyl hydroperoxide does not occur when mitochondria are incubated in anaerobic medium or in the presence of o-phenanthroline. In addition, it is also shown that the yield of stable methyl radical adducts, obtained from rat liver mitochondria treated with t-butyl hydroperoxide and the spin trap DMPO, is reduced by addition of EGTA and increases by addition of Ca2+ ions. These data support the hypothesis that Ca2+ ions stimulate electron leakage from the respiratory chain, increasing the mitochondrial production of reactive oxygen species.
At low drug levels, sodium salicylate and other drugs which uncouple oxidative phosphorylation in liver and muscle mitochondria selectively inhibit (i) the incorporation of inorganic phosphate into organic phosphates and (ii) the incorporation of inorganic sulphate into polysaccharide sulphates by bovine cartilage in vitro, without inhibiting the oxidation of glucose or octanoate by this tissue. Processes (i) and (ii) are also inhibited by respiratory inhibitors (nitrogen atmosphere, cyanide). It is concluded that salicylate will uncouple oxidative phosphorylation in connective tissues, as well as in muscle, kidney, liver etc.The relationship of chemical structure to activity in uncoupling phosphorylation in cartilage and in rat liver mitochondria, was studied for 80 salicylate analogues. The partial structures given in figure 1 were essential for uncoupling activity: within this structural requirement, potency was governed by lipophilic character.The following compounds were notably more active than salicylate in vitro: o.hydroxynaphthoates, phenylsalicylates, 2-mercaptobenzoate and lipophilic N-substituted derivatives of PAS, salicylamide and anthranilic acid. N-Salicyloylanthranilate, 3,5-diiodosalicylate and 5-phenylazosalicylate were the most active drugs in vitro uncovered in this survey. Nitrosalicylates were much less active than the corresponding nitrophenols.
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